US20050163782A1 - Modified binding molecules comprising connecting peptides - Google Patents

Modified binding molecules comprising connecting peptides Download PDF

Info

Publication number
US20050163782A1
US20050163782A1 US10/880,028 US88002804A US2005163782A1 US 20050163782 A1 US20050163782 A1 US 20050163782A1 US 88002804 A US88002804 A US 88002804A US 2005163782 A1 US2005163782 A1 US 2005163782A1
Authority
US
United States
Prior art keywords
domain
binding
composition
polypeptide
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/880,028
Other languages
English (en)
Inventor
Scott Glaser
Mitchell Reff
Tzung-Horng Yang
Xiufeng Wu
Paul Chinn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biogen MA Inc
Original Assignee
Biogen Idec MA Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biogen Idec MA Inc filed Critical Biogen Idec MA Inc
Priority to US10/880,028 priority Critical patent/US20050163782A1/en
Assigned to BIOGEN IDEC MA INC. reassignment BIOGEN IDEC MA INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YANG, TZUNG-HORNG, CHINN, PAUL, GLASER, SCOTT, REFF, MITCHELL, WU, XIUFENG
Publication of US20050163782A1 publication Critical patent/US20050163782A1/en
Assigned to BIOGEN IDEC MA INC. reassignment BIOGEN IDEC MA INC. CORRECTIVE COVERSHEET TO CORRECT ATTORNEY DOCKET NUMBER IDV-005 THAT WAS PREVIOUSLY RECORDED ON REEL 016395 FRAME 0885. Assignors: YANG, TZUNG-HORNG, CHINN, PAUL, GLASER, SCOTT, REFF, MITCHELL, WU, XIUFENG
Priority to US12/100,375 priority patent/US8603473B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/624—Disulfide-stabilized antibody (dsFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Antibodies are dimeric molecules; each monomer making up the dimer comprises one light and one heavy chain. Solutions of antibody molecules exist in two forms associated with hinge heterogeneity. Using SDS-PAGE analysis of purified Mab MAb, typically the two forms are observed as two protein bands, a major band (MW approximately 150-160 kDa) and a minor band (MW approximately 75-80 kDa). This latter form is typically observed after SDS-PAGE analysis of purified IgG4 preparations, but can be identified at much lower frequencies in all IgG isotypes, including purified, recombinant MAbs (Angal et al. 1993 . Mol. Immunol . 30:105; Norderhaug et al. 1990 . Eur.
  • Form A contains covalent interchain disulfide bonds at positions corresponding to 239 and 242, Kabat numbering system (positions 226 and 229, EU numbering system) (Kabat, E, Wu, T T, Perry, H M, Gottesman, K S, Foeller, C: Sequences of Proteins of Immunological Interest. Bethesda, US Department of Health and Human Services, NIH, 1991).
  • the second isoform, Form B is thought to contain no covalent linkages between the two heavy chains and an intrachain disulfide bond between the two neighboring cysteine residues as evidenced by the 75-80 kDa seen in non-reducing SDS-PAGE electrophoresis.
  • the two heavy chains of Form B are presumably held together by strong non-covalent (e.g., ionic) interactions associated with the CH3 domain region of the molecule.
  • These mixtures of A and B forms are not present in solutions of MAb fragments that contain an intact hinge, but lack a CH3 domain, such as, for example, F(ab) 2 fragments.
  • genetically engineered or enzymatically digested F(ab) 2 MAb preparations lack the B-form, since the molecule lacks the necessary domains for maintaining non-covalent interactions (e.g., hydrogen bonding). However, they are present in MAb preparations that do contain a CH3 domain, such as IgG4, CH2 domain deleted MAb fragments (e.g., as described in 02/060955 A2) and minibodies (see, e.g., Hu et al. 1996. Cancer Research 56:3055).
  • CH2 domain deleted antibodies have a molecular mass of approximately 120 kDa and have been shown to penetrate tumors significantly better than full length IgG.
  • Minibodies which also have deletion of the CH2 domain, have similar characteristics. These domain deleted molecules accumulate at tumor sites more efficiently than other MAb fragments, such as F(ab)′ 2 s, but without the unfavorable pharmacodynamic profiles seen with intact IgG antibody.
  • CH2 domain deleted antibodies consist of a VLCL light chain and a VH1 heavy chain domain and a portion of the hinge region (e.g., the upper and middle hinge) genetically fused (either directly or through a modified peptide spacer) to a CH3 domain.
  • CH2 domain deleted ddCC49 a domain deleted antibody that recognizes the tumor associated TAG72 antigen expressed on a variety of human carcinomas
  • a and B isoforms in approximately 50:50 distribution in cell cultures.
  • Cells engineered to express alternative forms of CH2 domain deleted antibodies for example, tetravalent CH2 domain deleted antibodies, minibodies, or tetravalent minibodies also express a mixture consisting of A and B isoforms and/or monomeric half-mer molecules.
  • Form A and Form B are extremely difficult to separate even after MAb purification, since they are composed of identical amino acids and, therefore, have identical molecular weight and similar physical and chemical properties. They cannot be separated by standard gel filtration, affinity chromatography, or ion exchange chromatography typically used to purify antibody molecules, including recombinant MAb proteins. Current manufacturing processes discard at least 50% of the total antibody produced, having a negative impact on overall yield. Moreover, the presence of the two isoforms increases efforts required for downstream processing. Thus, a method of separating forms A and B or of increasing biosynthesis of one or the other form of antibody would be of great benefit.
  • the invention is based, at least in part, on the finding that in a composition comprising a mixture of dimeric polypeptide molecules comprising different isoforms (molecules comprising two heavy chain portions in which a fraction of the molecules comprise two heavy chain portions that are linked via at least one interchain disulfide linkage (Form A) and a portion of the molecules comprise two heavy chain portions that are not linked via at least one interchain disulfide linkage (Form B)) one form or the other can be preferentially obtained, e.g., by separation using hydrophobic interaction chromatography or by inclusion of synthetic connecting peptides which result in the preferential biosynthesis of either Form A or Form B.
  • a composition comprising a mixture of dimeric polypeptide molecules comprising different isoforms (molecules comprising two heavy chain portions in which a fraction of the molecules comprise two heavy chain portions that are linked via at least one interchain disulfide linkage (Form A) and a portion of the molecules comprise two heavy chain portions that are not linked via at least one
  • the binding molecules of the invention are tetravalent.
  • the connecting peptides of the invention can be included in any dimeric molecule that tends to form both Form A and Form B, e.g., antibody molecules, domain deleted antibody molecules (e.g., lacking all or part of a CH2 domain), minibodies, diabodies, fusion proteins, etc.
  • Form A is enhanced.
  • the invention pertains to a composition
  • a composition comprising polypeptide dimers comprising at least four binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain portion and a synthetic connecting peptide, and wherein greater than about 50% of the dimers are linked via at least one interchain disulfide linkage.
  • greater than about 90% of the dimers are linked via at least one interchain disulfide linkage.
  • At least one of the polypeptide chains comprises a CH3 domain linked to a VL, VH or CH1 domain via the connecting peptide.
  • polypeptide chains lack all or part of a CH2 domain.
  • the dimers are linked via two or more interchain disulfide linkages.
  • the heavy chain portion is derived from an antibody of an isotype selected from the group consisting of: IgG1, IgG2, IgG3, and IgG4.
  • the heavy chain portion comprises an amino acid sequence derived from a hinge region selected from the group consisting of: a ⁇ 1 hinge, a ⁇ 2 hinge, a ⁇ 3 hinge, and a ⁇ 4 hinge.
  • the molecules are bispecific.
  • the molecules comprise at least one binding site specific for a soluble ligand.
  • the molecules comprise at least one binding site specific for a cell surface molecule.
  • the molecules comprise two binding sites specific for a tumor cell antigen and two binding sites specific for a prodrug.
  • the binding sites specific for the prodrug are catalytic.
  • the synthetic connecting peptide comprises a proline residue at position 243, Kabat numbering system.
  • the synthetic connecting peptide further comprises an alanine residue at position 244 and a proline residue at position 245, Kabat numbering system.
  • the heavy chain portion comprises a chimeric hinge.
  • the synthetic connecting peptide comprises at least a portion of an IgG1 hinge domain, at least a portion of an IgG3 hinge domain.
  • the connecting peptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8-15 and 48.
  • the invention provides a method of treating a subject that would benefit from treatment with an antigen binding molecule comprising administering to the subject a composition comprising a mixture of dimeric polypeptide molecules comprising different isoforms, wherein one isoform or the other is preferentially obtained, such that treatment occurs.
  • the subject is suffering from cancer.
  • the subject is suffering from lymphoma.
  • the subject is suffering from an autoimmune disease or disorder.
  • the subject is suffering from an inflammatory disease or disorder.
  • the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide molecule comprising different isoforms (molecules comprising two heavy chain portions in which a fraction of the molecules comprise two heavy chain portions that are linked via at least one interchain disulfide linkage (Form A) and a portion of the molecules comprise two heavy chain portions that are not linked via at least one interchain disulfide linkage (Form B)) such that one form or the other can be preferentially obtained.
  • a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide molecule comprising different isoforms (molecules comprising two heavy chain portions in which a fraction of the molecules comprise two heavy chain portions that are linked via at least one interchain disulfide linkage (Form A) and a portion of the molecules comprise two heavy chain portions that are not linked via at least one interchain disulfide linkage (Form B)) such that one form or the other can be preferentially obtained
  • the polypeptide dimers comprise four polypeptide chains and wherein two of the polypeptide chains comprise at least one heavy chain portion and a synthetic connecting peptide.
  • the nucleic acid molecule comprises a nucleotide sequence shown in FIG. 8B (SEQ ID NO: 17).
  • the nucleic acid molecule comprises a nucleotide sequence shown in FIG. 8C (SEQ ID NO: 18).
  • the nucleic acid molecule comprises a nucleotide sequence shown in FIG. 10B (SEQ ID NO: 23).
  • the nucleic acid molecule comprises a nucleotide sequence shown in FIG. 12A (SEQ ID NO:26).
  • the nucleic acid molecule comprises a nucleotide sequence shown in FIG. 12B (SEQ ID NO:27).
  • the nucleic acid molecule comprises a nucleotide sequence shown in FIG. 14 (SEQ ID NO:30).
  • the nucleic acid molecule comprises a nucleotide sequence shown in FIG. 15 (SEQ ID NO:31).
  • the nucleic acid molecule is in a vector.
  • the vector is in a host cell.
  • the invention provides binding molecules comprising an amino acid sequence encoding a polypeptide molecule comprising different isoforms (molecules comprising two heavy chain portions in which a fraction of the molecules comprise two heavy chain portions that are linked via at least one interchain disulfide linkage (Form A) and a portion of the molecules comprise two heavy chain portions that are not linked via at least one interchain disulfide linkage (Form B)) such that one form or the other can be preferentially obtained.
  • a polypeptide molecule comprising different isoforms molecules comprising two heavy chain portions in which a fraction of the molecules comprise two heavy chain portions that are linked via at least one interchain disulfide linkage (Form A) and a portion of the molecules comprise two heavy chain portions that are not linked via at least one interchain disulfide linkage (Form B)
  • the binding molecule comprises the amino acid sequence of FIG. 9B (SEQ ID NO: 20).
  • the binding molecule comprises the amino acid sequence of FIG. 9C (SEQ ID NO: 21).
  • the binding molecule comprises the amino acid sequence of FIG. 11B (SEQ ID NO: 25).
  • the binding molecule comprises the amino acid sequence of FIG. 13A (SEQ ID NO: 28).
  • the binding molecule comprises the amino acid sequence of FIG. 13B (SEQ ID NO: 29).
  • the binding molecule comprises the amino acid sequence of FIG. 16 (SEQ ID NO: 32).
  • the binding molecule comprises the amino acid sequence of FIG. 17 (SEQ ID NO: 33).
  • the invention provides a composition comprising a mixture of dimeric polypeptide molecules comprising different isoforms (molecules comprising two heavy chain portions in which a fraction of the molecules comprise two heavy chain portions that are linked via at least one interchain disulfide linkage (Form A) and a portion of the molecules comprise two heavy chain portions that are not linked via at least one interchain disulfide linkage (Form B)) one form or the other can be preferentially obtained, and wherein said binding sites are individually selected from the group consisting of: an antigen binding site, a ligand binding portion of a receptor, and a receptor binding portion of a ligand.
  • the polypeptide chains have at least one binding site derived from an antibody selected from the group consisting of: 2B8, Lym 1, Lym 2, LL2, Her2, B1, MB1 BH3, B4, B72.3, CC49, 5E8, B3F6, and 5E10.
  • polypeptide dimers are tetravalent minibody molecules.
  • polypeptide dimers are tetravalent domain deleted antibody molecules.
  • polypeptide dimers are diabodies.
  • the invention provides a composition comprising minibody molecules comprising two polypeptide chains, wherein the polypeptide chains comprise a heavy chain portion and a synthetic connecting peptide, wherein the polypeptide chains lack all or part of a CH2 domain, and wherein greater than about 50% of the molecules are present in a form in which one of the polypeptide chains are linked via at least one interchain disulfide linkage.
  • greater than about 90% of the dimers are linked via at least one interchain disulfide linkage.
  • At least one of the polypeptide chains comprises a CH3 domain genetically fused to a VL, VH or CH1 domain via the connecting peptide.
  • polypeptide chains lack the entire CH2 domain.
  • the dimers are linked via two or more interchain disulfide linkages.
  • the heavy chain portion is derived from an antibody of an isotype selected from the group consisting of: IgG1, IgG2, IgG3, and IgG4.
  • the heavy chain portion comprises an amino acid sequence is derived from a hinge region selected from the group consisting of: a ⁇ 1 hinge, a ⁇ 2 hinge a ⁇ 3 hinge, and a ⁇ 4 hinge.
  • the binding sites are individually selected from the group consisting of: an antigen binding site, a ligand binding portion of a receptor, and a receptor binding portion of a ligand.
  • the molecules are bispecific.
  • the connecting peptide comprises a proline residue at position 243, Kabat numbering system.
  • the synthetic connecting peptide comprises a chimeric hinge.
  • the synthetic connecting peptide comprises at least a portion of an IgG1 hinge domain, at least a portion of an IgG3 hinge domain.
  • the invention provides a method of treating a subject that would benefit from treatment with an antigen binding molecule comprising administering to the subject a composition comprising minibody molecules comprising two polypeptide chains, wherein the polypeptide chains comprise a heavy chain portion and a synthetic connecting peptide, wherein the polypeptide chains lack all or part of a CH2 domain, and wherein greater than about 50% of the molecules are present in a form in which one of the polypeptide chains are linked via at least one interchain disulfide linkage, such that treatment occurs.
  • the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain comprising a heavy chain portion and a synthetic connecting peptide, wherein the polypeptide chains lack all or part of a CH2 domain, and wherein greater than about 50% of the molecules are present in a form in which one of the polypeptide chains are linked via at least one interchain disulfide linkage.
  • the invention provides a composition comprising polypeptide dimers having at least four binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain portion and lacks all or part of a CH2 domain, wherein greater than 50% of the polypeptide dimers are linked via at least one interchain disulfide linkage.
  • FIG. 1 shows Form A which appears as a 120 kDa dimer and Form B which appears as a 60 kDa monomer in domain deleted antibodies.
  • FIG. 2 shows a schematic diagram of an exemplary two chain dimeric minibody and an exemplary two chain dimeric tetravalent minibody each comprising a hinge connecting peptide (HCP).
  • HCP hinge connecting peptide
  • the two chain dimeric tetravalent minibody comprising a connecting peptide (HCP) can also be constructed to be bispecific.
  • the orientation of the VH and VL domains in the scFv may be changed.
  • FIG. 3 shows a schematic diagram of a four chain dimeric diabody comprising a hinge connecting peptide (HCP).
  • HCP hinge connecting peptide
  • the four chain dimeric diabody comprising a hinge connecting peptide (HCP) can also be constructed to be bispecific. The orientation of the VH and VL domains may be changed.
  • FIG. 4 shows a schematic diagram of a four chain dimeric tetravalent scFv antibody (C-scFv tetravalent antibody) and a four chain tetravalent scFv CH2 domain deleted antibody (C-scFv tetravalent CH2 domain deleted antibody) each comprising a scFv appended to the carboxyl terminus of CH3 and a hinge connecting peptide.
  • the orientation of the VH and VL domains in the scFv may be changed.
  • FIG. 5 shows a schematic diagram of a four chain dimeric CH2 domain deleted tetravalent (N L -scFv CH2 domain deleted tetravalent) antibody comprising a hinge connecting peptide (HCP).
  • the four chain dimeric CH2 domain deleted tetravalent antibody comprising a connecting peptide (HCP) can also be constructed to be bispecific. The orientation of the VH and VL domains in the scFv appended to the light chain may be changed.
  • FIG. 6 shows a schematic diagram of a four chain dimeric CH2 domain deleted tetravalent (N H -scFv CH2 domain deleted tetravalent) antibody comprising a hinge connecting peptide (HCP).
  • the four chain dimeric CH2 domain deleted tetravalent antibody comprising a connecting peptide (HCP) can also be constructed to be bispecific. The orientation of the VH and VL domains in the scFv appended to the heavy chain may be changed.
  • FIG. 7 shows a schematic diagram of a two chain dimeric tetravalent minibody (C-scFv tetravalent minibody) comprising a hinge connecting peptide (HCP).
  • the two chain dimeric tetravalent minibody comprising a connecting peptide (HCP) can also be constructed to be bispecific. The orientation of the VH and VL domains in both scFvs may be changed.
  • FIG. 8A shows the single-stranded DNA sequence of heavy chain CH2 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) huCC49 gene.
  • FIG. 8B shows the single-stranded DNA sequence of heavy chain tetravalent CH2 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) huCC49 gene containing the synthetic G1/G3:/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 16 shows the single-stranded DNA sequence of heavy chain CH2 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) huCC49 gene containing the synthetic G1/G3:/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • SEQ ID NO: 18 shows the single-stranded DNA sequence of light chain CH2 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) huCC49.
  • FIG. 9A shows the amino acid sequence of heavy chainCH2 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) huCC49.
  • FIG. 9B shows the amino acid sequence of heavy chain CH2 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) huCC49 containing the synthetic G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 9C shows the amino acid sequence of light chain CH2 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) huCC49
  • FIG. 10A shows the single-stranded DNA sequence of CH2 domain-deleted huCC49 tetravalent (N-scFv tetravalent) minibody gene.
  • FIG. 10B shows the single-stranded DNA sequence of tetravalent CH2 domain-deleted (N-scFv tetravalent) huCC49 tetravalent minibody gene containing the synthetic G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 11A shows the amino acid sequence of tetravalent CH2 domain-deleted (N-scFv tetravalent) huCC49 minibody.
  • FIG. 11B shows the amino acid sequence of tetravalent CH2 domain-deleted (N-scFv tetravalent) huCC49 minibody containing the synthetic G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 12A shows the single-stranded DNA sequence of heavy chain tetravalent CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 (C-scFv tetravalent CH2 domain deleted) antibody gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 12B shows the single-stranded DNA sequence of light chain tetravalent CH2 domain-deleted (C-scFv tetravalent CH2 domain deleted) PRIMATIZED® p5E8 sc(Fv)2 gene.
  • FIG. 13A shows the amino acid sequence of heavy chain tetravalent CH2 domain-deleted (C-scFv tetravalent CH2 domain deleted) PRIMATIZED® p5E8 sc(Fv)2 antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 13B shows the amino acid sequence of light chain tetravalent CH2 domain-deleted (C-scFv tetravalent CH2 domain deleted) PRIMATIZED® p5E8 sc(Fv)2 antibody.
  • FIG. 14 shows the single-stranded DNA sequence of CH2 domain-deleted PRIMATIZED® p5E8 VL/VH minibody gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 15 shows the single-stranded DNA sequence of CH2 domain-deleted PRIMATIZED® p5E8 VH/VL minibody gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 16 shows the amino acid sequence of CH2 domain-deleted PRIMATIZED® p5E8 VL/VH minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 17 shows the amino acid sequence of CH2 domain-deleted PRIMATIZED® p5E8 VH/VL minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 18 shows a Western Blot of supernatants from five independent clones producing huCC49 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) antibody. Each supernatant was electrophoresed under both reducing and non-reducing conditions.
  • FIG. 19 shows a Coomassie Blue stained gel of purified Forms A and B huCC49 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) antibodies. Each antibody was electrophoresed under reducing and non-reducing conditions.
  • FIG. 20 shows that purified Form A huCC49 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) antibody predominantly elutes as a single peak by HPLC size-exclusion chromatography.
  • FIG. 21 shows the results of a competitive binding assay of Form A huCC49 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) antibody to bovine submaxillary mucine, a source of the TAG-72 antigen, by time-resolved fluorometic immunoassay using a Delphia fluorimeter (Wallac Inc, Gaithersburg, Md.).
  • FIG. 22 shows a Western Blot of supernatant from a representative clone producing huCC49 CH2 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 23 shows a Coomassie Blue stained gel of purified Form A huCC49 CH2 domain-deleted sc(Fv)2 tetravalent G1/G3/PAP (C-scFv tetravalent CH2 domain deleted) antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 24 shows that purified Form A huCC49 CH2 domain-deleted sc(Fv)2 tetravalent G1/G3/PAP (C-scFv tetravalent CH2 domain deleted) antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide predominantly elutes as a single peak by HPLC size-exclusion chromatography.
  • FIG. 25 shows the results of a competitive binding assay of Form A huCC49 CH2 domain-deleted sc(Fv)2 tetravalent G1/G3/PAP (C-scFv tetravalent CH2 domain deleted) antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide to bovine submaxillary mucine, a source of the TAG-72 antigen, by time-resolved fluorometic immunoassay using a Delphia fluorimeter (Wallac Inc, Gaithersburg, Md.).
  • FIG. 26 shows Western Blots of supernatants from representative clones producing huCC49 minibody, huCC49 2 sc(Fv)2 tetravalent antibody (N-scFv tetravalent huCC49 minibody), and huCC49 CH2 domain deleted antibody.
  • a Western Blot was performed on supernatants that were electrophoresed under reducing and non-reducing conditions.
  • FIG. 27 shows a Western Blot of supernatants from representative clones producing huCC49 2 sc(Fv)2 tetravalent antibody (N-scFv tetravalent huCC49 minibody) and huCC49 2 sc(Fv)2 tetravalent antibody (N-scFv tetravalent huCC49 minibody) containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 28 shows a Coomassie Blue stained gel of purified Form A huCC49 domain-deleted sc(Fv)2 tetravalent antibody (N-scFv tetravalent minibody) containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 29 shows purified Form A huCC49 domain-deleted sc(Fv)2 tetravalent antibody (N-scFv tetravalent minibody) containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide predominantly elutes as a single peak by HPLC size-exclusion chromatography.
  • FIG. 30 shows the results of a competitive binding assay of huCC49 domain-deleted sc(Fv)2 tetravalent antibody (N-scFv tetravalent minibody) containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide, huCC49 minibody, huCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody (C-scFv tetravalent antibody) containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide, and control parent CH2 domain-deleted huCC49 (referred to as HuCC49 or IDEC 159) to bovine submaxillary mucine, a source of the TAG-72 antigen, by time-resolved fluorometic immunoassay using a Delphia fluorimeter (Wallac Inc, Gaithersburg, Md.).
  • FIG. 31 shows a Western Blot of supernatant from a cell line producing tetravalent CH2 domain-deleted (C-scFv tetravalent CH2 domain deleted) PRIMATIZED® p5E8 sc(Fv)2 antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 32 shows a Western Blot of supernatants from five independent cell lines producing CH2 domain-deleted huCC49 VL/VH minibody. Minibody samples were analyzed under non-reducing, denaturing conditions revealing the presence of Form A and Form B isoforms.
  • FIG. 33 show the results of an ELISA binding assay of PRIMATIZED® p5E8 VH/VL and VL/VH minibodies containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide binding to the CD23 antigen.
  • p5E8G1 is intact full length PRIMATIZED® IgG1.
  • FIG. 34 shows a Western Blot of supernatant from cell lines producing PRIMATIZED® p5E8 VH/VL and VL/VH minibodies containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 35A shows tumor retention for control parent huCC49 and huCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody as measured by % ID/gm.
  • FIG. 35B shows the same tumor retention data normalized to peak antibody accumulation.
  • Human immunoglobulins can exist in two forms that are associated with hinge heterogeneity. In native solutions, both of these forms are present as dimeric proteins (each monomer comprising one heavy chain and one light chain).
  • One immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond (Form A) and one comprises form in which the dimers are not linked via interchain disulfide bonds (Form B).
  • Form B also forms a stable dimer under native conditions, but can be identified under denaturing, non-reducing conditions, in which the heavy chains dissociate yielding a 75-80 kDa molecule.
  • the frequency of appearance of the B form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the MAb molecule.
  • a single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the B form (Angal et al. 1993. Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge.
  • applying this same amino acid substitution to MAb fragments in which the CH3 domain was retained did not eliminate Form B from preparations.
  • all recombinant CH2 domain deleted antibodies produced in cell cultures often result in hinge heterogeneity which is not corrected via similar molecular mutations in the hinge.
  • the instant invention advances the state of the art by providing methods of, e.g., separating a first dimeric polypeptide from a second dimeric polypeptide wherein the first and second polypeptides comprise at least two polypeptide chains and at least two of the polypeptide chains comprise at least one heavy chain portion.
  • the polypeptides of the invention lack all or part of a CH2 domain.
  • the monomers are linked via at least one interchain disulfide linkage (referred to herein as “Form A”) and the monomers of the second polypeptide are not linked via at least one interchain disulfide linkage (referred to herein as “Form B”). These forms can be separated from each other using hydrophobic interaction chromatography.
  • polypeptides that comprise connecting peptides.
  • the inclusion of certain connecting peptides results in the preferential biosynthesis of polypeptide dimers comprising polypeptide chains that are linked via at least one interchain disulfide linkage or that are not linked via at least one interchain disulfide linkage.
  • the polypeptides of the invention are binding molecules, i.e., polypeptide molecules or the nucleic acid molecules that encode them, that comprise at least one binding domain which comprises a binding site that specifically binds to a target molecule (such as an antigen or binding partner).
  • a binding molecule of the invention comprises an immunoglobulin antigen binding site or the portion of a receptor molecule responsible for ligand binding or the portion of a ligand molecule that is responsible for receptor binding.
  • the binding molecules of the invention are polypeptides or the nucleic acid molecules which encode them.
  • the binding molecules comprise at least two binding sites. In one embodiment, the binding molecules comprise two binding sites. In one embodiment, the binding molecules comprise three binding sites. In another embodiment, the binding molecules comprise four binding sites.
  • the polypeptides of the invention are multimers.
  • the polypeptides of the invention are dimers.
  • the dimers of the invention are homodimers, comprising two identical monomeric subunits.
  • the dimers of the invention are heterodimers, comprising two non-identical monomeric subunits.
  • the subunits of the dimer may comprise one or more polypeptide chains.
  • the dimers comprise at least two polypeptide chains.
  • the dimers comprise two polypeptide chains.
  • the dimers comprise four polypeptide chains (e.g., as in the case of antibody molecules).
  • polypeptides of the invention comprise at least one amino acid sequence derived from an immunoglobulin domain.
  • a polypeptide or amino acid sequence “derived from” a designated protein refers to the origin of the polypeptide.
  • the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
  • binding polypeptides comprise an amino acid sequence derived from a human amino acid sequence.
  • binding polypeptides may comprise one or more amino acids from another mammalian species.
  • a primate heavy chain portion, hinge portion, or binding site may be included in the subject binding polypeptides and/or connecting polypeptides.
  • one or more murine amino acids may be present in a binding polypeptide, e.g., in an antigen binding site of a binding molecule.
  • Preferred binding molecules of the invention are not immunogenic.
  • binding molecules of the invention may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule from which they were derived. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at “non-essential” amino acid residues may be made.
  • An isolated nucleic acid molecule encoding a non-natural variant of a polypeptide derived from an immunoglobulin can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • mutations may be introduced randomly along all or part of the immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into polypeptides of the invention and screened for their ability to bind to the desired antigen.
  • a polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • a polypeptide of the invention comprises a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain.
  • a polypeptide of the invention comprises a polypeptide chain comprising a CH1 domain and a CH3 domain.
  • a polypeptide of the invention comprises a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain. In another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. In one embodiment, a polypeptide of the invention lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). In another embodiment, a polypeptide of the invention comprises a complete Ig heavy chain. As set forth above, it will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain portions) may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
  • At least two of the polypeptide chains of a binding molecule of the invention comprise at least one heavy chain portion derived from an antibody or immunoglobulin molecule.
  • at least two heavy chain portions of a polypeptide of the invention are present on different polypeptide chains and interact, e.g., via at least one disulfide linkage (Form A) or via non-covalent interactions (Form B) to form a dimeric polypeptide, each monomer of the dimer comprising at least one heavy chain portion.
  • the heavy chain portions of one polypeptide chain of a dimer are identical to those on a second polypeptide chain of the dimer.
  • the monomers (or half-mers) of a dimer of the invention are identical to each other. In another embodiment, they are not identical. For example, each monomer may comprise a different target binding site.
  • a dimer of the invention is held together by covalent interactions, e.g., disulfide bonds. In one embodiment, a dimer of the invention is held together by one or more disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably two disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably three disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably four disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably five disulfide bonds.
  • a dimer of the invention is held together by one or more, preferably six disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably seven disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably eight disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably nine disulfide bonds. In another embodiment, a dimer of the invention is held together by one or more, preferably ten disulfide bonds. In a further embodiment, a dimer of the invention is not held together by disulfide bonds, but is held together, e.g., by non-covalent interactions.
  • the heavy chain portions of a polypeptide may be derived from different immunoglobulin molecules.
  • a heavy chain portion of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule.
  • a heavy chain portion can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule.
  • a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.
  • the term “light chain portion” includes amino acid sequences derived from an immunoglobulin light chain.
  • the light chain portion comprises at least one of a VL or CL domain.
  • a polypeptide of the invention comprises an amino acid sequence or one or more moieties not derived from an Ig molecule. Exemplary modifications are described in more detail below.
  • a polypeptide of the invention may comprise a flexible linker sequence.
  • a polypeptide may be modified to add a functional moiety (e.g., PEG, a drug, or a label).
  • a binding polypeptide of the invention is a fusion protein.
  • Fusion proteins are chimeric molecules which comprise a binding domain comprising at least one target binding site and at least one heavy chain portion.
  • a fusion protein further comprises a synthetic connecting peptide.
  • a “chimeric” protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature.
  • the amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide.
  • a chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • Exemplary chimeric polypeptides include fusion proteins and the chimeric hinge connecting peptides of the invention.
  • heterologous as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
  • a heterologous polynucleotide or antigen may be derived from a different species origin, different cell type, or the same type of cell of distinct individuals.
  • ligand binding domain or “ligand binding portion of a receptor” as used herein refers to any native receptor (e.g., cell surface receptor) or any region or derivative thereof retaining at least a qualitative ligand binding ability, and preferably the biological activity of a corresponding native receptor.
  • receptor binding domain or “receptor binding portion of a ligand” as used herein refers to any native ligand or any region or derivative thereof retaining at least a qualitative receptor binding ability, and preferably the biological activity of a corresponding native ligand.
  • a binding molecule of the invention is a fusion protein.
  • a fusion protein of the invention is a chimeric molecule that comprises a binding domain (which comprises at least one binding site) and a dimerization domain (which comprises at least one heavy chain portion).
  • the heavy chain portion may be from any immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.
  • a binding molecule is an “antibody-fusion protein chimera.”
  • Such molecules comprise a molecule which combines at least one binding domain of an antibody with at least one fusion protein.
  • the interface between the two polypeptides is a CH3 domain of an immunoglobulin molecule.
  • the binding molecules of the invention are “antibody” or “immunoglobulin” molecules, e.g., naturally occurring antibody or immunoglobulin molecules or genetically engineered antibody molecules that bind antigen in a manner similar to antibody molecules.
  • immunoglobulin includes a polypeptide having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity.
  • Antibodies refers to such assemblies which have significant known specific immunoreactive activity to an antigen of interest (e.g. a tumor associated antigen).
  • Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood.
  • immunoglobulin comprises five distinct classes of antibody that can be distinguished biochemically. All five classes of antibodies are clearly within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
  • immunoglobulins comprise two identical light polypeptide chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000-70,000. The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
  • variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
  • Light chains are classified as either kappa or lambda ( ⁇ , ⁇ ). Each heavy chain class may be bound with either a kappa or lambda light chain.
  • the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • heavy chains are classified as gamma, mu, alpha, delta, or epsilon, ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ 1- ⁇ 4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
  • the immunoglobulin subclasses e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discemable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention.
  • variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the V L domain and V H domain of an antibody combine to form the variable region that defines a three dimensional antigen binding site.
  • This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the V H and V L chains.
  • CDRs complementary determining regions
  • binding site or “binding domain” comprises a region of a polypeptide which is responsible for selectively binding to a target molecule of interest (e.g. an antigen, ligand, receptor, substrate or inhibitor).
  • exemplary binding domains include an antibody variable domain, a receptor binding domain of a ligand, a ligand binding domain of a receptor or an enzymatic domain.
  • the binding molecules have at least one binding site specific for a molecule targeted for reduction or elimination, e.g., a cell surface antigen or a soluble antigen.
  • the binding domain is an antigen binding site.
  • An antigen binding site is formed by variable regions that vary from one polypeptide to another.
  • the polypeptides of the invention comprise at least two antigen binding sites.
  • the term “antigen binding site” includes a site that specifically binds (immunoreacts with) an antigen (e.g., a cell surface or soluble antigen).
  • the antigen binding site includes an immunoglobulin heavy chain and light chain variable region and the binding site formed by these variable regions determines the specificity of the antibody.
  • an antigen binding molecule of the invention comprises at least one heavy or light chain CDR of an antibody molecule (e.g., the sequence of which is known in the art or described herein).
  • an antigen binding molecule of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least six CDRs from one or more antibody molecules. Exemplary antibody molecules comprising at least one CDR that can be included in the subject antigen binding molecules are known in the art and exemplary molecules are described herein.
  • polypeptides comprising two heavy chain portions disclosed herein may be linked to form two associated Ys so there will be four binding sites forming a “tetravalent” molecule (see e.g., WO02/096948A2)).
  • tetravalent minibodies or domain deleted antibodies can be made.
  • polypeptide includes the number of potential binding sites which specifically bind (e.g., immunoreact with) a given target.
  • a polypeptide may be monospecific and contain one or more binding sites which specifically bind a target or a polypeptide may be multispecific and contain two or more binding sites which specifically bind the same or different targets.
  • a binding molecule of the invention is a bispecific molecule (e.g., antibody, minibody, domain deleted antibody, or fusion protein having binding specificity for more than one molecule, e.g., more than one antigen or more than one epitope on the same antigen.
  • the bispecific molecules have at least one target binding site specific for a molecule targeted for reduction or elimination and a targeting molecule on a cell.
  • the bispecific molecules have at least one target binding site specific for a molecule targeted for reduction or elimination and at least one target binding site specific for a drug.
  • the bispecific molecules have at least one target binding site specific for a molecule targeted for reduction or elimination and at least one target binding site specific for a prodrug.
  • the bispecific molecules are tetravalent antibodies that have two target binding sites specific for one target and two target binding sites specific for the second target.
  • a tetravalent bispecific molecule may be bivalent for each specificity. Further description of bispecific molecules is provided below.
  • valency refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site may specifically bind the same or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes on the same antigen).
  • the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding site as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the heavy and light variable domains show less inter-molecular variability in amino acid sequence and are termed the framework regions.
  • the framework regions largely adopt a ⁇ -sheet conformation and the CDRs form loops which connect, and in some cases form part of, the ⁇ -sheet structure. Thus, these framework regions act to form a scaffold that provides for positioning the six CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope.
  • the position of CDRs can be readily identified by one of ordinary skill in the art.
  • VH domain includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain.
  • CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
  • CH2 domain includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system,; and Kabat E A et al. Sequences of Proteins of Immunological Interest. Bethesda, US Department of Health and Human Services, NIH. 1991).
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.
  • Hinge region includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al. J. Immunol. 1998 161:4083).
  • a binding molecule of the invention comprises a connecting peptide.
  • the connecting peptides of the invention are synthetic.
  • synthetic with respect to polypeptides includes polypeptides which comprise an amino acid sequence that is not naturally occurring.
  • non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion) or which comprise a first amino acid sequence (which may or may not be naturally occurring) that is linked in a linear sequence of amino acids to a second amino acid sequence (which may or may not be naturally occurring) to which it is not naturally linked in nature.
  • Connecting peptides of the invention connect two domains (e.g., a binding domain and a dimerization domain) of a binding molecule of the invention.
  • connecting peptides connect a heavy chain portion to a binding domain comprising a binding site.
  • a connecting peptide connects two heavy chain constant region domains, such as CH1 and CH2 domains; CH1 and CH3 domains; hinge and CH1 domains; hinge and CH3 domains; VH and hinge domains, or a CH3 domain and a non-immunoglobulin polypeptide) in a linear amino acid sequence of a polypeptide chain.
  • connecting peptides provide flexibility to the polypeptide molecule and facilitate dimerization via disulfide bonding.
  • the connecting peptides of the invention are used to replace one or more heavy chain domains (e.g., at least a portion of a constant region domain (e.g., at least a portion of a CH2 domain) and/or at least a portion of the hinge region (e.g., at least a portion of the lower hinge region domain) in a domain deleted construct).
  • a VH domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the VH domain).
  • a VL domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the VL domain.
  • a CH1 domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the CH1 domain).
  • a synthetic connecting peptide comprises a portion of a constant region domain.
  • a connecting peptide that replaces a CH2 domain can comprise a portion of the CH2 domain.
  • a connecting peptide comprises or consists of a gly-ser linker.
  • gly-ser linker refers to a peptide that consists of glycine and serine residues
  • An exemplary gly/ser linker comprises the amino acid sequence GGGSSGGGSG (SEQ ID NO:1).
  • a connecting peptide of the invention comprises at least a portion of an upper hinge region (e.g., derived from an IgG1, IgG3, or IgG4 molecule), at least a portion of a middle hinge region (e.g., derived from an IgG1, IgG3, or IgG4 molecule) and a series of gly/ser amino acid residues (e.g., a gly/ser linker such as GGGSSGGGSG (SEQ ID NO:1)).
  • the connecting peptide comprises a substitution of one or more amino acids as compared to naturally occurring IgG1 or IgG3 hinge regions.
  • a connecting peptide comprises an amino acid sequence such as described in WO 02/060955. Connecting peptides are described in more detail below.
  • disulfide bond includes the covalent bond formed between two sulfur atoms.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
  • the constant region mediates several effector functions. For example, binding of the C1 component of complement to antibodies activates the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Further, antibodies bind to cells via the Fc region, with a Fc receptor site on the antibody Fc region binding to a Fc receptor (FcR) on a cell.
  • Fc receptor Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the Fc portion may be mutated to decrease effector function using techniques known in the art.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization.
  • constant region modifications consistent with the instant invention moderate compliment binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin.
  • Yet other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. More generally, those skilled in the art will realize that antibodies modified as described herein may exert a number of subtle effects that may or may not be readily appreciated. However the resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor localization, biodistribution and serum half-life, may easily be measured and quantified using well know immunological techniques without undue experimentation.
  • modified forms of antibodies can be made from a whole precursor or parent antibody using techniques known in the art. Exemplary techniques are discussed in more detail below. In particularly preferred embodiments both the variable and constant regions of polypeptides of the invention are human.
  • fully human antibodies can be made using techniques that are known in the art. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make antibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art.
  • a polypeptide comprising a heavy chain portion may or may not comprise other amino acid sequences or moieties not derived from an immunoglobulin molecule. Such modifications are described in more detail below.
  • a polypeptide of the invention may comprise a flexible linker sequence.
  • a polypeptide may be modified to add a functional moiety such as PEG.
  • polypeptides of the instant invention comprise at least two binding sites that provide for the association of the polypeptide with the selected target molecule.
  • a binding molecule of the invention comprises an antibody molecule, e.g., an intact antibody molecule, or a fragment of an antibody molecule.
  • binding molecule of the invention is a modified or synthetic antibody molecule.
  • a binding molecule of the invention comprises all or a portion of (e.g., at least one antigen binding site from, at least one CDR from, or at least one heavy chain portion from) a monoclonal antibody, a humanized antibody, a chimeric antibody, or a recombinantly produced antibody.
  • variable region may or be derived from any type of animal that can be induced to mount a humoral response and generate immunoglobulins against the desired antigen.
  • the variable region of the polypeptides may be, for example, of mammalian origin e.g., may be human, murine, non-human primate (such as cynomolgus monkeys, macaques, etc.), lupine, camelid (e.g., from camels, llamas and related species).
  • the variable region may be condricthoid in origin (e.g., from sharks).
  • polypeptides of the invention can be made using techniques that are known in the art.
  • the polypeptides of the invention are antibody molecules that have been “recombinantly produced,” i.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules are discussed in more detail below.
  • the polypeptides of the invention are modified antibodies.
  • modified antibody includes synthetic forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain portions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen); heavy chain molecules joined to scFv molecules and the like. ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019.
  • a binding molecule of the invention is a fusion protein comprising at least one heavy chain portion lacking a CH2 domain and comprising a binding domain of a polypeptide comprising the binding portion of one member of a ligand and its receptor.
  • the term, “modified antibody” according to the present invention includes immunoglobulins, antibodies, or immunoreactive fragments or recombinants thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, or reduced serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity.
  • the polypeptides of the present invention are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains. More preferably, one entire domain of the constant region of the modified antibody will be deleted and even more preferably all or part of the CH2 domain will be deleted.
  • a polypeptide of the invention will not elicit a deleterious immune response in a human.
  • Modifications to the constant region compatible with the instant invention comprise additions, deletions or substitutions of one or more amino acids in one or more domains. That is, the polypeptides of the invention disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or to the light chain constant region domain (CL).
  • the invention pertains to a modified antibody molecule comprising at least one CC49 binding site (specific for Tag72).
  • FIG. 8A shows the single-stranded DNA sequence of sc(Fv)2 heavy chain tetravalent CH 2 domain-deleted huCC49 gene.
  • FIG. 8B shows the single-stranded DNA sequence of sc(Fv)2 heavy chain tetravalent CH2 domain-deleted huCC49 gene containing the synthetic G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 16 shows the single-stranded DNA sequence of sc(Fv)2 heavy chain tetravalent CH 2 domain-deleted huCC49 gene.
  • FIG. 8B shows the single-stranded DNA sequence of sc(Fv)2 heavy chain tetravalent CH2 domain-deleted huCC49 gene containing the synthetic G1/G3/Pro243Ala244Pro245+[
  • FIG. 8C shows the single-stranded DNA sequence of sc(Fv)2 light chain CH 2 domain-deleted huCC49.
  • FIG. 9A shows the amino acid sequence of heavy chain sc(Fv)2 tetravalent CH 2 domain-deleted huCC49.
  • FIG. 9B shows the amino acid sequence of heavy chain tetravalent CH 2 domain-deleted sc(Fv)2 huCC49 containing the synthetic G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 9A shows the amino acid sequence of heavy chain tetravalent CH 2 domain-deleted sc(Fv)2 huCC49 containing the synthetic G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 10A shows the single-stranded DNA sequence of tetravalent CH 2 domain-deleted 2sc(Fv)2 huCC49 minibody gene.
  • FIG. 10B shows the single-stranded DNA sequence of tetravalent CH 2 domain-deleted sc(Fv)2 huCC49 minibody gene containing the synthetic G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 10A shows the single-stranded DNA sequence of tetravalent CH 2 domain-deleted sc(Fv)2 huCC49 minibody gene containing the synthetic G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 11A shows the amino acid sequence of tetravalent CH 2 domain-deleted sc(Fv)2 huCC49 minibody.
  • FIG. 11B shows the amino acid sequence of tetravalent CH 2 domain-deleted sc(Fv)2 huCC49 minibody containing the synthetic G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • the invention pertains to a modified antibody molecule comprising at least one p5E8 binding site (specific for CD23).
  • FIG. 12A shows the single-stranded DNA sequence of heavy chain tetravalent CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 antibody gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 12B (SEQ ID NO:27) shows the single-stranded DNA sequence of light chain tetravalent CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 gene.
  • FIG. 12A shows the single-stranded DNA sequence of heavy chain tetravalent CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 13A shows the amino acid sequence of heavy chain tetravalent CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 13B shows the amino acid sequence of light chain tetravalent CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 antibody.
  • FIG. 13B shows the amino acid sequence of light chain tetravalent CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 antibody.
  • FIG. 14 shows the single-stranded DNA sequence of CH2 domain-deleted PRIMATIZED® p5E8 VL/VH minibody gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 15 shows the single-stranded DNA sequence of CH2 domain-deleted PRIMATIZED® p5E8 VH/VL minibody gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 30 shows the single-stranded DNA sequence of CH2 domain-deleted PRIMATIZED® p5E8 VL/VH minibody gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 16 shows the amino acid sequence of CH2 domain-deleted PRIMATIZED® p5E8 VL/VH minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • FIG. 17 shows the amino acid sequence of CH2 domain-deleted PRIMATIZED® p5E8 VH/VL minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge connecting peptide.
  • the polypeptides of the invention may be modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies or polypeptides of the invention can be humanized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans.
  • CDRs complementarity determining regions
  • De-immunization can also be used to decrease the immunogenicity of an antibody.
  • the term “de-immunization” includes alteration of an antibody to modify T cell epitopes (see, e.g., WO9852976A1, WO0034317A2).
  • VH and VL sequences from the starting antibody are analyzed and a human T cell epitope “map” from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence.
  • CDRs complementarity-determining regions
  • VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of polypeptides of the invention that are tested for function.
  • Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody.
  • the antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
  • the binding molecule comprises a chimeric antibody.
  • chimeric antibodies will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant invention) is obtained from a second species.
  • the target binding region or site will be from a non-human source (e.g. mouse) and the constant region is human.
  • the variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence changing.
  • the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and preferably from an antibody from a different species. It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site. Given the explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional antibody with reduced inmunogenicity.
  • the term “properly folded polypeptide” includes polypeptides (e.g., antigen binding molecules such as antibodies) in which all of the functional domains comprising the polypeptide are distinctly active.
  • the term “improperly folded polypeptide” includes polypeptides in which at least one of the functional domains of the polypeptide is not active.
  • a properly folded polypeptide comprises polypeptide chains linked by at least one disulfide bond and, conversely, an improperly folded polypeptide comprises polypeptide chains not linked by at least one disulfide bond.
  • malignancy refers to a non-benign tumor or a cancer.
  • cancer includes a malignancy characterized by deregulated or uncontrolled cell growth. Exemplary cancers include: carcinomas, sarcomas, leukemias, and lymphomas.
  • primary malignant tumors e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor
  • secondary malignant tumors e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor.
  • a binding molecule of the invention binds to a tumor cell.
  • Exemplary antibodies which comprise antigen binding sites that bind to antigens expressed on tumor cells are known in the art and one or more CDRs from such antibodies can be included in a binding molecule of the invention.
  • Exemplary antibodies include: 2B8, Lym 1, Lym 2, LL2, Her2, B1, MB1, BH3, B4, B72.3, 5E8, B3F6 and 5E10.
  • a polypeptide of the invention is a C2B8 antibody which binds to CD20.
  • a polypeptide of the invention is a CC49 antibody which recognizes TAG72.
  • a binding molecule of the invention binds to a molecule which is useful in treating an autoimmune or inflammatory disease or disorder.
  • autoimmune disease or disorder refers to disorders or conditions in a subject wherein the immune system attacks the body's own cells, causing tissue destruction.
  • Autoimmune diseases include general autoimmune diseases, i.e., in which the autoimmune reaction takes place simultaneously in a number of tissues, or organ specific autoimmune diseases, ie., in which the autoimmune reaction targets a single organ.
  • autoimmune diseases that can be diagnosed, prevented or treated by the methods and compositions of the present invention include, but are not limited to, Crohn's disease; Inflammatory bowel disease (IBD); systemic lupus erythematosus; ulcerative colitis; rheumatoid arthritis; goodpasture's syndrome; Grave's disease; Hashimoto's thyroiditis; pemphigus vulgaris ; myasthenia gravis; scleroderma; autoimmune hemolytic anemia; autoimmune thrombocytopenic purpura; polymyositis and dermatomyositis; pernicious anemia; Sjögren's syndrome; ankylosing spondylitis; vasculitis; type I diabetes mellitus; neurological disorders, multiple sclerosis, and secondary diseases caused as a result of autoimmune diseases.
  • IBD Inflammatory bowel disease
  • systemic lupus erythematosus ulcerative colitis
  • rheumatoid arthritis good
  • inflammatory disease or disorder includes diseases or disorders which are caused, at least in part, or exacerbated by inflammation, e.g., increased blood flow, edema, activation of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis).
  • inflammation e.g., increased blood flow, edema, activation of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis).
  • Exemplary disorders include those in which inflammation or inflammatory factors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF, interleukins, plasma proteins, cellular defense systems, cytokines, lipid metabolites, proteases, toxic radicals, mitochondria, apoptosis, adhesion molecules, etc.) are involved or are present in an area in aberrant amounts, e.g., in amounts which may be advantageous to alter, e.g., to benefit the subject.
  • MMPs matrix metalloproteinases
  • NO nitric oxide
  • TNF interleukins
  • plasma proteins e.g., plasma proteins
  • cellular defense systems e.g., cytokines, lipid metabolites, proteases, toxic radicals, mitochondria, apoptosis, adhesion molecules, etc.
  • cytokines e.g., cytokines, lipid metabolites, proteases, toxic radicals, mitochondria, apopto
  • Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders.
  • Acute inflammatory disorders are generally of relatively short duration, and last for from about a few minutes to about one to two days, although they may last several weeks.
  • the main characteristics of acute inflammatory disorders include increased blood flow, exudation of fluid and plasma proteins (edema) and emigration of leukocytes, such as neutrophils.
  • Chronic inflammatory disorders generally, are of longer duration, e.g., weeks to months to years or even longer, and are associated histologically with the presence of lymphocytes and macrophages and with proliferation of blood vessels and connective tissue.
  • Recurrent inflammatory disorders include disorders which recur after a period of time or which have periodic episodes. Examples of recurrent inflammatory disorders include asthma and multiple sclerosis. Some disorders may fall within one or more categories.
  • Inflammatory disorders are generally characterized by heat, redness, swelling, pain and loss of function.
  • causes of inflammatory disorders include, but are not limited to, microbial infections (e.g., bacterial, viral and fungal infections), physical agents (e.g., burns, radiation, and trauma), chemical agents (e.g., toxins and caustic substances), tissue necrosis and various types of immunologic reactions.
  • inflammatory disorders include, but are not limited to, osteoarthritis, rheumatoid arthritis, acute and chronic infections (bacterial, viral and fungal); acute and chronic bronchitis, sinusitis, and other respiratory infections, including the common cold; acute and chronic gastroenteritis and colitis; acute and chronic cystitis and urethritis; acute respiratory distress syndrome; cystic fibrosis; acute and chronic dermatitis; acute and chronic conjunctivitis; acute and chronic serositis (pericarditis, peritonitis, synovitis, pleuritis and tendinitis); uremic pericarditis; acute and chronic cholecystis; acute and chronic vaginitis; acute and chronic uveitis; drug reactions; and burns (thermal, chemical, and electrical).
  • the term “medium that separates polypeptides based on hydrophobic interaction” includes a medium comprising hydrophobic ligands (e.g., alkyl or aryl groups) covalently attached to a matrix. Such a medium can be used to separate polypeptides based on interaction between a solvent and accessible non-polar groups on the surface of the polypeptides and the hydrophobic ligands of the medium.
  • An exemplary medium is Phenyl 5PW-HR available from Tosoh Bioscience.
  • conductivity includes electrical conductivity of a solution as measured in microSiemens/cm (formerly micromhos/cm). The greater the ion content of a solution, the greater the conductivity of the solution. Conductivity can be readily measured using techniques that are well known in the art (e.g., by measuring the current passing between two electrodes).
  • the separation methods of the invention can be used with solutions having a pH ranging from acid to neutral, e.g., from about pH 3.5 to approximately neutral.
  • approximately neutral pH includes pH values of approximately 7.
  • a separation method of the invention can be performed using a solution (e.g., a buffer) having a pH of about 3, about 4, about 5, about 6, about 7, or about 8.
  • the pH of the solution is about 6 or about 7.
  • the pH of the solution is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.
  • affinity matrix includes a matrix, such as agarose, controlled pore glass, or poly(styrenedivinyl)benzene to which an affinity ligand is attached.
  • the affinity ligand binds to the desired polypeptide and the contaminating polypeptides are not bound to the affinity ligand.
  • the desired polypeptide can be eluted from the affinity matrix using known protocols.
  • the term “engineered” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).
  • the binding molecules of the invention are engineered, e.g., to express a connecting peptide of the invention.
  • the terms “linked,” “fused” or “fusion” are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means.
  • An “in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs.
  • ORFs open reading frames
  • the resulting recombinant fusion protein is a single protein containing two ore more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.)
  • the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence.
  • a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
  • the phrase “subject that would benefit from administration of a binding molecule” includes subjects, such as mammalian subjects, that would benefit from administration of a binding molecule used, e.g., for detection of an antigen recognized by a binding molecule (e.g., for a diagnostic procedure) and/or from treatment with a binding molecule to reduce or eliminate the target recognized by the binding molecule.
  • the subject may benefit from reduction or elimination of a soluble or particulate molecule from the circulation or serum (e.g., a toxin or pathogen) or from reduction or elimination of a population of cells expressing the target (e.g., tumor cells).
  • the binding molecule can be used in unconjugated form or can be conjugated, e.g., to a drug, prodrug, or an isotope.
  • At least one polypeptide chain of a dimer of the invention can comprise a synthetic connecting peptide of the invention.
  • at least two chains of a dimer of the invention comprise a connecting peptide.
  • two chains of a dimer of the invention comprise a connecting peptide.
  • connecting peptides can be used to join two heavy chain portions in frame in a single polypeptide chain.
  • a connecting peptide of the invention can be used to fuse a CH3 domain (or synthetic CH3 domain) to a hinge region (or synthetic hinge region).
  • a connecting peptide of the invention can be used to fuse a CH3 domain (or synthetic CH3 domain) to a CH1 domain (or synthetic CH1 domain).
  • a connecting peptide can act as a peptide spacer between the hinge region (or synthetic hinge region) and a CH2 domain (or a synthetic CH2 domain).
  • a CH3 domain can be fused to an extracellular protein domain (e.g., a VL domain (or synthetic domain), a VH domain (or synthetic domain), a CH1 domain (or synthetic domain), a hinge domain (or synthetic hinge), or to the ligand binding portion of a receptor or the receptor binding portion of a ligand).
  • a VH or VL domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the VH or VL domain).
  • a CH1 domain is fused to a CH3 domain via a connecting peptide (the C-terminus of the connecting peptide is attached to the N-terminus of the CH3 domain and the N-terminus of the connecting peptide is attached to the C-terminus of the CH1 domain).
  • a connecting peptide of the invention can be used to fuse a CH3 domain (or synthetic CH3 domain) to a hinge region (or synthetic hinge region) or portion thereof.
  • a connecting peptide can act as a peptide spacer between the hinge region (or synthetic hinge region) and a CH2 domain (or a synthetic CH2 domain).
  • a connecting peptide can comprise or consist of a gly/ser spacer.
  • a domain deleted CC49 construct having a short amino acid spacer GGSSGGGGSG (SEQ. ID No. 1) substituted for the CH2 domain and the lower hinge region (CC49. 66 CH2 [gly/ser]) can be used.
  • a connecting peptide comprises the amino acid sequence IGKTISKKAK (SEQ ID NO:36).
  • connecting peptide can comprise at least a portion of an immunoglobulin hinge region.
  • chimeric hinge domains can be constructed which combine hinge elements derived from different antibody isotypes.
  • a connecting peptide comprises at least a portion of an IgG1 hinge region.
  • a connecting peptide can comprise at least a portion of an IgG3 hinge region.
  • a connecting peptide can comprise at least a portion of an IgG1 hinge region and at least a portion of an IgG3 hinge region.
  • a connecting peptide can comprise an IgG1 upper and middle hinge and a single IgG3 middle hinge repeat motif.
  • connecting peptides comprising an amino acid sequence derived from an immunoglobulin hinge region
  • the numbering of amino acid positions in these molecules is given using Kabat numbering see, e.g., Table 2).
  • Table 1 shows naturally occurring hinge sequence for IgG1, IgG3, and IgG4 molecules.
  • Table 2 shows Kabat numbering for portions of these hinge molecules and also shows Kabat numbering for connecting peptide amino acid residues presented in that table.
  • a connecting peptide of the invention comprises a non-naturally occurring immunoglobulin hinge region domain, e.g., a hinge region domain that is not naturally found in the polypeptide comprising the hinge region domain and/or a hinge region domain that has been altered so that it differs in amino acid sequence from a naturally occurring immunoglobulin hinge region domain.
  • mutations can be made to hinge region domains to make a connecting peptide of the invention.
  • a connecting peptide of the invention comprises a hinge domain which does not comprise a naturally occurring number of cysteines, i.e., the connecting peptide comprises either fewer cysteines or a greater number of cysteines than a naturally occurring hinge molecule.
  • incorporation of a connecting peptide into a polypeptide results in a composition in which greater than 50%, 60%, 70%, 80% or 90% of the dimeric molecules present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • a connecting peptide comprises hinge region domain comprising a proline residue at an amino acid position corresponding to amino acid position 243 in the Kabat numbering system (position 230, EU numbering system).
  • a connecting peptide comprises an alanine residue at an amino acid position corresponding to position 244, Kabat numbering system (position 246, EU numbering system).
  • a connecting peptide of the invention comprises a proline residue at an amino acid position corresponding to position 245 (Kabat numbering system; position 247, EU numbering system)).
  • a connecting peptide comprises a cysteine residue at an amino acid position corresponding to position 239, Kabat numbering system (position 226, EU numbering system).
  • a connecting peptide comprises a serine residue at an amino acid position corresponding to position 239, Kabat numbering system (position 226, EU numbering system). In one embodiment, a connecting peptide comprises a cysteine residue at an amino acid position corresponding to position 242, Kabat numbering system (position 229, EU numbering system). In one embodiment, a connecting peptide comprises a serine residue at an amino acid position corresponding to position 242, Kabat numbering system (position 229, EU numbering system).
  • the connecting peptide can be chosen to result in the preferential synthesis of a particular isoform of polypeptide, e.g., in which the two heavy chain portions are linked via disulfide bonds or are not linked via disulfide bonds.
  • the G1/G3/Pro243+[gly/ser] linker (SEQ ID NO: 8), G1/G3/Pro243Ala244Pro245+[gly/ser] linker (SEQ ID NO: 9), Pro243+[gly/ser] linker (SEQ ID NO: 15), and Pro243Ala244Pro245+[gly/ser] linker (SEQ ID NO: 14), connecting peptides resulted in the production of only Form A CH2 domain-deleted antibody with no detectable Form B.
  • IgG1 CH3 domain is 98.13% homologous to IgG2 CH3, 97.20% homologous to IgG3 CH3, and 96.26% homologous to IgG4 CH3).
  • the parentheticals referring to connecting peptides and various binding molecules of the invention represent equivalent terminology unless otherwise indicated.
  • a connecting peptide of the invention comprises a hinge region domain followed by a flexible gly/ser linker.
  • Exemplary connecting peptides are shown in Table 2 and in SEQ ID NOs: 8-15, 48, and 49. It will be understood that variant forms of these exemplary connecting peptides can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding a connecting peptide such that one or more amino acid substitutions, additions or deletions are introduced into the connecting peptide. For example, mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more non-essential amino acid residues such that the ability of the connecting peptide to preferentially enhance synthesis of Form A or Form B is not altered.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • Connecting peptides of the invention can be of varying lengths. In one embodiment, a connecting peptide of the invention is from about 15 to about 50 amino acids in length. In another embodiment, a connecting peptide of the invention is from about 20 to about 45 amino acids in length. In another embodiment, a connecting peptide of the invention is from about 25 to about 40 amino acids in length. In another embodiment, a connecting peptide of the invention is from about 30 to about 35 amino acids in length. In another embodiment, a connecting peptide of the invention is from about 24 to about 27amino acids in length. In another embodiment, a connecting peptide of the invention is from about 40 to about 42 amino acids in length.
  • Connecting peptides can be introduced into polypeptide sequences using techniques known in the art. For example, in one embodiment, the Splicing by Overlap Extension (SOE) method (Horton, R. M. 1993 Methods in Molecular Biology, Vol 15: PCR Protocols: Current Methods and applications. Ed. B. A. White) can be used. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells for stable production of the polypeptides produced.
  • SOE Splicing by Overlap Extension
  • incorporation of one of the subject connecting peptides into a polypeptide yields a composition comprising polypeptide molecules having at least two binding sites and at least two polypeptide chains, wherein at least two of the polypeptide chains comprise a synthetic connecting peptide and wherein greater than 50% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • greater than 60% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • greater than 70% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • greater than 80% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than 90% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • incorporation of one of the subject connecting peptides into an IgG4 molecule yields a composition in which greater than 95% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • the polypeptides of the invention comprise at least two binding sites which bind to a target molecule of interest.
  • Exemplary binding sites include, e.g., sites which bind to an antigen (antigen binding sites), sites which bind to a receptor (receptor binding sites), or sites which bind to a ligand (ligand binding sites).
  • the binding molecules comprise at least two binding sites.
  • the binding molecules comprise two binding sites.
  • the binding molecules comprise three binding sites.
  • the binding molecules comprise four binding sites.
  • the binding molecules have at least one target binding site specific for a molecule which mediates a biological effect (e.g., which modulates cellular activation (e.g., by binding to a cell surface receptor and resulting in transmission or inhibition of an activating or inhibitory signal), which results in death of the cell (e.g., by complement fixation or exposure to a payload present on the binding molecule), or which modulates a disease or disorder in a subject (e.g., by promoting lysis of a fibrin clot or promoting clot formation, or by modulating the amount of a substance which is bioavailable (e.g., by enhancing or reducing the amount of a ligand such as TNF ⁇ in the subject)).
  • a biological effect e.g., which modulates cellular activation (e.g., by binding to a cell surface receptor and resulting in transmission or inhibition of an activating or inhibitory signal), which results in death of the cell (e.g., by complement fixation or exposure to a payload
  • the binding molecules have at least one target binding site specific for an molecule targeted for reduction or elimination, e.g., a cell surface antigen or a soluble antigen. In one embodiment, binding of the binding molecule to the target results in reduction or elimination of the target, e.g., from a tissue or from the circulation. In another embodiment, the binding molecules have at least one binding site specific for a molecule that can be used to detect the presence of a target molecule (e.g., to detect a contaminant or diagnose a condition or disorder). In yet another embodiment, a binding molecule of the invention comprises at least one binding site that targets the binding molecule to a specific site in a subject (e.g., to a tumor cell or blood clot).
  • Exemplary binding sites that can be included in the binding domain of a binding molecule of the invention include: the receptor binding portion of a ligand, the ligand binding portion of a receptor, the substrate binding portion of an enzyme, the enzyme binding portion of a substrate, or one or more antigen binding portions of an antibody.
  • At least one target binding site of a binding molecule is catalytic (Shokat and Schultz. 1990. Annu. Rev. Immunol. 8:335).
  • a heavy chain variable portion and a light chain variable portion of a binding molecule are present in the same polypeptide, e.g., as in a single chain antibody or a minibody (see e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1).
  • the heavy chain portion and the light chain portion of a polypeptide are present in different polypeptide chains, e.g., as in antibody molecules.
  • the target binding polypeptides of the invention are multimeric molecules.
  • the target binding polypeptides are dimers.
  • the dimers of the invention are homodimers, comprising two identical monomeric subunits.
  • the dimers of the invention are heterodimers, comprising two non-identical monomeric subunits.
  • the dimers comprise at least two polypeptide chains.
  • the binding molecules comprise two polypeptide chains.
  • the binding molecules comprise three polypeptide chains.
  • the binding molecules comprise four polypeptide chains.
  • a binding molecule of the invention comprises at least one CDR of an antibody, e.g., an antibody known in the art to bind to a target of interest.
  • a binding molecule of the invention comprises at least two CDRs.
  • a binding molecule of the invention comprises at least three CDRs.
  • a binding molecule of the invention comprises at least four CDRs.
  • a binding molecule of the invention comprises at least five CDRs.
  • a binding molecule of the invention comprises at least six CDRs.
  • a binding molecule of the invention comprises at least one VH domain of an antibody, e.g., an antibody known in the art to bind to a target of interest.
  • a binding molecule of the invention comprises at least one VL domain.
  • a binding molecule of the invention comprises at least one VH domain and one VL domain of an antibody.
  • an antigen binding site consists of a VH domain, e.g., derived from camelids, which is stable in the absence of a VL chain (Hamers-Casterman et al. 1993. Nature 363:446; Desmyter et al. 1996. Nat. Struct. Biol. 3:803; Desmyter, A., 1996. Nat. Struct. Biol. 3:803; Decanniere, K., et al. 1999. Structure 7:361; Davies et al. 1996. Protein Eng. 9:531; Kortt et al. 1995. J. Protein Chem. 14:167).
  • VH domain e.g., derived from camelids
  • the invention also pertains to binding molecules which comprise one or more immunoglobulin domains.
  • the fusion proteins of the invention comprise a binding domain (which comprises at least one binding site) and a dimerization domain (which comprises at least one heavy chain portion).
  • the subject fusion proteins may be bispecific (with one binding site for a first target and a second binding site for a second target) or may be multivalent (with two binding sites for the same target).
  • Exemplary fusion proteins reported in the literature include fusions of the T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989); Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990)); L-selectin (homing receptor) (Watson et al., J. Cell. Biol.
  • CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley et al., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp. Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144 (1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci.
  • a fusion protein combines the binding domain(s) of the ligand or receptor (e.g. the extracellular domain (ECD) of a receptor) with at least one heavy chain domain and a synthetic connecting peptide.
  • nucleic acid encoding the binding domain of the ligand or receptor will be fused C-terminally to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence. N-terminal fusions are also possible.
  • a fusion protein includes a CH2 and a CH3 domain. Fusions may also be made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CH1 of the heavy chain or the corresponding region of the light chain.
  • the sequence of the ligand or receptor domain is fused to the N-terminus of the Fc domain of an immunoglobulin molecule. It is also possible to fuse the entire heavy chain constant region to the sequence of the ligand or receptor domain. In one embodiment, a sequence beginning in the hinge region just upstream of the papain cleavage site which defines IgG Fc chemically (i.e. residue 216, taking the first residue of heavy chain constant region to be 114), or analogous sites of other immunoglobulins is used in the fusion. The precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion, or binding characteristics of the molecule. Methods for making fusion proteins are known in the art.
  • the fusion proteins are assembled as multimers, and particularly as heterodimers or heterotetramers.
  • these assembled immunoglobulins will have known unit structures.
  • a basic four chain structural unit is the form in which IgG, IgD, and IgE exist.
  • a four chain unit is repeated in the higher molecular weight immunoglobulins; IgM generally exists as a pentamer of four basic units held together by disulfide bonds.
  • IgA globulin, and occasionally IgG globulin may also exist in multimeric form in serum. In the case of multimer, each of the four units may be the same or different.
  • Additional exemplary ligands and their receptors that may be included in the subject fusion proteins include the following:
  • Cytokines have pleiotropic effects on the proliferation, differentiation, and functional activation of lymphocytes.
  • Various cytokines, or receptor binding portions thereof, can be utilized in the fusion proteins of the invention.
  • Exemplary cytokines include the interleukins (e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, and IL-18), the colony stimulating factors (CSFs) (e.g.
  • G-CSF granulocyte CSF
  • GM-CSF granulocyte-macrophage CSF
  • M-CSF monocyte macrophage CSF
  • TNF tumor necrosis factor alpha and beta
  • interferons such as interferon- ⁇ , ⁇ , or ⁇ (U.S. Pat. Nos. 4,925,793 and 4,929,554).
  • Cytokine receptors typically consist of a ligand-specific alpha chain and a common beta chain.
  • exemplary cytokine receptors include those for GM-CSF, IL-3 (U.S. Pat. No. 5,639,605), IL-4 (U.S. Pat. No. 5,599,905), IL-5 (U.S. Pat. No. 5,453,491), IFN ⁇ (EP0240975), and the TNF family of receptors (e.g., TNF ⁇ (e.g. TNFR-1 (EP 417,563), TNFR-2 (EP 417,014) lymphotoxin beta receptor).
  • TNF ⁇ e.g. TNFR-1 (EP 417,563), TNFR-2 (EP 417,014) lymphotoxin beta receptor
  • Adhesion molecules are membrane-bound proteins that allow cells to interact with one another.
  • Various adhesion proteins including leukocyte homing receptors and cellular adhesion molecules, of receptor binding portions thereof, can be incorporated in a fusion protein of the invention.
  • Leucocyte homing receptors are expressed on leucocyte cell surfaces during inflammation and include the ⁇ -1 integrins (e.g. VLA-1, 2, 3, 4, 5, and 6) which mediate binding to extracellular matrix components, and the ⁇ 2-integrins (e.g. LFA-1, LPAM-1, CR3, and CR4) which bind cellular adhesion molecules (CAMs) on vascular endothelium.
  • exemplary CAMs include ICAM-1, ICAM-2, VCAM-1, and MAdCAM-1.
  • Other CAMs include those of the selectin family including E-selectin, L-selectin, and P-selectin.
  • Chemokines which stimulate the migration of leucocytes towards a site of infection, can also be incorporated into a fusion protein of the invention.
  • exemplary chemokines include Macrophage inflammatory proteins (MIP-1- ⁇ and MIP-1- ⁇ ), neutrophil chemotactic factor, and RANTES (regulated on activation normally T-cell expressed and secreted).
  • Growth factors or their receptors may be incorporated in the fusion proteins of the invention.
  • Exemplary growth factors include Vascular Endothelial Growth Factor (VEGF) and its isoforms (U.S. Pat. No. 5,194,596); Fibroblastic Growth Factors (FGF), including aFGF and bFGF; atrial natriuretic factor (ANF); hepatic growth factors (HGFs; U.S. Pat. Nos.
  • neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF- ⁇ platelet-derived growth factor (PDGF) (U.S. Pat. Nos. 4,889,919, 4,845,075, 5,910,574, and 5,877,016); transforming growth factors (TGF) such as TGF-alpha and TGF-beta (WO 90/14359), osteoinductive factors including bone morphogenetic protein (BMP); insulin-like growth factors-I and -II (IGF-I and IGF-II; U.S. Pat.
  • BDNF bone-derived neurotrophic factor
  • NT-3, NT-4, NT-5, or NT-6 neurotrophin-3, -4, -5, or NT-6
  • a nerve growth factor such as NGF- ⁇ platelet-derived growth factor (PDGF)
  • PDGF NGF
  • EPO Erythropoietin
  • SCF stem-cell factor
  • c-Mpl ligand thrombopoietin
  • Wnt polypeptides U.S. Pat. No. 6,159,462
  • Exemplary growth factor receptors which may be used as targeting receptor domains of the invention include EGF receptors; VEGF receptors (e.g. Flt1 or Flk1/KDR), PDGF receptors (WO 90/14425); HGF receptors (U.S. Pat. Nos.
  • LNGFR low affinity receptor
  • p75 NTR or p75 which binds NGF, BDNF, and NT-3
  • high affinity receptors that are members of the trk family of the receptor tyrosine kinases (e.g. trkA, trkb (EP 455,460), trkC (EP 522,530)).
  • Exemplary growth hormones for use as targeting agents in the fusion proteins of the invention include renin, human growth hormone (HGH; U.S. Pat. No. 5,834,598), N-methionyl human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone (PTH); thyroid stimulating hormone (TSH); thyroxine; proinsulin and insulin (U.S. Pat. Nos.
  • HGH human growth hormone
  • PTH parathyroid hormone
  • TSH thyroid stimulating hormone
  • thyroxine proinsulin and insulin
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • leptin leptin
  • glucagons bombesin
  • somatropin mullerian-inhibiting substance
  • relaxin and prorelaxin gonadotropin-associated peptide
  • prolactin placental lactogen
  • OB protein mullerian-inhibiting substance
  • Exemplary blood coagulation factors for use as targeting agents in the fusion proteins of the invention include the clotting factors (e.g., factors V, VII, VIII, X, IX, XI, XII and XIII, von Willebrand factor); tissue factor (U.S. Pat. Nos. 5,346,991, 5,349,991, 5,726,147, and 6,596,84); thrombin and prothrombin; fibrin and fibrinogen; plasmin and plasminogen; plasminogen activators, such as urokinase or human urine or tissue-type plasminogen activator (t-PA).
  • clotting factors e.g., factors V, VII, VIII, X, IX, XI, XII and XIII, von Willebrand factor
  • tissue factor U.S. Pat. Nos. 5,346,991, 5,349,991, 5,726,147, and 6,596,84
  • thrombin and prothrombin thrombin and prothrom
  • exemplary fusion proteins are taught, e.g., in WO0069913A1 and WO0040615A2.
  • Another exemplary molecule that may be included in a fusion protein of the invention is IGSF9.
  • Fusion proteins can be prepared using methods that are well known in the art (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Ordinarily, the ligand or receptor domain is fused C-terminally to the N-terminus of the constant region of the heavy chain (or heavy chain portion) and in place of the variable region. Any transmembrane regions or lipid or phospholipids anchor recognition sequences of ligand binding receptor are preferably inactivated or deleted prior to fusion. DNA encoding the ligand or receptor domain is cleaved by a restriction enzyme at or proximal to the 5′ and 3′ ends of the DNA encoding the desired ORF segment.
  • DNA encoding the fusion protein is then readily inserted into DNA encoding a heavy chain constant region.
  • the precise site at which the fusion is made may be selected empirically to optimize the secretion or binding characteristics of the soluble fusion protein.
  • DNA encoding the fusion protein is then transfected into a host cell for expression.
  • a binding molecule e.g., antigen binding molecule
  • a binding molecule is an antibody molecule.
  • antibodies are preferably raised in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g., purified tumor associated antigens or cells or cellular extracts comprising such antigens) and an adjuvant. This immunization typically elicits an immune response that comprises production of antigen-reactive antibodies from activated splenocytes or lymphocytes.
  • the resulting antibodies may be harvested from the serum of the animal to provide polyclonal preparations, it is often desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood to provide homogenous preparations of monoclonal antibodies (MAbs).
  • the lymphocytes are obtained from the spleen.
  • lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line (e.g. a myeloma cell line), thus, producing hybrid cells or “hybridomas” which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • an immortal tumor cell line e.g. a myeloma cell line
  • hybrid cells or “hybridomas” which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • the resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed “monoclonal.”
  • Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established.
  • culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen.
  • the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro assay, such as a radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice , pp 59-103 (Academic Press, 1986)).
  • the monoclonal antibodies secreted by the subclones may be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
  • DNA encoding a desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the isolated and subcloned hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins.
  • the isolated DNA (which may be modified as described herein) may be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al., U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody may be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.
  • DNA encoding antibodies or antibody fragments may also be derived from antibody phage libraries, e.g., using pd phage or Fd phagemid technology. Exemplary methods are set forth, for example, in EP 368 684 B1; U.S. Pat. No. 5,969,108, Hoogenboom, H. R. and Chames. 2000 . Immunol. Today 21:371; Nagy et al. 2002 . Nat. Med . 8:801; Huie et al. 2001 . Proc. Natl. Acad. Sci. USA 98:2682; Lui et al. 2002 . J. Mol. Biol .
  • Ribosomal display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al. 2000 . Nat. Biotechnol . 18:1287; Wilson et al. 2001 . Proc. Natl. Acad. Sci. USA 98:3750; or Irving et al. 2001 J. Immunol.
  • cell surface libraries can be screened for antibodies (Boder et al. 2000 . Proc. Natl. Acad. Sci. USA 97:10701; Daugherty et al. 2000 J. Immunol. Methods 243:211. Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.
  • Yet other embodiments of the present invention comprise the generation of human or substantially human antibodies in transgenic animals (e.g., mice) that are incapable of endogenous immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference).
  • transgenic animals e.g., mice
  • the homozygous deletion of the antibody heavy-chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin gene array to such germ line mutant mice will result in the production of human antibodies upon antigen challenge.
  • SCID mice Another preferred means of generating human antibodies using SCID mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated herein by reference. It will be appreciated that the genetic material associated with these human antibodies may also be isolated and manipulated as described herein.
  • lymphocytes can be selected by micromanipulation and the variable genes isolated.
  • peripheral blood mononuclear cells can be isolated from an immunized mammal and cultured for about 7 days in vitro. The cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated.
  • Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay.
  • Ig-producing B cells can be micromanipulated into a tube and the Vh and Vl genes can be amplified using, e.g., RT-PCR.
  • the VH and VL genes can be cloned into an antibody expression vector and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.
  • genetic sequences useful for producing the polypeptides of the present invention may be obtained from a number of different sources. For example, as discussed extensively above, a variety of human antibody genes are available in the form of publicly accessible deposits. Many sequences of antibodies and antibody-encoding genes have been published and suitable antibody genes can be chemically synthesized from these sequences using art recognized techniques. Oligonucleotide synthesis techniques compatible with this aspect of the invention are well known to the skilled artisan and may be carried out using any of several commercially available automated synthesizers. In addition, DNA sequences encoding several types of heavy and light chains set forth herein can be obtained through the services of commercial DNA synthesis vendors. The genetic material obtained using any of the foregoing methods may then be altered or modified to provide obtain polypeptides of the present invention.
  • antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a variety of laboratory manuals and primary publications. In this respect, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology , Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.
  • RNA may be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
  • Variable and constant region domains can be obtained from any source and be incorporated into a modified antibody of the invention.
  • mRNA can be isolated from hybridoma, spleen, or lymph cells, reverse transcribed into DNA, and antibody genes amplified by PCR.
  • PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences.
  • PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
  • primer sets suitable for amplification of antibody genes are known in the art (e.g., 5′ primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250); or based on known variable region framework amino acid sequences from the Kabat (Kabat et al. 1991. Sequences of Proteins of Immunological Interest. Bethesda, Md: JS Dep. Health Hum. Serv.
  • Constant region domains can be selected having a particular effector function (or lacking a particular effector function) or with a particular modification to reduce immunogenicity.
  • Variable and constant domains can be cloned, e.g., using the polymerase chain reaction and primers which are selected to amplify the domain of interest. PCR amplification methods are described in detail in U.S. Pat. Nos.
  • V domains can be obtained from libraries of V gene sequences from an animal of choice. Libraries expressing random combinations of domains, e.g., VH and VL domains, can be screened with a desired antigen to identify elements which have desired binding characteristics. Methods of such screening are well known in the art. For example, antibody gene repertoires can be cloned into a ⁇ bacteriophage expression vector (Huse, W D et al. 1989. Science 2476:1275). In addition, cells (Boder and Wittrup. 1997. Nat. Biotechnol. 15:553; Daugtherty, P. et al. 2000. J. Immunol. Methods. 243:211; Francisco et al. 1994. Proc.
  • VL and VH domains from a non-human source may also be used.
  • non-human V domains can be altered to reduce their immunogenicity using art recognized techniques.
  • mutations can be made to immunoglobulin domains to create a library of nucleic acid molecules having greater heterogeneity (Thompson, J., et al. 1996. J. Mol. Biol. 256:77; Lamminmaki, U. et al. 1999. J. Mol. Biol. 291:589; Caldwell, R. C. and Joyce G F. 1992. PCR Methods Appl. 2:28; Caldwell R C and Joyce G F. 1994. PCR Methods Appl. 3:S136. Standard screening procedures can be used to select high affinity variants.
  • changes to VH and VL sequences can be made to increase antibody avidity, e.g., using information obtained from crystal structures using techniques known in the art.
  • Exemplary constructs include, e.g., minibodies, diabodies, diabodies fused to CH3 molecules, tetravalent antibodies, intradiabodies (e.g., Jendreyko et al. 2003. J. Biol. Chem. 278:47813), bispecific antibodies, fusion proteins (e.g., antibody cytokine fusion proteins, proteins fused to at least a portion of an Fc receptor), bispecific antibodies.
  • Other immunoglobulins (Ig) and certain variants thereof are described, for example in U.S. Pat. No.
  • a polypeptide of the invention comprises an immunoglobulin heavy chain having deletion or substitution of at least one amino acid.
  • the mutation of one or more single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase tumor localization.
  • Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
  • a binding molecule of the invention lacks all or part of a CH2 domain.
  • the constant regions of the disclosed antibodies may be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct.
  • Yet other preferred embodiments may comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it may be desirable to insert or replicate specific sequences derived from selected constant region domains.
  • mutations to naturally occurring hinge regions can be made.
  • modifications to the constant region in accordance with the instant invention may easily be made using well known biochemical or molecular engineering techniques well within the skill of the art.
  • polypeptides of the invention comprise modified constant regions wherein one or more domains are partially or entirely deleted (“domain deleted antibodies”).
  • domain deleted antibodies are partially or entirely deleted.
  • compatible modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed. A variety of modified antibody constructs are described in more detail below.
  • the modified antibodies of the invention are minibodies.
  • Minibodies are dimeric molecules made up of two polypeptide chains each comprising an ScFv molecule (a single polypeptide comprising one or more antigen binding sites, e.g., a VL domain linked by a flexible linker to a VH domain fused to a CH3 domain via a connecting peptide.
  • An exemplary minibody construct is shown in FIG. 2 .
  • a CH3 domain is fused at its N-terminus to a connecting peptide which is fused at its N-terminus to a VH domain which is fused via its N-terminus to a flexible linker which is fused at its N-terminus to a VL domain.
  • ScFv molecules can be constructed in a VH-linker-VL orientation or VL-linker-VH orientation.
  • the flexible hinge that links the VL and VH domains that make up the antigen binding site preferably comprises from about 10 to about 50 amino acid residues.
  • An exemplary connecting peptide for this purpose is (Gly4Ser)3 (SEQ ID NO:35) (Huston et al. 1988. Proc. Natl. Acad. Sci. USA 85:5879). Other connecting peptides are known in the art.
  • Minibodies can be made by constructing an ScFv component and connecting peptide-CH3 component using methods described in the art (see, e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1). These components can be isolated from separate plasmids as restriction fragments and then ligated and recloned into an appropriate vector. Appropriate assembly can be verified by restriction digestion and DNA sequence analysis.
  • a minibody of the invention comprises a connecting peptide.
  • the connecting peptide comprises a gly/ser linker, e.g., GGGSSGGGSGG (SEQ ID NO: 1).
  • a tetravalent minibody in another embodiment, can be constructed. Tetravalent minibodies can be constructed in the same manner as minibodies, except that two ScFv molecules are linked using a flexible linker, e.g., having an amino acid sequence (G4S) 4 G3AS (SEQ ID NO: 36). An exemplary tetravalent minibody is illustrated in FIG. 2 .
  • the modified antibodies of the invention are CH2 domain deleted antibodies.
  • Domain deleted constructs can be derived from a vector (e.g., from IDEC Pharmaceuticals, San Diego) encoding an IgG 1 human constant domain (see, e.g., WO 02/060955A2 and WO02/096948A2).
  • the vector was engineered to delete the CH2 domain and provide a modified vector expressing a domain deleted IgG 1 constant region.
  • Genes encoding the murine variable region of the C2B8 antibody, 5E8 antibody, B3F6 antibody, or the variable region of the humanized CC49 antibody were then inserted in the modified vector and cloned.
  • these vectors When expressed in transformed cells, these vectors provided C2B8. ⁇ CH2, 5E8. ⁇ CH2, B3F6. ⁇ CH2 or huCC49. ⁇ CH2 or respectively. These constructs exhibit a number of properties that make them particularly attractive candidates for monomeric subunits.
  • the antibodies of the present invention can be engineered to partially delete or substitute of a few amino acids or even a single amino acid.
  • the mutation of a single amino acid in selected areas of the C H 2 domain may be enough to substantially reduce Fc binding and thereby increase tumor localization.
  • Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Creation of a C H 2 domain deleted version can be accomplished by way of overlapping PCR mutagenesis.
  • the gamma 1 constant domain begins with a plasmid encoded NheI site with is in translational reading frame with the immunoglobulin sequence.
  • a 5′ PCR primer was constructed encoding the NheI site as well as sequence immediately downstream.
  • a 3′ PCR primer mate was constructed such that it anneals with the 3′ end to the immunoglobulin hinge region and encodes in frame the first several amino acids of the gamma 1 CH3 domain.
  • a second PCR primer pair consisted of the reverse complement of the 3′ PCR primer from the first pair (above) as the 5′ primer and a 3′ primer that anneals at a loci spanning the BsrGI restriction site within the C H 3 domain.
  • the resultant products were utilized as template with the NheI and BsrGI 5′ and 3′, respectively primers.
  • the amplified product was then cloned back into N5KG1 to create the plasmid N5KG1 ⁇ C H 2.
  • This construction places the intact CH3 domain immediately downstream and in frame with the intact hinge region.
  • a similar procedure can be used to create a domain deleted construct in which the CH3 domain is immediately downstream of a connecting peptide.
  • a domain deleted version of the C2B8 antibody was created in this manner as described in U.S. Pat. Nos. 5,648,267 and 5,736,137 each of which is incorporated herein by reference.
  • tetravalent domain-deleted antibodies can be produced by combining a DNA sequence encoding a domain deleted antibody with a ScFv molecule. For example, in one embodiment, these sequences are combined such that the ScFv molecule is linked at its N-terminus to the CH3 domain of the domain deleted antibody via a flexible linker (e.g., a gly/ser linker such as (Gly4Ser) 3 (SEQ ID NO: 35).
  • a flexible linker e.g., a gly/ser linker such as (Gly4Ser) 3 (SEQ ID NO: 35).
  • a tetravalent antibody in another embodiment can be made by fusing an ScFv molecule to a connecting peptide, which is fused to a CH1 domain to construct an ScFv-Fab tetravalent molecule.
  • a binding molecule of the invention is a diabody fused to at least one heavy chain portion. In a preferred embodiment, a binding molecule of the invention is a diabody fused to a CH3 domain.
  • a binding molecule comprises a tetravalent or bispecific tetravalent CH2 domain-deleted antibody with a scFv appended to the N-terminus of the light chain.
  • a binding molecule comprises a a tetravalent or bispecific tetravalent CH2 domain-deleted antibody with a scFv appended to the N-terminus of the heavy chain.
  • the attachment of the scFv to the N-terminus results in reduced aggregation of the molecules as compared to molecules in which the scFv is attached at the carboxy-terminus.
  • less than about 30% aggregates are present in a composition of binding molecules produced by cells expressing a N-terminal fusion construct. In one embodiment, less than about 20% aggregates are present in a composition of binding molecules produced by cells expressing a N-terminal fusion construct. In one embodiment, less than about 10% aggregates are present in a composition of binding molecules produced by cells expressing a N-terminal fusion construct. In one embodiment, less than about 5% aggregates are present in a composition of binding molecules produced by cells expressing a N-terminal fusion construct.
  • modified antibodies are also within the scope of the instant invention (e.g., WO 02/02781 A1; U.S. Pat. Nos. 5,959,083; 6,476,198 B1; US 2002/0103345 A1; WO 00/06605; Byrn et al. 1990. Nature. 344:667-70; Chamow and Ashkenazi. 1996. Trends Biotechnol. 14:52).
  • At least one binding specificity of a modified antibody molecule of the invention is catalytic.
  • Catalytic binding specificities can be made using art recognized techniques (see, e.g., U.S. Pat. No. 6,590,080, U.S. Pat. No. 5,658,753).
  • Catalytic binding specificities can work by a number of basic mechanisms similar to those identified for enzymes to stabilize the transition state, thereby reducing the free energy of activation. For example, general acid and base residues can be optimally positioned for participation in catalysis within catalytic active sites; covalent enzyme-substrate intermediates can be formed; catalytic antibodies can also be in proper orientation for reaction and increase the effective concentration of reactants by at least seven orders of magnitude (Fersht, A.
  • catalytic antibodies can convert the energy obtained upon substrate binding to distort the reaction towards a structure resembling the transition state.
  • acid or base residues can be brought into the binding site by using a complementary charged molecule as an immunogen.
  • a complementary charged molecule as an immunogen. This technique proved successful for elicitation of antibodies with a hapten containing a positively-charged ammonium ion (Shokat, et al., Chem. Int. Ed. Engl. 27 (1988):269-271).
  • antibodies can be elicited to stable compounds that resemble the size, shape, and charge of the transition state of a desired reaction (i.e., transition state analogs). See U.S. Pat. No. 4,792,446 and U.S. Pat. No. 4,963,355 which describe the use of transition state analogues to immunize animals and the production of catalytic antibodies. Both of these patents are hereby incorporated by reference.
  • such molecules can be administered as part of an immunoconjugate, e.g., with an immunogenic carrier molecule, such as KLH.
  • Exemplary catalytic binding specificities can have, e.g., esterase activity (involving a charged transition state whose electrostatic and shape characteristics can be mimicked by a phosphonate structure; Jacobs, et al., J. Am. Chem. Soc. 109 (1987):2174-2176; Durfor, et al., J. Am. Chem. Soc. 110 (1988):8713-8714; Tramontano, et al., J. Am. Chem. Soc. 110 (1988):2282; Pollack, et al., J. Am. Chem. Soc.
  • catalytic B cells can be selected, e.g., as described in U.S. Pat. No. 6,590,080 using a molecule can be constructed which facilitates selection of catalytic B cells.
  • catalytic binding specificities can be developed as part of a two step process.
  • Catalytic antibodies can be selected only if displaying the following binding features: binding both the substrate and a reactive group in such a way that the two groups are in a reactive position towards each other.
  • the selected antibodies can be chemically engineered by covalently binding a reactive group into the binding pocket of the antibody. J Immunol Methods. 2002. 269:81-98.
  • a catalytic binding specificity is specific for a prodrug.
  • a binding specificity can be used to catalyze the conversion of a prodrug into a drug which is effective in vivo.
  • the reaction catalyzed is one that cannot be accomplished by natural enzymes in vivo.
  • prodrug activation by antibodies are known in the art (see, e.g., Miyashita et al. 1993. Proc. Natl. Acad. Sci. USA 90:5337).
  • a modified antibody molecule of the invention comprises at least one binding specificity for a target cell and at least one binding specificity for a prodrug.
  • an modified antibody molecule of the invention comprises at least one binding specificity for a tumor cell and at least one binding specificity for a prodrug which can be converted to cytotoxic drug.
  • a modified antibody of the invention comprises a binding specificity for a carbamate prodrug 4-[N,N,-bis(2-chloroethyl)]aminophenyl-N-[(1S-(1,3-dicarboxy)propyl]carbamate and generates the corresponding cytotoxic nitrogen mustard (Wentworth et al. 1996. Proc Natl. Acad. Sci. USA. 93:799).
  • the modified antibody is administered prior to administration of the prodrug to allow accumulation at the site of the target cell.
  • exemplary prodrugs are known in the art. Prodrugs can also be synthesized by incorporating a portion designed to be released by catalytic action, e.g., by sequential retro-aldol/retro-Michael reactions catalyzed by an antibody with aldolase activity. (Shabat et al. 2001. Proc. Natl. Acad. Sci. USA 98:7428). Such drug masking portions can be made, e.g., by modification of hydroxyl or thiol groups of drugs.
  • a binding molecule of the invention is multispecific, i.e., has at least one binding site that binds to a first molecule or epitope of a molecule and at least one second binding site that binds to a second molecule of epitope of a molecule.
  • a binding molecule of the invention is bispecific.
  • Bispecific molecules can bind to two different target sites, e.g., on the same target molecule or on different target molecules.
  • bispecific molecules can bind to two different epitopes, e.g., on the same antigen or on two different antigens.
  • Bispecific molecules can be used, e.g., in diagnostic and therapeutic applications. For example, they can be used to immobilize enzymes for use in immunoassays. They can also be used in diagnosis and treatment of cancer, e.g., by binding both to a tumor associated molecule and a detectable marker (e.g., a chelator which tightly binds a radionuclide.
  • a detectable marker e.g., a chelator which tightly binds a radionuclide.
  • Bispecific molecules can also be used for human therapy, e.g., by directing cytotoxicity to a specific target (for example by binding to a pathogen or tumor cell and to a cytotoxic trigger molecule, such as the T cell receptor or the Fc ⁇ receptor.
  • a cytotoxic trigger molecule such as the T cell receptor or the Fc ⁇ receptor.
  • Bispecific antibodies can also be used, e.g., as fibrinolytic agents or vaccine adjuvants.
  • bispecific binding molecules include those with at least two arms directed against tumor cell antigens; bispecific binding molecules with at least one arm directed against a tumor cell antigen and the at least one arm directed against a cytotoxic trigger molecule (such as anti-Fc.gamma.RI/anti-CD15, anti-p185.sup.HER2/Fc.gamma.RIII (CD16), anti-CD3/anti-malignant B-cell (1D10), anti-CD3/anti-p185.sup.HER2, anti-CD3/anti-p97, anti-CD3/anti-renal cell carcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma), anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGF receptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19, anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM)/anti-CD3, anti-folate binding protein (FBP)/anti-cyto
  • bispecific binding molecules for use in therapy of infectious diseases such as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cell receptor: CD3 complex/anti-influenza, anti-Fc.gamma.R/anti-HIV; bispecific binding molecules for tumor detection in vitro or in vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA, anti-p185HER2/anti-hapten); bispecific binding molecules as vaccine adjuvants (see Fanger et al., supra); and bispecific binding molecules as diagnostic tools (such as anti-rabbit IgG/anti-ferritin, anti-horse radish peroxidase (HRP)/anti-hormone, anti-somatostatin/anti-substance P, anti-HRP/anti-FITC, anti-CEA/anti-.beta.-galactosi
  • infectious diseases such as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cell receptor: CD
  • a bispecific molecule of the invention binds to CRIPTO-I.
  • Bispecific molecules may be monovalent for each specificity or be multivalent for each specificity.
  • an antibody molecule or fusion protein may comprise one binding site that reacts with a first target molecule and one binding site that reacts with a second target molecule or it may comprise two binding sites that react with a first target molecule and two binding sites that react with a second target molecule.
  • Methods of producing bispecific molecules are well known in the art.
  • recombinant technology can be used to produce bispecific molecules, e.g., diabodies, single-chain diabodies, tandem scFvs, etc.
  • Exemplary techniques for producing bispecific molecules are known in the art (e.g., Kontermann et al. Methods in Molecular Biology Vol.
  • a multimeric bispecific molecules are prepared using methods such as those described e.g., in US 2003/0207346 A1 or U.S. Pat. No. 5,821,333, or US2004/0058400.
  • multispecific fusion protein designates fusion proteins (as hereinabove defined) having at least two binding specificities (i.e. combining two or more binding domains of a ligand or receptor).
  • Multispecific fusion proteins can be assembled as heterodimers, heterotrimers or heterotetramers, essentially as disclosed in WO 89/02922 (published Apr. 6, 1989), in EP 314,317 (published May 3, 1989), and in U.S. Pat. No. 5,116,964 issued May 2, 1992.
  • Preferred multispecific fusion proteins are bispecific. Examples of bispecific fusion proteins include CD4-IgG/TNFreceptor-IgG and CD4-IgG/L-selectin-IgG.
  • the last mentioned molecule combines the lymph node binding function of the lymphocyte homing receptor (LHR, L-selectin), and the HIV binding function of CD4, and finds potential application in the prevention or treatment of HIV infection, related conditions, or as a diagnostic.
  • exemplary binding sites include one or more epitopes of a tumor antigen.
  • Other exemplary target molecules include one or more epitopes of, e.g., heparin sulfate, growth factors or their receptors (e.g., epidermal growth factor receptor, insulin-like growth factor receptor, hepatocyte growth factor (HGF/SF) receptor, MORE (See, e.g., Cao et al. Proc. Natl. Acad. Sci 2001. 98:7443; Lu et al. 2004. J. Biol. Chem. 279:2856).
  • the invention pertains to bispecific molecules, e.g., antibodies, which incorporate at least one binding site that binds to a known target and at least one binding site which recognizes an unknown target (for example, in one embodiment, the bispecific molecule incorporates binding sites selected from a semi-synthetic antibody phage display library).
  • bispecific molecules e.g., antibodies, which incorporate at least one binding site that binds to a known target and at least one binding site which recognizes an unknown target
  • the bispecific molecule incorporates binding sites selected from a semi-synthetic antibody phage display library.
  • one of ordinary skill in the art could start with a single chain antibody of known specificity and build a Fab library using techniques known in the art or, alternatively, the skilled artisan could start with an Fab fragment of known specificity and build a single chain library using techniques known in the art.
  • libraries from nonimmunized sources and prepared by synthetic recombination of V-gene sequences preferably recombination of VH with, DH and JH, and VL with JL sequences
  • VH with, DH and JH, and VL with JL sequences can be used to isolate antibodies to any antigen.
  • patent application WO92/01047 teaches that antibody fragments can be displayed on the surface of bacteriophage and that they will bind antigen.
  • Antibody fragments e.g., Fab, Fv, ScFv and VH
  • Antibody fragments can be directly selected using this characteristic.
  • Other methods known in the art include those taught, e.g., in U.S. Pat. No. 5,698,426; 6,291,159; 5,658,727; 5,667,988; and 5,969,108.
  • scFv which recognize a known target can be dimerized with scFv isolated from a semi-synthetic human phage antibody display library. (see, e.g., Kruif and Logtenberg 1996. J. Biol. Chem. 271:7630).
  • the subject bispecific molecule is expressed in any expression system used to express antibody molecules, for example mammalian cells, yeast such as Picchia, E. coli , Bacculovirus, etc.
  • the subject bispecific molecule is expressed in the NEOSPLA vector system (see, e.g., U.S. Pat. No. 6,159,730).
  • This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence.
  • the subject bispecific molecules comprise a synthetic connecting peptide.
  • bispecific molecules have one or more binding sites for a known target and express a library at one or more binding sites.
  • Such bispecific molecules can be used, e.g., to identify molecules in close proximity to or associated with the known target.
  • the skilled artisan could use the subject bispecific molecules in an assay to select for those that induce a particular response, e.g., apoptosis or cellular activation, using screening methods well known in the art.
  • the bispecific molecule identified as producing the response screened for can then be identified and its specificity determined. Using such methods it is possible to identify molecules in close association with particular targets of interest, e.g., T cell markers or other signaling molecules (such as CRIPTO-I, death domain molecules, or molecules involved in apoptosis).
  • the proximity of the known target and the molecule newly identified as a “nearest neighbor” can be confirmed using immunoprecipitation or other techniques known to those of skill in the art. Using these methods it is possible to identify molecules as targets for modulating a particular cellular response.
  • a polypeptide molecule of the invention comprises an amino acid sequence encoded by a nucleic acid molecule comprising a nucleic acid sequence shown in FIG. 8A (SEQ ID NO:16), FIG. 8B (SEQ ID NO:17), FIG. 8C (SEQ ID NO:18), FIG. 10A (SEQ ID NO:22), FIG. 10B (SEQ ID NO:23).
  • a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 8A (SEQ ID NO:16), FIG. 8B (SEQ ID NO:17), FIG. 8C (SEQ ID NO:18), FIG. 10A (SEQ ID NO:22), FIG. 10B (SEQ ID NO:23).
  • a polypeptide of the invention comprises an amino acid sequence shown in FIG. 9A (SEQ ID NO:19), FIG. 9B (SEQ ID NO:20), FIG. 9C (SEQ ID NO:21), FIG. 11A (SEQ ID NO:24), or FIG. 11B (SEQ ID NO:25).
  • a polypeptide of the invention is encoded by a nucleic acid molecule comprising the nucleotide sequence shown in FIG. 12A (SEQ ID NO:26), FIG. 12B (SEQ ID NO:27), FIG. 14 (SEQ ID NO:30), or FIG. 15 (SEQ ID NO:31).
  • a nucleic acid molecule of the invention comprises a nucleotide sequence shown in FIG. 12A (SEQ ID NO:26), FIG. 12B (SEQ ID NO:27), FIG. 14 (SEQ ID NO:30), or FIG. 15 (SEQ ID NO:31).
  • a polypeptide molecule of the invention comprises an amino acid sequence shown in FIG. 13A (SEQ ID NO:28), FIG. 13B (SEQ ID NO:29), FIG. 16 (SEQ ID NO:32), or FIG. 17 (SEQ ID NO:33).
  • the genes are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of modified antibody that, in turn, provides the claimed polypeptides.
  • vector or “expression vector” is used herein for the purposes of the specification and claims, to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a cell.
  • vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses.
  • vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
  • vector systems may be employed.
  • one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus.
  • Others involve the use of polycistronic systems with internal ribosome binding sites.
  • Exemplary vectors include those taught in U.S. Pat. Nos. 6,159,730 or 6,413,777 or US 2003 0157641 A1).
  • cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper.
  • biocide resistance e.g., antibiotics
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation.
  • an inducible expression system can be employed.
  • Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.
  • a secretion signal e.g., any one of several well characterized bacterial leader peptides (e.g., pelB, phoA, or ompA), can be fused in-frame to the N terminus of a polypeptide of the invention to obtain optimal secretion of the polypeptide.
  • a secretion signal e.g., any one of several well characterized bacterial leader peptides (e.g., pelB, phoA, or ompA)
  • pelB e.g., phoA, or ompA
  • a vector can be used which comprises a nucleic acid sequence encoding a peptide linker.
  • the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human) modified as discussed above.
  • this is effected using a proprietary expression vector of IDEC, Inc., referred to as NEOSPLA see U.S. Pat. No. 6,159,730.
  • This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence.
  • this vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification.
  • Vector systems are also taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated by reference in its entirety herein. This system provides for high expression levels, e.g., >30 pg/cell/day.
  • Other exemplary vector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.
  • polypeptides of the invention of the instant invention may be expressed using polycistronic constructs such as those disclosed in United States provisional application No. 60/331,481 filed Nov. 16, 2001 and incorporated herein in its entirety.
  • polycistronic constructs such as those disclosed in United States provisional application No. 60/331,481 filed Nov. 16, 2001 and incorporated herein in its entirety.
  • multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct.
  • IRES internal ribosome entry site
  • Compatible IRES sequences are disclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of polypeptides disclosed in the instant application.
  • the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed.
  • Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. “ Mammalian Expression Vectors ” Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds.
  • plasmid introduction into the host is via electroporation.
  • the transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis.
  • exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or flourescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
  • transformation shall be used in a broad sense to refer to any introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.
  • host cells refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.
  • the terms “cell” and “cell culture” are used interchangeably to denote the source of antibody unless it is clearly specified otherwise.
  • recovery of polypeptide from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
  • the host cell line used for protein expression is most preferably of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein.
  • Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney).
  • CHO cells are particularly preferred. Host cell lines are typically available from commercial services, the
  • a host cell is a prokaryotic cell, e.g., a strain which allows the formation of disulfide bonds (Derman, A I, et al. 1993. Science. 262:1744; Bessette, P H. Et al. 1999. Proc. Natl. Acad. Sci. USA 96:13703).
  • tissue culture conditions include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges.
  • the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a modified hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
  • customary chromatography methods for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a modified hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
  • Genes encoding the polypeptide of the invention can also be expressed non-mammalian cells such as bacteria or yeast or plant cells.
  • various unicellular non-mammalian microorganisms such as bacteria can also be transformed; i.e. those capable of being grown in cultures or fermentation.
  • Bacteria which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella ; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus , and Haemophilus influenzae .
  • the polypeptides typically become part of inclusion bodies.
  • the polypeptides must be isolated, purified and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits will then self-assemble into tetravalent antibodies (WO02/096948A2).
  • eukaryotic microbes may also be used. Saccharomyces cerevisiae , or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available.
  • Saccharomyces cerevisiae or common baker's yeast
  • the plasmid YRp7 for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used.
  • This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)).
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • the invention pertains to separation of molecules having two heavy chain portions from a mixture, where a fraction of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage and a fraction of the molecules comprise heavy chain portions that are not linked via at least one disulfide linkage by hydrophobic interaction chromatography.
  • Hydrophobic interaction chromatography was first developed following the observation that proteins could be retained on affinity gels which comprised hydrocarbon spacer arms but lacked the affinity ligand. Elution from HIC supports can be effected by alterations in solvent, pH, ionic strength, or by the addition of chaotropic agents or organic modifiers, such as ethylene or propylene glycol. A description of the general principles of hydrophobic interaction chromatography can be found e.g., in U.S. Pat. No. 3,917,527 and in U.S. Pat. No. 4,000,098.
  • HIC in the context of high performance liquid chromatography (HPLC) has been used to separate antibody fragments lacking heavy chain portions (e.g., F(ab′) 2 ) from intact antibody molecules in a single step protocol.
  • HPLC high performance liquid chromatography
  • the separation method of the invention can be performed on an unpurified population of polypeptides (e.g., culture supernatants or preparations or preparations of polypeptides isolated from prokaryotic inclusion bodies).
  • the instant separation methods can be used on polypeptide mixtures obtained after one or more initial purification steps, e.g., after a preparation comprising forms A and B has been eluted from an affinity matrix.
  • the binding molecules subjected to HIC chromatography comprise a connecting peptide of the invention.
  • HIC can be applied to mixtures that have been partially purified by other protein purification procedures.
  • the term “partially purified” as used herein includes a protein preparation in which the protein of interest is present in at least 5% by weight, more preferably at least 10% and most preferably at least 45%.
  • Initial or subsequent purification steps can be used to remove, e.g., immunoglobulin aggregates, misfolded species, host cell protein, residue material from preceding chromatographic steps (such as Protein A when employed).
  • HIC can be performed on polypeptides comprising a connecting peptide of the invention. Accordingly, the application of HIC can also be appreciated in the context of an overall purification protocol.
  • Exemplary purification steps that can be used prior to or subsequent to HIC include: affinity chromatography (for example, PROSEP-A® (BioProcessing Ltd., U.K.) which consists of Protein A covalently coupled to controlled pore glass or Protein A SEPHAROSE® Fast Flow (Pharmacia) or TOYOPEARL 650M Protein A (TosoHaas)). Protein A is preferred for human ⁇ 1, ⁇ 2, or ⁇ 4 heavy chains and protein G for mouse isotypes. Bakerbond ABXtm resin can be used if the molecule comprises a CH3 domain. In addition or alternatively, ion exchange chromatography may be employed.
  • affinity chromatography for example, PROSEP-A® (BioProcessing Ltd., U.K.) which consists of Protein A covalently coupled to controlled pore glass or Protein A SEPHAROSE® Fast Flow (Pharmacia) or TOYOPEARL 650M Protein A (TosoHaas)
  • Protein A is preferred
  • anionic or cationic substituents may be attached to matrices in order to form anionic or cationic supports for chromatography.
  • Anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups.
  • Cationic exchange substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).
  • Cellulose ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K.
  • SEPHADEX®-based and -locross-linked ion exchangers are also known.
  • DEAE-, QAE-, CM-, and SP-SEPHADEX® and DEAE-, Q-, CM-and S-SEPHAROSE® and SEPHAROSE® Fast Flow are all available from Pharmacia AB.
  • both DEAE and CM derivitized ethylene glycol-methacrylate copolymer such as TOYOPEARL DEAE-650S or M and TOYOPEARL CM-650S or M are available from Toso Haas Co., Philadelphia, Pa.
  • HIC ionic exchange chromatographic step or other salt mediated purification step
  • Additional purification protocols may be added including but not necessarily limited to: further ionic exchange chromatography, size exclusion chromatography, viral inactivation, concentration and freeze drying, hydroxylapatite chromatography, gel electrophoresis, dialysis, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEQHAROSETM, chromatofocusing, or ammonium sulfate precipitation.
  • the composition comprising the mixture of polypeptides to be separated will preferably be placed in a buffer of acidic or approximately neutral pH. This can be done, for example, by adding concentrated buffer, resuspending the sample in the buffer, exchanging the buffer (e.g., using dialysis or ultrafiltration). Alternatively, the pH of the sample buffer can simply be adjusted to be within the desired range.
  • Hydrophobic interactions are strongest at high ionic strength, therefore, this form of separation is conveniently performed following salt precipitations or ion exchange procedures.
  • Adsorption of the proteins to a HIC column is favored by high salt concentrations, but the actual concentrations can vary over a wide range depending on the nature of the protein and the particular HIC ligand chosen.
  • Various ions can be arranged in a so-called soluphobic series depending on whether they promote hydrophobic interactions (salting-out effects) or disrupt the structure of water (chaotropic effect) and lead to the weakening of the hydrophobic interaction.
  • Cations are ranked in terms of increasing salting out effect as Ba ++ ⁇ ; Ca ++ ⁇ ; Mg ++ ⁇ ; Li + ⁇ ; Cs + ⁇ ; Na + ⁇ ; K + ⁇ ; Rb + ⁇ ; NH 4 + , while anions may be ranked in terms of increasing chaotropic effect as PO ⁇ ⁇ ; SO 4 ⁇ ⁇ ; CH 3 COOO ⁇ ⁇ ; Cl ⁇ ⁇ ; Br ⁇ ⁇ ; NO 3 ⁇ ⁇ ; ClO 4 ⁇ ⁇ ; I ⁇ ⁇ ; SCN ⁇
  • Na, K or NH 4 sulfates effectively promote ligand-protein interaction in HIC.
  • Salts may be formulated that influence the strength of the interaction as given by the following relationship: (NH 4 ) 2 SO 4 >; Na 2 SO 4 >; NaCl>; NH 4 Cl>; NaBr>; NaSCN.
  • salt concentrations of between about 0.75 and about 2M ammonium sulfate or between about 1 and 4M NaCl are useful.
  • a number of chromatographic supports may be employed in the preparation of HIC columns, the most extensively used are agarose, silica and organic polymer or co-polymer resins.
  • the hydrophobic interaction material is generally a base matrix (e.g., a hydrophilic carbohydrate (such as cross-linked agarose) or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled.
  • the preferred HIC material comprises an agarose resin substituted with phenyl groups.
  • Exemplary HIC material includes: phenyl SEPHAROSETM, FAST FLOW with low or high substitution (Pharmacia LKB Biotechnology, AB, Sweden); phenyl SEPHAROSETM High Performance column; phenyl or butyl-SEPHAROSE® CL-4B, butyl-SEPHAROSE® FF, octyl-SEPHAROSE® FF and phenyl-SEPHAROSE® FF (Pharmacia LKB Biotechnology AB, Sweden); FractogelTM EMD Propyl or FRACTOGELTM EMC Phenyl columns (E.
  • HIC materials are also available from Tosoh Corporation, Tokyo, Japan under the product names TOYOPEARL ether 650, phenyl 650, butyl 650 (Fractogel), ether-5PW-HR, or phenyl-5PW-HR; Miles-Yeda, Rehovot, Israel under the product name alkyl-agarose, wherein the alkyl group contains from 2-10 carbon atoms, and J.T. Baker, Phillipsburg, N.J.
  • a particular gel can be determined by the skilled artisan. In general the strength of the interaction of the protein and the HIC ligand increases with the chain length of the alkyl ligands but ligands having from about 4 to about 8 carbon atoms are suitable for most separations.
  • a phenyl group has about the same hydrophobicity as a pentyl group, although the selectivity can be different owing to the possibility of pi-pi orbital interaction with aromatic groups on the protein. Selectively may also be affected by the chemistry of the supporting resin.
  • Ligand density is an important parameter in that it influences not only the strength of the interaction but the capacity of the column as well.
  • the ligand density of the commercially available phenyl or octyl phenyl gels is on the order of 40 pmoles/ml gel bed.
  • Gel capacity is a function of the particular protein in question as well as pH, temperature and salt type and concentration but generally can be expected to fall in the range of 3-20 mg/ml of gel.
  • the polypeptides of the invention can be eluted isocratically.
  • isocratic elution all compounds begin migration through the column at onset. However, each migrates at a different rate, resulting in faster or slower elution rate.
  • form A can be eluted with the flow through of the column.
  • one or more polypeptides of the invention can be bound to the column and eluted, e.g., using stepwise elution or gradient elution.
  • Elution can be accomplished in a variety of ways: (a) by changing the salt concentration, (b) by changing the polarity of the solvent or (c) by adding detergents.
  • By decreasing salt concentration adsorbed proteins are eluted in order of increasing hydrophobicity. Changes in polarity may be affected by additions of solvents such as ethylene or propylene glycol or (iso)propanol, thereby decreasing the strength of the hydrophobic interactions.
  • Detergents function as displacers of proteins and have been used primarily in connection with the purification of membrane proteins
  • the polypeptide mixture can be contacted with the HIC material e.g., using a batch purification technique or using a column.
  • HIC purification it may be desirable to remove any chaotropic agents or very hydrophobic substances, e.g., by passing the mixture through a precolumn.
  • HIC material is prepared in or equilibrated to the desired starting buffer.
  • a slurry of the HIC material is obtained.
  • the polypeptide solution is contacted with the slurry to adsorb at least one of the polypeptides to be separated to the HIC material.
  • the solution containing the polypeptides that do not bind to the HIC material is separated from the slurry, e.g., by allowing the slurry to settle and removing the supernatant.
  • the slurry can be subjected to one or more washing steps.
  • the slurry can be contacted with a solution of lower conductivity to desorb polypeptides that have bound to the HIC material. In order to elute bound polypeptides, the salt concentration can be decreased.
  • the HIC material can be packed in a column.
  • a mixture comprising the polypeptides to be separated can be applied to the column allowing at least one of the polypeptides to be separated to adsorb to the column.
  • the polypeptides that do not adsorb to the column pass through and can be collected.
  • the salt concentration can be decreased, e.g., in a step-wise fashion or using a salt gradient.
  • form B Since form B is more hydrophobic than form A, it adsorbs irreversibly to the stationary phase using approximately 0.7 M (e.g., 0.73M)Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0 to pH 8.0 as the mobile phase.
  • Form A binds to a lesser extent to the stationary phase under these conditions and is therefore eluted isocratically, i.e. it leaves the column with the flowthrough fraction. Subsequent to the isocratic elution of form A, omitting Ammonium sulfate from the mobile phase desorbs form B.
  • the HIC material is equilibrated in a buffer comprising a salt concentration yielding a conductivity of from between about 160 to about 110, preferably from between about 140 to about 115, even more preferably from between about 130 or about 120 to about 117 mS/cm.
  • a starting solution comprises a salt concentration of approximately 1M to 0.7M, e.g., 1M to 0.7M ammonium sulfate.
  • the solution comprising the mixture of polypeptides to be separated is also brought to the same, or approximately the same conductivity (e.g., using a concentrated stock solution of salt).
  • Form A is eluted from the column at a conductivity of about 120 mS/cm.
  • a stepwise or linear gradient of reducing ammonium sulfate content can be applied to the column.
  • Form B elutes at a conductivity of approximately 115 to approximately 100 mS/cm.
  • the subject purification method yields a composition comprising polypeptide molecules having at least two target binding sites and two heavy chain portions, wherein the heavy chain portions lack CH2 domains and wherein greater than about 50% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • greater than about 60% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • greater than about 70% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • greater than about 80% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage. In another embodiment, greater than about 90% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • the subject purification method yields a composition comprising recombinant polypeptide molecules having at least two target binding sites and two heavy chain portions, wherein greater than about 99% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage.
  • the subject purification method yields a composition comprising polypeptide molecules having at least two target binding sites and two heavy chain portions, wherein greater than about 95% of the molecules are present in a form in which the two heavy chain portions are linked via at least one interchain disulfide linkage, and wherein the heavy chain portions of the polypeptides are derived from an antibody of the IgG4 isotype.
  • the subject purification method yields a composition comprising polypeptide molecules having two light chain portions and two heavy chain portions, wherein the heavy chain portions lack CH2 domains and wherein greater than about 80% of the molecules are present in a form in which the two heavy chain portions are not linked via at least one interchain disulfide linkage.
  • the instant invention also provides methods for monitoring the results of purification and/or preferential biosynthesis comprising measuring the relative amounts of Form A and Form B in a composition.
  • Form A and Form B can be measured, e.g., as described herein using non-reducing SDS polyacrylamide gel electrophoresis or mass spectrometry.
  • polypeptide molecules of the present invention may be used in non-conjugated form or may conjugated to at least one of a variety of molecules, e.g., to facilitate antigen detection or for imaging or therapy of the patient.
  • the polypeptides of the invention can be labeled or conjugated either before or after purification, when purification is performed.
  • the polypeptides of the present invention may be conjugated to cytotoxins (such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or antibodies wherein the resulting molecule binds to both the neoplastic cell and an effector cell such as a T cell), or PEG.
  • cytotoxins such as radioisotopes, cytotoxic drugs, or toxins
  • therapeutic agents such as radioisotopes, cytotoxic drugs, or toxins
  • cytostatic agents such as radioisotopes, cytotoxic drugs, or toxins
  • Still other embodiments of the present invention comprise the use of polypeptides of the invention conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin or diphtheria toxin.
  • cytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin or diphtheria toxin.
  • the selection of which conjugated or unconjugated polypeptide to use will depend on the type and stage of cancer, use of adjunct treatment (e.g., chemotherapy or external radiation) and patient condition. It will be appreciated that one skilled in the art could readily make such a selection in view of the teachings herein.
  • radioisotopes include: 90 Y, 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re and 188 Re.
  • the radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death.
  • the isotopes used to produce therapeutic conjugates typically produce high energy ⁇ - or ⁇ -particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.
  • polypeptides of the invention may be directly labeled (such as through iodination) or may be labeled indirectly through the use of a chelating agent.
  • a chelating agent is covalently attached to a molecule and at least one radionuclide is associated with the chelating agent.
  • Such chelating agents are typically referred to as bifinctional chelating agents as they bind both the polypeptide and the radioisotope.
  • Particularly preferred chelating agents comprise 1-isothiocycmatobenzyl-3-methyldiothelene triaminepentaacetic acid (“MX-DTPA”) and cyclohexyl diethylenetriamine pentaacetic acid (“CHX-DTPA”) derivatives.
  • Other chelating agents comprise P-DOTA and EDTA derivatives.
  • Particularly preferred radionuclides for indirect labeling include 111 In and 90 Y.
  • direct labeling and “direct labeling approach” both mean that a radionuclide is covalently attached directly to a polypeptide (typically via an amino acid residue). More specifically, these linking technologies include random labeling and site-directed labeling. In the latter case, the labeling is directed at specific sites on the polypeptide, such as the N-linked sugar residues present only on the Fc portion of the conjugates. Further, various direct labeling techniques and protocols are compatible with the instant invention.
  • Technetium-99m labeled polypeptides may be prepared by ligand exchange processes, by reducing pertechnate (TcO 4 ⁇ ) with stannous ion solution, chelating the reduced technetium onto a Sephadex column and applying the polypeptides to this column, or by batch labeling techniques, e.g. by incubating pertechnate, a reducing agent such as SnCl 2 , a buffer solution such as a sodium-potassium phthalate-solution, and the molecules.
  • a reducing agent such as SnCl 2
  • a buffer solution such as a sodium-potassium phthalate-solution
  • Polypeptides according to the invention may be derived, for example, with radioactive sodium or potassium iodide and a chemical oxidizing agent, such as sodium hypochlorite, chloramine T or the like, or an enzymatic oxidizing agent, such as lactoperoxidase, glucose oxidase and glucose.
  • a chemical oxidizing agent such as sodium hypochlorite, chloramine T or the like
  • an enzymatic oxidizing agent such as lactoperoxidase, glucose oxidase and glucose.
  • the indirect labeling approach is particularly preferred.
  • Patents relating to chelators and chelator conjugates are known in the art. For instance, U.S. Pat. No. 4,831,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation. U.S. Pat. Nos.
  • Compatible chelators including the specific bifunctional chelator used to facilitate chelation in co-pending application Ser. Nos. 08/475,813, 08/475,815 and 08/478,967, are preferably selected to provide high affinity for trivalent metals, exhibit increased tumor-to-non-tumor ratios and decreased bone uptake as well as greater in vivo retention of radionuclide at target sites, i.e., B-cell lymphoma tumor sites.
  • target sites i.e., B-cell lymphoma tumor sites.
  • other bifunctional chelators that may or may not possess all of these characteristics are known in the art and may also be beneficial in tumor therapy.
  • polypeptides may be conjugated to different radiolabels for diagnostic and therapeutic purposes.
  • “In2B8” conjugate comprises a murine monoclonal antibody, 2B8, specific to human CD20 antigen, that is attached to 111 In via a bifunctional chelator, i.e., MX-DTPA (diethylenetriaminepentaacetic acid), which comprises a 1:1 mixture of 1-isothiocyanatobenzyl-3-methyl-DTPA and 1-methyl-3-isothiocyanatobenzyl-DTPA.
  • MX-DTPA diethylenetriaminepentaacetic acid
  • 111 In is particularly preferred as a diagnostic radionuclide because between about 1 to about 10 mCi can be safely administered without detectable toxicity; and the imaging data is generally predictive of subsequent 90 Y-labeled antibody distribution.
  • Most imaging studies utilize 5 mCi 111 In-labeled antibody, because this dose is both safe and has increased imaging efficiency compared with lower doses, with optimal imaging occurring at three to six days after antibody administration. See, for example, Murray, J. Nuc. Med . 26: 3328 (1985) and Carraguillo et al., J. Nuc. Med . 26: 67 (1985).
  • 131 I is a well known radionuclide used for targeted immunotherapy.
  • the clinical usefulness of 131 I can be limited by several factors including: eight-day physical half-life; dehalogenation of iodinated antibody both in the blood and at tumor sites; and emission characteristics (e.g., large gamma component) which can be suboptimal for localized dose deposition in tumor.
  • emission characteristics e.g., large gamma component
  • 90 Y provides several benefits for utilization in radioimmunotherapeutic applications: the 64 hour half-life of 90 Y is long enough to allow accumulation by tumor and, unlike e.g., 131 I, 90 Y is a pure beta emitter of high energy with no accompanying gamma irradiation in its decay, with a range in tissue of 100 to 1,000 cell diameters. Furthermore, the minimal amount of penetrating radiation allows for outpatient administration of 90 Y-labeled molecules. Additionally, internalization of labeled polypeptides is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells lacking the target antigen.
  • conjugates with biotin are prepared e.g. by reacting the polypeptides with an activated ester of biotin such as the biotin N-hydroxysuccinimide ester.
  • conjugates with a fluorescent marker may be prepared in the presence of a coupling agent, e.g. those listed above, or by reaction with an isothiocyanate, preferably fluorescein-isothiocyanate.
  • Conjugates of the polypeptides of the invention with cytostatic/cytotoxic substances and metal chelates are prepared in an analogous manner.
  • cytotoxic drugs particularly those which are used for cancer therapy.
  • a cytotoxin or cytotoxic agent means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or destroy a cell or malignancy.
  • cytotoxins include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents, prodrugs, immunologically active ligands and biological response modifiers such as cytokines. Any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells is within the scope of the present invention.
  • Exemplary cytotoxins include, in general, cytostatic agents, alkylating agents, antimetabolites, anti-proliferative agents, tubulin binding agents, hormones and hormone antagonists, and the like.
  • Exemplary cytostatics that are compatible with the present invention include alkylating substances, such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, also nitrosourea compounds, such as carmustine, lomustine, or semustine.
  • alkylating substances such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone
  • nitrosourea compounds such as carmustine, lomustine, or semustine.
  • Other preferred classes of cytotoxic agents include, for example, the maytansinoid family of
  • cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins.
  • Particularly useful members of those classes include, for example, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, aminopterin, methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, ftorafur, 6-mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like.
  • cytotoxins that are compatible with the teachings herein include taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Hormones and hormone antagonists such as corticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesterone or medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens, e.g.
  • tamoxifen, androgens e.g. testosterone
  • aromatase inhibitors e.g. aminogluthetimide
  • one skilled in the art may make chemical modifications to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
  • cytotoxins comprise members or derivatives of the enediyne family of anti-tumor antibiotics, including calicheamicin, esperamicins or dynemicins. These toxins are extremely potent and act by cleaving nuclear DNA, leading to cell death. Unlike protein toxins which can be cleaved in vivo to give many inactive but immunogenic polypeptide fragments, toxins such as calicheamicin, esperamicins and other enediynes are small molecules which are essentially non-immunogenic. These non-peptide toxins are chemically-linked to the dimers or tetramers by techniques which have been previously used to label monoclonal antibodies and other molecules. These linking technologies include site-specific linkage via the N-linked sugar residues present only on the Fc portion of the constructs. Such site-directed linking methods have the advantage of reducing the possible effects of linkage on the binding properties of the constructs.
  • compatible cytotoxins may comprise a prodrug.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form.
  • Prodrugs compatible with the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate containing prodrugs, peptide containing prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to the more active cytotoxic free drug.
  • Further examples of cytotoxic drugs that can be derivatized into a prodrug form for use in the present invention comprise those chemotherapeutic agents described above.
  • polypeptides can also be associated with a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme.
  • a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme.
  • cytokines such as lymphokines and interferons.
  • radiosensitizing drugs that may be effectively directed to tumor or immunoreactive cells. Such drugs enhance the sensitivity to ionizing radiation, thereby increasing the efficacy of radiotherapy.
  • An conjugate internalized by the tumor cell would deliver the radiosensitizer nearer the nucleus where radiosensitization would be maximal.
  • the unbound radiosensitizer linked polypeptides of the invention would be cleared quickly from the blood, localizing the remaining radiosensitization agent in the target tumor and providing minimal uptake in normal tissues.
  • adjunct radiotherapy would be administered in one of three ways: 1.) external beam radiation directed specifically to the tumor, 2.) radioactivity directly implanted in the tumor or 3.) systemic radioimmunotherapy with the same targeting molecule.
  • a potentially attractive variation of this approach would be the attachment of a therapeutic radioisotope to the radiosensitized immunoconjugate, thereby providing the convenience of administering to the patient a single drug.
  • a moiety that enhances the stability or efficacy of the polypeptide can be conjugated.
  • PEG can be conjugated to the polypeptides of the invention to increase their half-life in vivo. Leong, S. R., et al. 2001 . Cytokine 16:106; 2002 ; Adv. in Drug Deliv. Rev . 54:531; or Weir et al. 2002. Biochem. Soc. Transactions 30:512.
  • the route of administration of the polypeptide of the invention may be oral, parenteral, by inhalation or topical.
  • parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration.
  • the intravenous, intraarterial, subcutaneous and intramuscular forms of parenteral administration are generally preferred. While all these forms of administration are clearly contemplated as being within the scope of the invention, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip.
  • a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc.
  • a buffer e.g. acetate, phosphate or citrate buffer
  • a surfactant e.g. polysorbate
  • a stabilizer agent e.g. human albumin
  • Preparations for parenteral administration includes sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline.
  • Intravenous vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • sterile injectable solutions can be prepared by incorporating an active compound (e.g., a polypeptide by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • an active compound e.g., a polypeptide by itself or in combination with other active agents
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in co-pending U.S. Ser. No. 09/259,337 and U.S. Ser. No. 09/259,338 each of which is incorporated herein by reference. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to autoimmune or neoplastic disorders.
  • Effective doses of the compositions of the present invention, for the treatment of the above described conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human but non-human mammals including transgenic mammals can also be treated.
  • Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
  • the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg.
  • Doses intermediate in the above ranges are also intended to be within the scope of the invention.
  • Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis.
  • An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
  • Polypeptides of the invention can be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of polypeptide or antigen in the patient. In some methods, dosage is adjusted to achieve a plasma polypeptide concentration of 1-1000 ⁇ g/ml and in some methods 25-300 ⁇ g/ml. Alternatively, binding molecules can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the molecule in the patient. In general, humanized antibodies show the longest half-life, followed by chimeric antibodies and nonhuman antibodies.
  • the binding molecules of the invention can be administered in unconjugated form, In another embodiment, the polypeptides of the invention can be administered multiple times in conjugated form. In still another embodiment, the binding molecules of the invention can be administered in unconjugated form, then in conjugated form, or vise versa.
  • compositions containing the present polypeptides or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance. Such an amount is defined to be a “prophylactic effective dose.”
  • prophylactic effective dose the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose.
  • a relatively low dosage is administered at relatively infrequent intervals over a long period of time.
  • a relatively high dosage e.g., from about 1 to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molecules
  • a relatively high dosage e.g., from about 1 to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molecules
  • the patent can be administered a prophylactic regime.
  • a subject can be treated with a nucleic acid molecule encoding a polypeptide of the invention (e.g., in a vector).
  • Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ⁇ g to 10 mg, or 30-300 ⁇ g DNA per patient.
  • Doses for infectious viral vectors vary from 10-100, or more, virions per dose.
  • Therapeutic agents can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment.
  • the most typical route of administration of an immunogenic agent is subcutaneous although other routes can be equally effective.
  • the next most common route is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles.
  • agents are injected directly into a particular tissue where deposits have accumulated, for example intracranial injection. Intramuscular injection or intravenous infusion are preferred for administration of molecules.
  • particular therapeutic molecules are injected directly into the cranium.
  • molecules are administered as a sustained release composition or device, such as a MedipadTM device.
  • Agents of the invention can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).
  • Effective single treatment dosages (i.e., therapeutically effective amounts) of 90 Y-labeled polypeptides of the invention range from between about 5 and about 75 mCi, more preferably between about 10 and about 40 mCi.
  • Effective single treatment non-marrow ablative dosages of 131 I-labeled antibodies range from between about 5 and about 70 mCi, more preferably between about 5 and about 40 mCi.
  • Effective single treatment ablative dosages (i.e., may require autologous bone marrow transplantation) of 131 I-labeled antibodies range from between about 30 and about 600 mCi, more preferably between about 50 and less than about 500 mCi.
  • an effective single treatment non-marrow ablative dosages of iodine-131 labeled chimeric antibodies range from between about 5 and about 40 mCi, more preferably less than about 30 mCi. Imaging criteria for, e.g., the 111 In label, are typically less than about 5 mCi.
  • radiolabels are known in the art and have been used for similar purposes. Still other radioisotopes are used for imaging.
  • additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to, 123 I, 125 I, 32 P, 57 Co, 64 Cu, 67 Cu, 77 Br, 81 Rb, 81 Kr, 87 Sr, 113 In, 127 Cs, 129 Cs, 132 I, 197 Hg, 203 Pb, 206 Bi, 177 Lu, 186 Re, 212 Pb, 212 Bi, 47 Sc, 105 Rh, 109 Pd, 153 Sm, 188 Re, 199 Au, 225 Ac, 211 At, and 213 Bi.
  • radionuclides are compatible with a selected course of treatment without undue experimentation.
  • additional radionuclides which have already been used in clinical diagnosis include 125 I, 123 I, 99 Tc, 43 K, 52 Fe, 67 Ga, 68 Ga, as well as 111 In.
  • Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Peirersz et al. Immunol. Cell Biol . 65: 111-125 (1987)).
  • These radionuclides include 188 Re and 186 Re as well as 199 Au and 67 Cu to a lesser extent.
  • U.S. Pat. No. 5,460,785 provides additional data regarding such radioisotopes and is incorporated herein by reference.
  • a major advantage of the present invention is the ability to use these polypeptides in myelosuppressed patients, especially those who are undergoing, or have undergone, adjunct therapies such as radiotherapy or chemotherapy. That is, the beneficial delivery profile (i.e. relatively short serum dwell time, high binding affinity and enhanced localization) of the polypeptides makes them particularly useful for treating patients that have reduced red marrow reserves and are sensitive to myelotoxicity. In this regard, the unique delivery profile of the polypeptides make them very effective for the administration of radiolabeled conjugates to myelosuppressed cancer patients.
  • the polypeptides of the invention are useful in a conjugated or unconjugated form in patients that have previously undergone adjunct therapies such as external beam radiation or chemotherapy.
  • the polypeptides (again in a conjugated or unconjugated form) may be used in a combined therapeutic regimen with chemotherapeutic agents.
  • chemotherapeutic agents Those skilled in the art will appreciate that such therapeutic regimens may comprise the sequential, simultaneous, concurrent or coextensive administration of the disclosed molecules and one or more chemotherapeutic agents.
  • Particularly preferred embodiments of this aspect of the invention will comprise the administration of a radiolabeled polypeptide.
  • polypeptides may be administered as described immediately above, it must be emphasized that in other embodiments conjugated and unconjugated polypeptides may be administered to otherwise healthy patients as a first line therapeutic agent. In such embodiments the polypeptides may be administered to patients having normal or average red marrow reserves and/or to patients that have not, and are not, undergoing adjunct therapies such as external beam radiation or chemotherapy.
  • selected embodiments of the invention comprise the administration of polypeptides to myelosuppressed patients or in combination or conjunction with one or more adjunct therapies such as radiotherapy or chemotherapy (i.e. a combined therapeutic regimen).
  • a combined therapeutic regimen means the sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the disclosed molecules.
  • the administration or application of the various components of the combined therapeutic regimen may be timed to enhance the overall effectiveness of the treatment.
  • chemotherapeutic agents could be administered in standard, well known courses of treatment followed within a few weeks by radioimmunoconjugates of the present invention.
  • cytotoxin associated polypeptides could be administered intravenously followed by tumor localized external beam radiation.
  • the polypeptide may be administered concurrently with one or more selected chemotherapeutic agents in a single office visit.
  • a skilled artisan e.g. an experienced oncologist
  • the combination of the polypeptide (with or without cytotoxin) and the chemotherapeutic agent may be administered in any order and within any time frame that provides a therapeutic benefit to the patient. That is, the chemotherapeutic agent and polypeptide may be administered in any order or concurrently.
  • the polypeptides of the present invention will be administered to patients that have previously undergone chemotherapy.
  • the polypeptides and the chemotherapeutic treatment will be administered substantially simultaneously or concurrently.
  • the patient may be given the binding molecule while undergoing a course of chemotherapy.
  • the binding molecule will be administered within 1 year of any chemotherapeutic agent or treatment.
  • the polypeptide will be administered within 10, 8, 6, 4, or 2 months of any chemotherapeutic agent or treatment. In still other preferred embodiments the polypeptide will be administered within 4, 3, 2 or 1 week of any chemotherapeutic agent or treatment. In yet other embodiments the polypeptide will be administered within 5, 4, 3, 2 or 1 days of the selected chemotherapeutic agent or treatment. It will further be appreciated that the two agents or treatments may be administered to the patient within a matter of hours or minutes (i.e. substantially simultaneously).
  • a myelosuppressed patient shall be held to mean any patient exhibiting lowered blood counts.
  • blood count parameters conventionally used as clinical indicators of myelosuppresion and one can easily measure the extent to which myelosuppresion is occurring in a patient.
  • Examples of art accepted myelosuppression measurements are the Absolute Neutrophil Count (ANC) or platelet count.
  • ANC Absolute Neutrophil Count
  • Such myelosuppression or partial myeloablation may be a result of various biochemical disorders or diseases or, more likely, as the result of prior chemotherapy or radiotherapy.
  • patients who have undergone traditional chemotherapy typically exhibit reduced red marrow reserves.
  • cytotoxin i.e. radionuclides
  • conjugated or unconjugated polypeptides of the present invention may be used to effectively treat patients having ANCs lower than about 2000/mm 3 or platelet counts lower than about 150,000/mm 3 . More preferably the polypeptides of the present invention may be used to treat patients having ANCs of less than about 1500/mm 3 , less than about 1000/mm 3 or even more preferably less than about 500/mm 3 . Similarly, the polypeptides of the present invention may be used to treat patients having a platelet count of less than about 75,000/mm 3 , less than about 50,000/mm 3 or even less than about 10,000/mm 3 . In a more general sense, those skilled in the art will easily be able to determine when a patient is myelosuppressed using government implemented guidelines and procedures.
  • the disclosed polypeptides may be used to treat disorders in patients exhibiting myelosuppression regardless of the cause.
  • the polypeptides of the instant invention may be used in conjunction or combination with any chemotherapeutic agent or agents (e.g. to provide a combined therapeutic regimen) that eliminates, reduces, inhibits or controls the growth of neoplastic cells in vivo.
  • any chemotherapeutic agent or agents e.g. to provide a combined therapeutic regimen
  • such agents often result in the reduction of red marrow reserves. This reduction may be offset, in whole or in part, by the diminished myelotoxicity of the compounds of the present invention that advantageously allow for the aggressive treatment of neoplasias in such patients.
  • the radiolabeled immunoconjugates disclosed herein may be effectively used with radiosensitizers that increase the susceptibility of the neoplastic cells to radionuclides.
  • radiosensitizing compounds may be administered after the radiolabeled binding molecule has been largely cleared from the bloodstream but still remains at therapeutically effective levels at the site of the tumor or tumors.
  • exemplary chemotherapeutic agents that are compatible with the instant invention include alkylating agents, vinca alkaloids (e.g., vincristine and vinblastine), procarbazine, methotrexate and prednisone.
  • alkylating agents e.g., vincristine and vinblastine
  • procarbazine methotrexate
  • prednisone methotrexate
  • MOPP mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazine and prednisone
  • ABVD e.g., adriamycin, bleomycin, vinblastine and dacarbazine
  • ChlVPP chlorambucil, vinblastine, procarbazine and prednisone
  • CABS lomustine, doxorubicin, bleomycin and streptozotocin
  • MOPP plus ABVD MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine and prednisone) combinations
  • Additional regimens include use of single alkylating agents such as cyclophosphamide or chlorambucil, or combinations such as CVP (cyclophosphamide, vincristine and prednisone), CHOP (CVP and doxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin and leucovorin), ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide and leucovorin plus standard MOPP), ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin
  • CVP cyclophos
  • CHOP has also been combined with bleomycin, methotrexate, procarbazine, nitrogen mustard, cytosine arabinoside and etoposide.
  • Other compatible chemotherapeutic agents include, but are not limited to, 2-chlorodeoxyadenosine (2-CDA), 2′ -deoxycoformycin and fludarabine.
  • Salvage therapies employ drugs such as cytosine arabinoside, cisplatin, etoposide and ifosfamide given alone or in combination.
  • IMVP-16 ifosfamide, methotrexate and etoposide
  • MIME methyl-gag, ifosfamide, methotrexate and etoposide
  • DHAP dexamethasone, high dose cytarabine and cisplatin
  • ESHAP etoposide, methylpredisolone, HD cytarabine, cisplatin
  • CEPP(B) cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin
  • CAMP lomustine, mitoxantrone, cytarabine and prednisone
  • chemotherapeutic agent used in combination with the polypeptides of the instant invention may vary by subject or may be administered according to what is known in the art. See for example, Bruce A Chabner et al., Antineoplastic Agents , in G OODMAN & G ILMAN'S T HE P HARMACOLOGICAL B ASIS OF T HERAPEUTICS 1233-1287 ((Joel G. Hardman et al., eds., 9 th ed. 1996).
  • polypeptides of the present invention may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders.
  • the disclosed binding molecules will be formulated so as to facilitate administration and promote stability of the active agent.
  • pharmaceutical compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
  • a pharmaceutically effective amount of the polypeptide, immunoreactive fragment or recombinant thereof, conjugated or unconjugated to a therapeutic agent shall be held to mean an amount sufficient to achieve effective binding to an antigen and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell.
  • the polypeptide will be preferably be capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells and provide for an increase in the death of those cells.
  • the pharmaceutical compositions of the present invention may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the polypeptide.
  • the polypeptides of the invention may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect.
  • the binding molecules of the invention can be administered to such human or other animal in a conventional dosage form prepared by combining the binding molecule of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktail comprising one or more species of polypeptides according to the present invention may prove to be particularly effective.
  • polypeptides of the invention can be used for diagnostic or therapeutic purposes.
  • Preferred embodiments of the present invention provide compounds, compositions, kits and methods for the diagnosis and/or treatment of disorders, e.g., neoplastic disorders in a mammalian subject in need of such treatment.
  • the subject is a human.
  • polypeptides of the instant invention will be useful in a number of different applications.
  • the subject binding molecules should be useful for reducing or eliminating cells bearing an epitope recognized by a binding molecule of the invention.
  • the subject binding molecules are effective in reducing the concentration of or eliminating soluble antigen in the circulation
  • tumor size inhibiting tumor growth and/or prolonging the survival time of tumor-bearing animals.
  • this invention also relates to a method of treating tumors in a human or other animal by administering to such human or animal an effective, non-toxic amount of polypeptide.
  • an effective, non-toxic amount of polypeptide One skilled in the art would be able, by routine experimentation, to determine what an effective, non-toxic amount of polypeptide would be for the purpose of treating malignancies.
  • a therapeutically active amount of a polypeptide may vary according to factors such as the disease stage (e.g., stage I versus stage IV), age, sex, medical complications (e.g., immunosuppressed conditions or diseases) and weight of the subject, and the ability of the molecule to elicit a desired response in the subject.
  • the dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram body weight per day and more preferably from about 0.5 to 10, milligrams per kilogram body weight per day.
  • mammal refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • the mammal is human.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease or disorder as well as those in which the disease or disorder is to be prevented. Hence, the mammal may have been diagnosed as having the disease or disorder or may be predisposed or susceptible to the disease.
  • the polypeptides of the present invention may be immunoreactive with one or more tumor antigens or antigens associated with immune disorders.
  • the antigen binding site i.e. the variable region or immunoreactive fragment or recombinant thereof
  • the disclosed polypeptides will bind to selected markers on the offending cells.
  • polypeptides useful in the present invention may be obtained or derived from any antibody (including those previously reported in the literature) that reacts with a molecule or marker associated with the selected condition.
  • the parent or precursor antibody, or fragment thereof, used to generate the disclosed polypeptides may be murine, human, chimeric, humanized, non-human primate or primatized.
  • the polypeptides of the present invention may comprise single chain antibody constructs (such as that disclosed in U.S. Pat. No. 5,892,019 which is incorporated herein by reference) having altered constant domains as described herein. Consequently, any of these types of antibodies modified in accordance with the teachings herein is compatible with the instant invention.
  • tumor associated antigens means any antigen which is generally associated with tumor cells, i.e., occurring at the same or to a greater extent as compared with normal cells. More generally, tumor associated antigens comprise any antigen that provides for the localization of immunoreactive antibodies at a neoplastic cell irrespective of its expression on non-malignant cells. Such antigens may be relatively tumor specific and limited in their expression to the surface of malignant cells. Alternatively, such antigens may be found on both malignant and non-malignant cells.
  • CD20 is a pan B antigen that is found on the surface of both malignant and non-malignant B cells that has proved to be an extremely effective target for immunotherapeutic antibodies for the treatment of non-Hodgkin's lymphoma.
  • pan T cell antigens such as CD2, CD3, CD5, CD6 and CD7 also comprise tumor associated antigens within the meaning of the present invention.
  • tumor associated antigens comprise but not limited to MAGE-1, MAGE-3, MUC-1, HPV 16, HPV E6 & E7, TAG-72, CEA, L6-Antigen, CD19, CD22, CD37, CD52, HLA-DR, EGF Receptor and HER2 Receptor.
  • immunoreative antibodies for each of these antigens have been reported in the literature. Those skilled in the art will appreciate that each of these antibodies may serve as a precursor for polypeptides of the invention in accordance with the present invention.
  • polypeptides of the present invention preferably associate with, and bind to, tumor or immune associated antigens as described above. Accordingly, as will be discussed in some detail below the polypeptides of the present invention may be derived, generated or fabricated from any one of a number of antibodies that react with tumor associated antigens. In preferred embodiments the polypeptides are modified or domain deleted antibodies that are derived using common genetic engineering techniques whereby at least a portion of one or more constant region domains are deleted or altered so as to provide the desired biochemical characteristics such as reduced serum half-life.
  • polypeptides of the invention may be expressed and produced on a clinical or commercial scale using well-established protocols.
  • Previously reported antibodies that react with tumor associated antigens may be altered as described herein to provide the polypeptides of the present invention.
  • Exemplary antibodies that may be used to provide antigen binding regions for, generate or derive the disclosed polypeptides include, but are not limited to 2B8 and C2B8 (Zevalin® and Rituxan®, IDEC Pharmaceuticals Corp., San Diego), Lym 1 and Lym 2 (Techniclone), LL2 (Immunomedics Corp., New Jersey), HER2 (Herceptin®, Genentech Inc., South San Francisco), B1 (Bexxar®, Coulter Pharm., San Francisco), Campath® (Millennium Pharmaceuticals, Cambridge) MB1, BH3, B4, B72.3 (Cytogen Corp.), CC49 (National Cancer Institute) and 5E10 (University of Iowa).
  • the polypeptides of the present invention will bind to the same tumor associated antigens as the antibodies enumerated immediately above.
  • the polypeptides will be derived from or bind the same antigens as 2B8, C2B8, CC49 and C5E10 and, even more preferably, will lack all or part of a CH2 domain.
  • a binding molecule of the invention binds to the CD23 (U.S. Pat. No. 6,011,138). In a preferred embodiment, a binding molecule of the invention binds to the same epitope as the 5E8 antibody. In another embodiment, a binding molecule of the invention comprises at least one CDR from an anti-CD23 antibody, e.g., the 5E8 antibody.
  • a binding molecule of the invention binds to the CRIPTO-I antigen (WO02/088170A2 or WO03/083041A2). In a preferred embodiment, a binding molecule of the invention binds to the same epitope as the B3F6 antibody. In another embodiment, a binding molecule of the invention comprises at least one CDR from an anti-CRIPTO-I antibody, e.g., the B3F6 antibody.
  • the polypeptide will bind to the same tumor associated antigen as Rituxan®.
  • Rituxan® also known as, rituximab, IDEC-C2B8 and C2B8
  • Y2B8 90Y labeled 2B8; Zevalin®; ibritumomab tiuxetan
  • C2B8 the murine parent of C2B8.
  • Rituxan® is a chimeric, anti-CD20 monoclonal antibody which is growth inhibitory and reportedly sensitizes certain lymphoma cell lines for apoptosis by chemotherapeutic agents in vitro.
  • the antibody efficiently binds human complement, has strong FcR binding, and can effectively kill human lymphocytes in vitro via both complement dependent (CDC) and antibody-dependent (ADCC) mechanisms (Romb al., Blood 83: 435-445 (1994)).
  • CDC complement dependent
  • ADCC antibody-dependent
  • Those skilled in the art will appreciate that dimeric variants (homodimers or heterodimers) of C2B8 or 2B8, modified according to the instant disclosure, may be used in conjugated or unconjugated forms to effectively treat patients presenting with CD20+ malignancies. More generally, it must be reiterated that the polypeptides disclosed herein may be used in either a “naked” or unconjugated state or conjugated to a cytotoxic agent to effectively treat any one of
  • the polypeptide of the invention will be derived from, or bind to, the same tumor associated antigen as CC49.
  • CC49 binds human tumor associated antigen TAG-72 which is associated with the surface of certain tumor cells of human origin, specifically the LS174T tumor cell line.
  • LS174T [American Type Culture Collection (herein ATCC) No. CL 188] is a variant of the LS180 (ATCC No. CL 187) colon adenocarcinoma line.
  • B72.3 is a murine IgG1 produced by hybridoma B72.3 (ATCC No. HB-8108).
  • B72.3 is a first generation monoclonal antibody developed using a human breast carcinoma extract as the immunogen (see Colcher et al., Proc. Natl. Acad. Sci. (USA), 78:3199-3203 (1981); and U.S. Pat. Nos. 4,522,918 and 4,612,282 each of which is incorporated herein by reference).
  • Other monoclonal antibodies directed against TAG-72 are designated “CC” (for colon cancer).
  • CC monoclonal antibodies are a family of second generation murine monoclonal antibodies that were prepared using TAG-72 purified with B72.3. Because of their relatively good binding affinities to TAG-72, the following CC antibodies have been deposited at the ATCC, with restricted access having been requested: CC49 (ATCC No. HB 9459); CC 83 (ATCC No. HB 9453); CC46 (ATCC No. HB 9458); CC92 (ATTCC No. HB 9454); CC30 (ATCC No. HB 9457); CC11 (ATCC No.
  • Still other preferred embodiments of the present invention comprise modified antibodies that are derived from or bind to the same tumor associated antigen as C5E10.
  • C5E10 is an antibody that recognizes a glycoprotein determinant of approximately 115 kDa that appears to be specific to prostate tumor cell lines (e.g. DU145, PC3, or ND1).
  • polypeptides e.g. CH2 domain-deleted antibodies
  • C5E10 antibodies could be produced and used in a conjugated or unconjugated form for the treatment of neoplastic disorders.
  • the modified antibody will be derived or comprise all or part of the antigen binding region of the C5E10 antibody as secreted from the hybridoma cell line having ATCC accession No. PTA-865.
  • the resulting modified antibody could then be conjugated to a radionuclide as described below and administered to a patient suffering from prostate cancer in accordance with the methods herein.
  • the disclosed invention may be used to prophylactically or therapeutically treat any neoplasm comprising an antigenic marker that allows for the targeting of the cancerous cells by the binding molecule.
  • exemplary cancers that may be treated include, but are not limited to, prostate, gastric carcinomas, such as colon, skin, breast, ovarian, lung and pancreatic.
  • the binding molecules of the instant invention may be used to treat Kaposi's sarcoma, CNS neoplasias (capillary hemangioblastomas, meningiomas and cerebral metastases), melanoma, gastrointestinal and renal sarcomas, rhabdomyosarcoma, glioblastoma (preferably glioblastoma multiforme), leiomyosarcoma, retinoblastoma, papillary cystadenocarcinoma of the ovary, Wilm's tumor or small cell lung carcinoma.
  • Kaposi's sarcoma CNS neoplasias (capillary hemangioblastomas, meningiomas and cerebral metastases), melanoma, gastrointestinal and renal sarcomas, rhabdomyosarcoma, glioblastoma (preferably glioblastoma multiforme), leiomyosarcoma, retino
  • hematologic malignancies that are amenable to treatment with the disclosed invention include Hodgkins and non-Hodgkins lymphoma as well as leukemias, including ALL-L3 (Burkitt's type leukemia), chronic lymphocytic leukemia (CLL) and monocytic cell leukemias.
  • B-cell lymphomas including low grade/follicular non-Hodgkin's lymphoma (NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL and Waldenstrom's Macroglobulinemia.
  • NHL low grade/follicular non-Hodgkin's lymphoma
  • FCC cell lymphoma
  • MCL mantle cell lymphoma
  • DLCL diffuse large cell lymphoma
  • SL small lymphocytic
  • lymphomas will often have different names due to changing systems of classification, and that patients having lymphomas classified under different names may also benefit from the combined therapeutic regimens of the present invention.
  • the disclosed invention may advantageously be used to treat additional malignancies bearing compatible tumor associated antigens.
  • the polypeptides of the instant invention are particularly effective in the treatment of autoimmune disorders or abnormal immune responses.
  • the polypeptide of the present invention may be used to control, suppress, modulate or eliminate unwanted immune responses to both external and autoantigens.
  • the antigen is an autoantigen.
  • the antigen is an allergen.
  • the antigen is an alloantigen or xenoantigen.
  • Use of the disclosed polypeptides to reduce an immune response to alloantigens and xenoantigens is of particular use in transplantation, for example to inhibit rejection by a transplant recipient of a donor graft, e.g. a tissue or organ graft or bone marrow transplant. Additionally, suppression or elimination of donor T cells within a bone marrow graft is useful for inhibiting graft versus host disease.
  • the polypeptides of the present invention may be used to treat immune disorders that include, but are not limited to, allergic bronchopulmonary aspergillosis; Allergic rhinitis Autoimmune hemolytic anemia; Acanthosis nigricans; Allergic contact dermatitis; Addison's disease; Atopic dermatitis; Alopecia areata; Alopecia universalis; Amyloidosis; Anaphylactoid purpura; Anaphylactoid reaction; Aplastic anemia; Angioedema, hereditary; Angioedema, idiopathic; Ankylosing spondylitis; Arteritis, cranial; Arteritis, giant cell; Arteritis, Takayasu's; Arteritis, temporal; Asthma; Ataxia-telangiectasia; Autoimmune oophoritis; Autoimmune orchitis; Autoimmune polyendocrine failure; Behcet's disease; Berg
  • the binding molecules of the invention can be used for pretargeting applications.
  • the same advantages will be apparent in pretargeting applications for chemotherapeutic drug delivery.
  • a tumor is pretargeted with a binding construct that has affinity for the tumor-associated antigen on the one hand and for, e.g., a radiolabeled hapten on the other.
  • the radiolabeled hapten is administered later, preferably after the binding molecule has cleared (see, e.g., Boerman et al. 2003. J. Nuclear Med. 44:400).
  • an antibody which is non-toxic, but has been derivitized to react with a drug or prodrug that is toxic only when bound by the binding molecule.
  • the binding molecules of the invention are well suited to use in pretargeting applications.
  • a clearing agent could be eliminated from the pretargeting methodology by using the instant binding molecules.
  • Solutions of antibody molecules comprise two different isoforms.
  • One form, Form A comprises heavy chain molecules that are linked via at least one disulfide linkage.
  • the other form, Form B comprises heavy chain molecules that are not linked via at least one disulfide linkage.
  • Form B does not appear or appears at a very low frequency in with intact gamma 1 MAbs, such as Rituxan®.
  • domain deleted (dd) constructs having a similar hinge, the frequency of Form B is much higher.
  • Hinge domains can be subdivided into three distinct regions: upper, middle, and lower hinge regions (Roux et al. J. Immunol. 1998 161:4083). Polypeptide sequences encompassing these regions for IgG1 and IgG3 hinges are shown in Table 1. The IgG3 hinge middle region contains, in addition to the two conserved cysteine residues, a 15 amino acid motif that repeats three times. Amino acid sequences from these regions were used to design synthetic IgG1/IgG3 connecting peptides.
  • IgG1 upper hinge residues corresponding to positions 226 through 238, an IgG1 middle hinge corresponding to positions 239 through 241, and a single IgG3 middle hinge repeat motif corresponding to positions 241 EE through 242 combined with either an added proline at position 243 or an added proline, alanine, proline at positions 243, 244, and 245, respectively (Kabat numbering system), followed by a flexible Gly/Ser spacer (Table 2).
  • novel connecting peptides were designed consisting of a serine amino acid residue substituted for the cysteine at positions 239 or 242 combined with either an added proline at position 243 or an added proline, alanine, proline at positions 243, 244, and 245, respectively (Kabat numbering system).
  • Pro243Ala244Pro245 and Pro 243 connecting peptides were also made.
  • the amino acid sequence of the parent CH2 domain deleted humanized CC49 connecting peptide beginning at the first residue of the IgG1 hinge (position 226, Kabat numbering system) to the last residue of the hinge/GlySer connecting peptide is shown in Table 2.
  • Nucleic acid sequences encoding the hinge region connecting peptides shown in Table 2 were introduced into CH2 domain deleted huCC49 gene sequences using the Splicing by Overlap Extension (SOE) method (Horton, R. M. 1993 Methods in Molecular Biology, Vol 15: PCR Protocols: Current Methods and applications. Ed. B. A. White). Correct modifications to the hinge region were confirmed by DNA sequence analysis. Plasmid DNA was used to transform CHO DG44 cells for stable production of antibody protein.
  • SOE Overlap Extension
  • CH2 domain deleted huCC49 antibodies containing the eight designed synthetic connecting peptides indicated in Table 2 were constructed and antibody produced in CHO DG44 cells.
  • Supernatants were collected from isolated cell lines and concentration of antibody in the culture supernatants determined by immunoassay.
  • Supernatants containing antibody ranging from 0 to 30 ng of total antibody protein from each cell line was analyzed by non-reducing SDS-PAGE electrophoresis followed by Western Blot with anti-human kappa HRP conjugated antibody to detect CH2 domain deleted huCC49 Form A and Form B isoforms. Under these conditions, Form A migrates as a single 120 kDa homodimer and Form B as a 60 kDa doublet.
  • a connecting peptide of the invention comprises a cysteine at at least one of position 239 or 242.
  • connecting peptides which increase the proportion of Form A produced will lead to a beneficial improvement in process, yield and/or stability.
  • These synthetic hinge region connecting peptides are useful for favoring synthesis of CH2 domain deleted antibody Form A isoform for any antibody isotype, e.g., IgG1, IgG2, IgG3, or IgG4, based on the extremely high degree of homology among the CH3 domains for all four human isotypes.
  • IgG1 CH3 domain is 98.13% homologous to IgG2 CH3, 97.20% homologous to IgG3 CH3, and 96.26% homologous to IgG4 CH3.
  • a Toso Biosep Phenyl 5PW-HR column was pre-equilibrated with 20 mM Phosphate pH 7.2; 1 M Ammonium Sulfate.
  • the Protein G eluate was titrated to 1 M Ammonium Sulfate using a 3.5 M Ammonium Sulfate pH 7.2 stock and loaded at a concentration of 2 mg/ml of gel bed.
  • Bound protein was washed with a 20 mM Phosphate pH 4 or 7.2 Ammonium Sulfate to adjust the conductivity to 116.4 mS/cm.
  • the material eluted from this condition has an apparent molecular weight about 120 kD (Form A) on a non-reducing SDS-PAGE.
  • the remaining bound antibody was further eluted with a linear gradient of reducing Ammonium Sulfate content in the Phosphate buffer. This method separates Forms A and B in two separate peaks. The latter eluted antibody apparently lacks the disulfide linkage between the heavy chains and its molecular weight is about 60 kDa (Form B).
  • Both of the above purified materials can be recaptured by bringing the ammonium sulfate concentration to 1M and reloading it onto the cleaned Phenyl 5PW-HR column.
  • Bound protein is eluted with 20 mM Phosphate pH 7.2 and dialyzed into 1 ⁇ PBS.
  • Forms A and B The stability of Forms A and B was also compared.
  • Purified ddCC49 molecules were concentrated to about 5 mg/ml by Amicon concentrator fitted with YM30 membrane (Millipore). The concentrated materials were equally divided into four portions for each isoforms and each fraction was put into 10K dialysis cassette (Pierce, cat #66410) for 16 h dialysis in the following buffers: 1) 10 mM Sodium Phosphate, pH3; 2) 10 mM Sodium acetate, pH 5; 3) 10 mM Sodium Phosphate, pH 7; and 4) 10 mM Sodium Borate, pH 9. After dialysis, the protein concentration of each solution was adjusted to 3 mg/ml.
  • Non-reducing SDS-PAGE was performed on for samples stored at 2-8° C., 20-25° C. and 38-42° C. for various time points. Both A and B form are relatively stable at pH 5 when stored at 2-8° C.. However, when formulated at pH 7 and 9, both A and B forms showed degradation as indicated by increasing in number of bands that were smaller than the original major bands (120 kDa for form A and 60 kDa for form B). It was noticed that, particularly for pH 7 and 9 samples stored at low and intermediate temperatures, the intensity and number of bands that were less than 55 kDa were higher in B-isoform than A. This indicated that under these conditions the A-isoform is more stable than B-isoform.
  • Table 4A through Table 4C list the turbidity data for ddCC49 stored at three different temperatures.
  • the turbidity measures both the soluble and non-soluble aggregates and it is based on the amount of light scattered by these particles. When present, aggregates will scatter light and result in an increase in A 320 .
  • Table 4A-C the turbidity of ddCC49 molecules stored at 2-8° C. increases as pH increased for both A and B isoforms, with the former being less turbid than the latter. This trend held true for samples stored for less than a month at higher temperatures (20-25° C. and 38-40° C.). As storage time reached 3 months, the turbidity increased significantly for samples at high pH and temperature, and the difference between A and B forms diminished.
  • Time A-isoform B-isoform Mixture (month) pH 3 5 7 9 3 5 7 9 3 5 7 9 0 0.030 0.038 0.044 0.056 0.034 0.042 0.046 0.066 0.036 0.042 0.051 0.061 1 ⁇ 2 0.029 0.029 0.046 0.045 0.030 0.038 0.048 0.058 0.034 0.033 0.043 0.055 1 0.033 0.039 0.035 0.055 0.033 0.035 0.044 0.059 0.032 0.040 0.039 0.066 2 0.042 0.022 0.042 0.044 0.039 0.037 0.055 0.067 0.042 0.024 0.040 0.058 3 0.035 0.047 0.051 0.050 0.038 0.041 0.066 0.081 0.027 0.048 0.051 0.065
  • Size exclusion chromatography is a powerful method for revealing the percent of intact molecules and the degraded products (both fragments and soluble aggregates) and is highly reproducible.
  • Table 4A-C the percent of intact monomer of A-isoform, B-isoform and the mixture stored at different temperatures are listed. For samples stored at 2-8° C., it is clear that Form A has a higher percentage of monomer as compared to Form B, and the mixture of Form A and Form B was somewhere in between. At this storage temperature, both forms were relatively stable at pH 3, 5 and 7 (with pH 5 being the most stable condition) for about three months. However, at pH 9 there was a significant decrease in percentage of monomer for Form B but only a slight decrease for Form A.
  • Time A-isoform B-isoform Mixture (month) pH 3 5 7 9 3 5 7 9 3 5 7 9 0 98.81 99.13 98.16 97.93 97.02 97.70 96.88 93.51 97.83 98.27 97.44 95.81 1 ⁇ 2 98.98 99.16 98.25 98.00 97.15 97.87 96.96 91.95 98.15 98.49 97.68 95.59 1 98.80 99.20 97.99 97.11 97.02 97.81 96.62 88.99 98.04 98.45 97.41 94.45 2 98.74 99.01 98.00 95.67 97.15 97.69 95.50 84.84 98.06 98.34 96.81 92.17 3 98.28 98.89 97.88 95.31 96.69 98.14 95.37 85.98 97.61 98.15
  • IDEC-159 (ddCC49) is a CH2 domain deleted monoclonal antibody directed against TAG-72 antigen, which is expressed on the surface of tumors. IDEC-159 contains two isoforms of the antibody, called Form A and Form B. The current cell culture process for IDEC-159 produces an approximate 50:50 ratio of Form A to Form B.
  • the Form A isoform is an antibody with a deleted CH2 region in the Fc portion of the heavy chain. In addition to having a deleted CH2 region, Form B also lacks the disulfide bond linkage across the Fc region and is only held together by hydrophobic interactions and salt bridges.
  • the third and final chromatography step in the IDEC-159 purification process was developed to separate the two isoforms of IDEC-159.
  • the separation is achieved by hydrophobic interaction chromatography (HIC), using a Phenyl TSKgel 5PW-HR adsorbent. Since form B is more hydrophobic than form A, it adsorbs irreversibly to the stationary phase using approximately 0.73 M Ammonium Sulfate/20 mM Sodium Phosphate, pH 4.0-pH 7.0 as the mobile phase. Form A binds to a lesser extent to the stationary phase under these conditions and is therefore eluted isocratically, i.e. it leaves the column with the flowthrough fraction. Subsequent to the isocratic elution of Form A, omitting Ammonium sulfate from the mobile phase desorbs Form B.
  • the following method was used to separate the two isoforms of IDEC-159:
  • FIGS. 13 A and B.
  • the separation of the two forms at a preparative scale (5 L column volume, total IDEC-159 load approximately 20 g) is shown in FIGS. 13 (A and B).
  • the first two peaks comprise the isocratic elution of Form A, the second peak shows the eluted Form B, while the third peak contains impurities, which are removed from the stationary phase during cleaning.
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody design was based on appending a huCC49 single chain Fv (scFv) to the carboxyl terminus of the HuCC49 CH2 domain-deleted antibody CH3 domain.
  • a schematic diagram of HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody is shown in FIG. 4 .
  • An equivalent reference to this design is C-scFv Tetravalent CH2 Domain Deleted antibody.
  • 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.
  • a CC49 scFv immunoglobulin-like antibody is described in U.S. Pat. No. 5,892,019.
  • the huCC49 scFv is comprised of a VL and a VH region sequence tethered by a short synthetic linker (VL ⁇ (Gly 4 Ser) 3 linker ⁇ VH orientation) and was synthesized by PCR amplification.
  • the 5′ VL PCR primer included a BamHI restriction endonuclease site followed by sequence encoding a (Gly 4 Ser) 2 linker peptide.
  • the 3′ VL PCR primer included sequence partially encoding a (Gly 4 Ser) 3 linker peptide used to connect the two VL and VH regions.
  • the 5′ VH PCR primer also included sequence partially encoding a (Gly 4 Ser) 3 linker peptide used to connect the VL and VH regions.
  • the 3′ VH PCR primer included a stop codon followed by a BamHI site. The two V regions were amplified with the two sets of PCR primers from plasmid DNA substrate containing huCC49 CH2 domain-deleted antibody and assembly of the scFv was accomplished in a second PCR reaction through the common overlapping sequences encoding the (Gly 4 Ser) 3 linker.
  • the huCC49 scFv gene fragment was gel isolated, digested with BamHI restriction endonuclease and cloned into a single BamHI site previously introduced into the huCC49 CH2 domain-deleted antibody Polycistronic expression vector. (See US 2003 0157641 A1.). Briefly, the vector was modified by removing an existing stop codon at the 3′ end of the gene coding for the CH3 domain and replacing with nucleotides coding for the amino acid sequence Ser-Gly-Gly-Gly immediately followed by a BamHI restriction endonuclease site (coding for Gly-Ser).
  • the BamHI digested huCC49 scFv fragment was cloned into the BamHI site of the vector resulting in a fusion product of the huCC49 scFv to the carboxyl terminus of the huCC49 CH2 domain-deleted antibody CH3 domain through a 16 amino acid Ser(Gly 4 Ser) 3 linker. Correct sequences were confirmed by DNA sequence analysis. Plasmid DNA was used to transform CHO DG44 cells for stable production of antibody protein. The engineered antibody was designated huCC49 domain-deleted sc(Fv)2 tetravalent antibody.
  • FIG. 8A shows the DNA sequence of heavy chain huCC49 domain-deleted sc(Fv)2 tetravalent antibody.
  • FIG. 8C shows the DNA sequence of light chain huCC49 domain-deleted sc(Fv)2 tetravalent antibody.
  • FIG. 9A shows the amino acid sequence of heavy chain huCC49 domain-deleted sc(Fv)2 tetravalent antibody.
  • FIG. 9C shows the amino sequence of light chain huCC49 domain-deleted sc(Fv)2 tetravalent antibody.
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody Form A was examined by size exclusion chromatography and found to elute primarily as a single peak (96%) indicating that there was no significant aggregation or decomposition of antibody product ( FIG. 20 ).
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody Form A was tested in a competitive binding assay for it's ability to bind to bovine submaxillary mucine, a source of the TAG-72 antigen, by time-resolved flourometric immunoassay using a Delphia fluorimeter (Wallac Inc., Gaithersburg, Md.).
  • Competitive binding curves are shown in FIG. 21 .
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody Form A and control parent CH2 domain-deleted huCC49 (referred to as HuCC49 or IDEC 159) antibodies were evaluated.
  • Relative binding activity for the tetravalent antibody was found to be 5-fold more avid than the control parent CC49 antibody (GraphPad Prism 4.0 for Windows, GraphPad, Software, San Diego Calif. USA. www.graphpad.com ), consistent with an expected increase in the number of antigen binding sites.
  • Biodistribution of 90 Y-radiolabeled huCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody was compared to results previously generated with control parent huCC49 antibody in athymic nude mice bearing LS-174T human tumor xenografts.
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody was conjugated with the chelator Chx-DTPA and binding activity assessed.
  • Conjugated huCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody showed a 5-6 fold higher avidity than the conjugated control parent huCC49 demonstrating that the tetravalent antibody could be derivatized without losing significant binding activity.
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody was labeled with 90 Y and a single dose of radiolabeled antibody was administered by i.v. tail vein injection to mice presenting pre-established tumors of approximately 250 mm 3 . Samples were harvested and processed for beta counting.
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent (C-scFv tetravalent CH2 domain deleted) antibody showed lower accumulation in the kidney than control huCC49 antibody, likely due to the increased molecular mass of the antibody. Tumor accumulation of huCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody was similar to that achieved with the control parent huCC49 (Table 6). However, as mentioned, these two biodistribution studies were not conducted simultaneously and are subject to experimental variation.
  • FIG. 35 A figure showing tumor retention for control parent huCC49 and huCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody as measured by % ID/gm is shown in FIG. 35 .
  • FIG. 35 also shows the same tumor retention data normalized to peak accumulation of radioactivity.
  • AUC analysis of the two normalized retention curves in FIG. 35 was calculated using GraphPad Prism 4.0 for Windows, GraphPad, Software, San Diego Calif. USA. www.graphpad.com .
  • Control parent huCC49 had a Total Peak Area of 2037 units and huCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody had a Total Peak Area of 2562 units representing a 25.8% increase in area for the tetravalent protein. Representation of the data in this format suggests that there is improved retention of tetravalent antibody in the tumor compartment compared to the parent divalent molecule.
  • the tetrameric HuCC49 construct affords certain advantages for pretargeting, e.g., radioimmunotherapeutic (RIT) pretargeting.
  • This advantage is defined by three observations derived from Table 6.
  • First, the tetrameric HuCC49 CH2 domain-deleted construct has a blood clearance rate comparable to that of the current CH2 domain-deleted HuCC49 construct.
  • This fast blood clearance may obviate the need for a “clearing agent” to expedite removal of the antibody from the blood prior to administration of the radiolabeled ligand that localizes to the tumor-bound antibody. Elimination of the clearing agent significantly reduces the complexity of this modality of treatment in the clinical setting.
  • Second, tumor retention for the tetrameric construct is comparable to the current dimeric construct.
  • a huCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody similar to the design shown in the bottom half of FIG. 4 but containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] synthetic connecting peptide indicated in Table 2 was constructed. Briefly, gene sequences coding for partial G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide was synthesized by PCR amplification using a 5′ connecting peptide PCR primer encoding a SalI restriction endonuclease site and a 3′ connecting peptide PCR primer encoding an XhoI site.
  • Plasmid DNA containing the gene sequence coding for the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide was used as substrate.
  • the PCR product encodes all of G1/G3/Pro243Ala244Pro245 and part of [Gly/Ser] connecting peptide.
  • the G1/G3/Pro243Ala244Pro245 hinge fragment was gel isolated, digested with SalI and XhoI restriction endonucleases and cloned into the SalI and XhoI vector sites reconstituting the full length G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide. Correct sequences were confirmed by DNA sequence analysis.
  • FIG. 8B shows the DNA sequence of heavy chain huCC49 domain-deleted sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 8C shows the DNA sequence of light chain huCC49 domain-deleted sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 8C shows the DNA sequence of light chain huCC49 domain-deleted sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 9B shows the amino acid sequence of heavy chain huCC49 domain-deleted sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 9C shows the amino sequence of light chain huCC49 domain-deleted sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • a cell line containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) connecting peptide introduced into the huCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody sequence was used for antibody production.
  • Antibody was produced and purified using methods described in Example 4 above.
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) connecting peptide was purified using only the Protein G column, eluted essentially as a single peak at 96% purity without further HIC purification.
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent G1/G3/Pro243Ala244Pro245 antibody was examined by size exclusion chromatography and found to essentially elute as a single peak containing 84% monomer. The fraction containing the residual aggregates was removed by preparative size exclusion chromatography with the resulting preparation containing essentially 95% monomer ( FIG.
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent G1I/G3/Pro243Ala244Pro245 antibody was tested in a competitive binding assay for it's ability to bind to bovine submaxillary mucine, a source of the TAG-72 antigen, by time-resolved flourometric immunoassay using a Delphia fluorimeter (Wallac Inc., Gaithersburg, Md.).
  • Competitive binding curves are shown in FIG. 25 .
  • HuCC49 CH2 domain-deleted sc(Fv)2 tetravalent antibody Form A and control parent CH2 domain-deleted huCC49 (referred to as HuCC49 or IDEC 159) antibodies were evaluated.
  • Relative binding activity for the tetravalent antibody was found to be 8-fold more avid than the control parent HuCC49 antibody (GraphPad Prism 4.0 for Windows, GraphPad, Software, San Diego Calif. USA. www.graphpad.com ), consistent with an expected increase in the number of antigen binding sites.
  • huCC49 minibody and a huCC49 2 sc(Fv)2 tetravalent antibody (N-scFv tetravalent minibody) as shown in FIG. 2 were both constructed.
  • huCC49 2 sc(Fv)2 tetravalent antibody (N-scFv tetravalent minibody) design was based on inserting a huCC49 single chain Fv (scFv) between the carboxyl end of the first scFv domain and the amino-end of the hinge connecting peptide in the huCC49 minibody.
  • the preparation of single polypeptide chain binding molecules of the Fv region, single-chain Fv molecules, is described in U.S. Pat. No.
  • a CC49 scFv immunoglobulin-like antibody is described in U.S. Pat. No. 5,892,019.
  • the preparation of a minibody construct is described in U.S. Pat. No. 5,837,821.
  • the huCC49 scFv is comprised of a VL and a VH region sequence tethered by a short synthetic linker (VL ⁇ (Gly 4 Ser) 3 linker ⁇ VH orientation) and was synthesized by PCR amplification and used to construct a huCC49 minibody.
  • the minibody vector was modified by adding a modified (Gly 4 Ser) 5 linker to the carboxyl end of the amino-terminal scFv domain of the huCC49 minibody by PCR amplification, replacing nucleotides coding for the last two amino acids residues of the linker with an Nhe I restriction endonuclease site.
  • a second huCC49 scFv was amplified using PCR from plasmid DNA substrate containing huCC49 scFv gene with the 5′ VL PCR primer encoding an Nhe I restriction endonuclease site and the 3′ VH PCR primer encoding a Sal I site.
  • the huCC49 scFv fragment was gel isolated, digested with Nhe I and Sal I restriction endonucleases and cloned into Nhe I and Sal I sites between the first scFv and the hinge connecting peptide. This results in a fusion product consisting of a leader peptide to two serial huCC49 scFvs to a hinge connecting peptide to a CH3 domain.
  • FIG. 10A shows the DNA sequence of huCC49 tetravalent (N-scFv tetravalent) minibody gene.
  • FIG. 11A shows the amino acid sequence of huCC49 tetravalent (N-scFv tetravalent) minibody.
  • huCC49 minibody Form A is expected to migrate as a single ⁇ 82 kDa homodimer and half molecules or Form B as a ⁇ 41 kDa doublet.
  • HuCC49 2 sc(Fv)2 tetravalent minibody Form A is expected to migrate as a single ⁇ 138 kDa homodimer and Form B as a ⁇ 69 kDa doublet in approximately a 50:50 ratio of A to B isoform.
  • huCC49 CH2 domain deleted antibody expected to migrate as a single ⁇ 120 kDa homodimer and Form B as a ⁇ 60 kDa doublet in approximately a 50:50 ratio of A to B isoform
  • FIG. 26 representative isolated minibody and 2 sc(Fv)2 tetravalent minibody clones were found to secrete both A and B isoforms consistent with the expected molecular masses under both non-reducing and reducing SDS-PAGE conditions.
  • a huCC49 2 sc(Fv)2 tetravalent antibody (N-scFv tetravalent minibody) similar to the design shown in the bottom of FIG. 2 but containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide indicated in Table 2 was constructed. Briefly, gene sequences coding for partial G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide was synthesized by PCR amplification using a 5′ connecting peptide PCR primer encoding a Sal I restriction endonuclease site and a 3′ connecting peptide PCR primer encoding an Xho I site.
  • Plasmid DNA containing the gene sequence coding for the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide was used as substrate.
  • the PCR product encodes all of G1/G3/Pro243Ala244Pro245 and part of [Gly/Ser] connecting peptide.
  • the G1/G3/Pro243Ala244Pro245 hinge fragment was gel isolated, digested with Sal I and Xho I restriction endonucleases and cloned into the Sal I and Xho I vector sites reconstituting the full length G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide. Correct sequences were confirmed by DNA sequence analysis.
  • FIG. 10B shows the DNA sequence of huCC49 2 sc(Fv)2 tetravalent antibody (N-scFv tetravalent minibody) gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 11B shows the amino acid sequence of huCC49 2 sc(Fv)2 tetravalent antibody (N-scFv tetravalent minibody) gene containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • Supernatants were collected from isolated cell lines and concentration of antibody in the culture supernatants determined by immunoassay. Supernatants were analyzed by non-reducing SDS-PAGE electrophoresis followed by Western Blot with anti-IgG-HRP conjugated antibody to anti-IgG-HRP conjugated antibody to detect huCC49 2 sc(Fv)2 tetravalent minibody (N-scFv tetravalent minibody) Form A and Form B isoforms.
  • huCC49 2 sc(Fv)2 tetravalent minibody Form A is expected to migrate as a single ⁇ 138 kDa homodimer and Form B as a ⁇ 69 kDa doublet in approximately a 50:50 ratio of A to B isoform.
  • a representative huCC49 2 sc(Fv)2 tetravalent minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide was found to produce essentially all Form A tetravalent minibody isoform.
  • a cell line containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) connecting peptide introduced into the huCC49 2 sc(Fv)2 tetravalent minibody sequence was used for antibody production. Because huCC49 2 sc(Fv)2 tetravalent minibody lacks a CH1 domain, protein cannot be purified using Protein G chromatography. Antibody was subsequently purified using a combination of anion exchange, hydrophobic interaction, and size exclusion chromatography methods.
  • HuCC49 2 sc(Fv)2 tetravalent minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) connecting peptide was purified essentially as a single peak at 97.6% purity. Reduced and non-reduced purified protein samples were analyzed by SDS-PAGE electrophoresis. Under non-reducing conditions, Form A is expected to migrate as a single ⁇ 138 kDa homodimer and Form B as a ⁇ 69 kDa doublet. The connecting peptide shown in SEQ ID NO: 9 was found to substantially increase the proportion of Form A produced. Exemplary results are shown in FIG. 28 .
  • HuCC49 2 sc(Fv)2 tetravalent G1/G3/Pro243Ala244Pro245 minibody, huCC49 minibody, huCC49 CH2 domain-deleted sc(Fv)2 tetravalent G1/G3/Pro243Ala244Pro245 antibody, and control parent CH2 domain-deleted huCC49 (referred to as HuCC49 or IDEC 159) antibodies were evaluated. Relative binding activities for both tetravalent antibodies were found to be more avid than the control parent huCC49 antibody or minibody (GraphPad Prism 4.0 for Windows, GraphPad, Software, San Diego Calif. USA. www.graphpad.com ), consistent with an expected increase in the number of antigen binding sites.
  • PRIMATIZED® p5E8G1 is a chimeric macaque/human (PRIMATIZED®) monoclonal antibody containing macaque heavy and light variable regions fused to human gamma 1 and kappa constant regions, respectively.
  • PRIMATIZED® p5E8G1 binds to human CD23, the low affinity receptor for IgE (Fc ⁇ RII) (Mavromatis and Cheson. 2003. J. Clin. Oncol. 21:1874; US Patent Application 20030059424).
  • CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] synthetic connecting peptide indicated in Table 2 (SEQ ID NO:9) was constructed using a similar strategy as that described in Example 10.
  • the PRIMATIZED® p5E8 scFv used for constructing the sc(Fv)2 tetravalent antibody is comprised of a p5E8 VL and a VH region sequence tethered by a short synthetic linker in the VL ⁇ (Gly 4 Ser) 3 linker ⁇ VH orientation and is described in greater detail in Example 12. Correct sequences were confirmed by DNA sequence analysis.
  • FIG. 12A shows the DNA sequence of heavy chain CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 12B shows the DNA sequence of light chain CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 tetravalent antibody.
  • FIG. 12A shows the DNA sequence of heavy chain CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 12B shows the DNA sequence of light chain CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 tetravalent antibody.
  • FIG. 13A shows the amino acid sequence of heavy chain CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 13B shows the amino sequence of light chain CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 tetravalent antibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • the connecting peptide shown in SEQ ID NO: 9 was found to substantially increase the proportion of Form A tetravalent antibody produced. Exemplary results are shown in FIG. 31 . These results show that the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide (SEQ ID NO:9) resulted in the production of essentially all Form A CH2 domain-deleted PRIMATIZED® p5E8 sc(Fv)2 tetravalent antibody with no detectable Form B, demonstrating that the utility of this hinge for producing the Form A isoform CH2 domain-deleted sc(Fv)2 tetravalent antibodies is generally applicable to antibodies of varying specificities.
  • Minibodies are single chain polypeptides consisting of an scFv fused to immunoglobulin hinge region and CH3 domain.
  • the preparation of minibody single polypeptide chain binding molecules is described in U.S. Pat. No. 5,837,821.
  • the cysteine residue at position 230 in the hinge region forms a covalent disulfide bond with the carboxyl-terminal light chain constant domain cysteine residue at position 214, joining heavy and light chain polypeptides (Kabat numbering system).
  • the light chain constant domain is absent, eliminating one of the two cysteines that participate in forming the disulfide bond.
  • the single remaining cysteine in the hinge region at position 230 has been proposed to participate in a homotypic interchain disulfide bond possibly contributing to hinge region structure and therefore was left in place.
  • Minibodies which lack a CH2 domain, are secreted from cells and accumulate in culture supernatant as a mixture containing Form A and Form B isoforms and perhaps unassembled half molecules as shown in FIG. 32 .
  • the ratio of these forms is highly variable and presents a challenge for preparing a reproducibly pure product.
  • the absence of the immunoglobulin CH1/CL domains precludes isolation of total minibody using Protein G immunoaffinity matrices.
  • the unassembled half molecules (MW ⁇ 40-45 kD) can be separated from the homodimers (MW ⁇ 80-90 kD) using a number of chromatographic strategies
  • the remaining intact A and B isoforms cannot be efficiently separated from one another using techniques such as HIC chromatography (described herein) also due to the absence of CH1/CL domains.
  • HIC chromatography described herein
  • isoforms also cannot be separated by size exclusion chromatography because the molecular masses of the two isoforms are nearly identical, prohibiting separation based on this characteristic.
  • the composition of minibody preparations may actually be complex, consisting of a mixture of A and B isoforms.
  • PRIMATIZED® p5E8 scFvs were constructed by PCR amplification. ScFvs were built in both orientations (VL ⁇ (Gly 4 Ser) 3 linker ⁇ VH (VL/VH) and VH ⁇ (Gly 4 Ser) 3 linker ⁇ VL (VH/VL)). Oligonucleotides used in the construction are shown the tables below:
  • the VL/VH scFv was constructed in two steps.
  • the 5′ VL PCR forward primer N-23VL-1F (SEQ ID NO:37) included a BspLU11 I restriction endonuclease site to permit joining of the scFv to the immunoglobulin signal peptide in the expression vector.
  • the 3′ VL PCR reverse primer 23-VL-1R (SEQ ID NO:38) included sequence partially encoding the (Gly 4 Ser) 3 linker peptide used to join the VL and VH regions.
  • the 5′ forward VH PCR primer 23-VH-2F also included sequence partially encoding a (Gly 4 Ser) 3 linker peptide used to connect the VL and VH regions and the 3′ VH PCR reverse primer N-23-VH-2R (SEQ ID NO:40) included a Sal I restriction endonuclease site to join the scFv to the hinge region.
  • the two V regions were amplified with the two sets of PCR primers from plasmid DNA substrate containing PRIMATIZED® p5E8G1 antibody and assembly of the scFv was accomplished in a second PCR reaction through the common overlapping sequences encoding the (Gly 4 Ser) 3 linker.
  • the PRIMATIZED® p5E8 scFv gene fragment was gel isolated, digested with BspLU11 I and Sal I restriction endonucleases and cloned into the BspLU11 I and Sal I double-digested CH2 domain-deleted antibody Polycistronic expression vector containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • the PRIMATIZED® p5E8 VH/VL scFv was constructed in a similar manner using PCR primer pairs N-23VH-1F (SEQ ID NO:41) and 23-VH-1R (SEQ ID NO:36) and 23-VL-2F (SEQ ID NO:43) and N-23-VL-2R (SEQ ID NO:44) shown in Tables 7 and 8. Correct sequences of both completed constructs were confirmed by DNA sequence analysis. Plasmid DNA was used to transform CHO DG44 cells for transient production of antibody protein. FIG.
  • FIG. 14 shows the DNA sequence of CH2 domain-deleted PRIMATIZED® p5E8 VL/VH minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide in the VL ⁇ VH orientation (VL/VH).
  • FIG. 15 shows the DNA sequence of CH2 domain-deleted PRIMATIZED® p5E8 VH/VL minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide in the VH ⁇ VL orientation (VH/VL).
  • FIG. 16 shows the amino acid sequence of CH2 domain-deleted PRIMATIZED® p5E8 VL/VH minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.
  • FIG. 17 shows the amino acid sequence of CH2 domain-deleted PRIMATIZED® p5E8 VH/VL minibody containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser] connecting peptide.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Diabetes (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Pulmonology (AREA)
  • Oncology (AREA)
  • Endocrinology (AREA)
  • Dermatology (AREA)
  • Neurology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Reproductive Health (AREA)
  • Urology & Nephrology (AREA)
  • Cardiology (AREA)
  • Obesity (AREA)
  • Communicable Diseases (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Neurosurgery (AREA)
  • Cell Biology (AREA)
  • Rheumatology (AREA)
  • Pain & Pain Management (AREA)
US10/880,028 2003-06-27 2004-06-28 Modified binding molecules comprising connecting peptides Abandoned US20050163782A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/880,028 US20050163782A1 (en) 2003-06-27 2004-06-28 Modified binding molecules comprising connecting peptides
US12/100,375 US8603473B2 (en) 2003-06-27 2008-04-09 Modified binding molecules comprising connecting peptides

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US48387703P 2003-06-27 2003-06-27
US50881003P 2003-10-03 2003-10-03
US51535103P 2003-10-28 2003-10-28
US51603003P 2003-10-30 2003-10-30
US10/880,028 US20050163782A1 (en) 2003-06-27 2004-06-28 Modified binding molecules comprising connecting peptides

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/100,375 Continuation US8603473B2 (en) 2003-06-27 2008-04-09 Modified binding molecules comprising connecting peptides

Publications (1)

Publication Number Publication Date
US20050163782A1 true US20050163782A1 (en) 2005-07-28

Family

ID=33556653

Family Applications (3)

Application Number Title Priority Date Filing Date
US10/880,028 Abandoned US20050163782A1 (en) 2003-06-27 2004-06-28 Modified binding molecules comprising connecting peptides
US10/880,320 Expired - Lifetime US7700097B2 (en) 2003-06-27 2004-06-28 Purification and preferential synthesis of binding molecules
US12/100,375 Expired - Fee Related US8603473B2 (en) 2003-06-27 2008-04-09 Modified binding molecules comprising connecting peptides

Family Applications After (2)

Application Number Title Priority Date Filing Date
US10/880,320 Expired - Lifetime US7700097B2 (en) 2003-06-27 2004-06-28 Purification and preferential synthesis of binding molecules
US12/100,375 Expired - Fee Related US8603473B2 (en) 2003-06-27 2008-04-09 Modified binding molecules comprising connecting peptides

Country Status (7)

Country Link
US (3) US20050163782A1 (enExample)
EP (2) EP1641826A2 (enExample)
JP (4) JP5068072B2 (enExample)
AU (3) AU2004252171B2 (enExample)
CA (2) CA2530388A1 (enExample)
NZ (2) NZ544924A (enExample)
WO (2) WO2005000898A2 (enExample)

Cited By (154)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090041758A1 (en) * 2003-06-27 2009-02-12 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
US20090048122A1 (en) * 2006-03-17 2009-02-19 Biogen Idec Ma Inc. Stabilized polypeptide compositions
WO2010040508A1 (en) 2008-10-08 2010-04-15 F. Hoffmann-La Roche Ag Bispecific anti-vegf/anti-ang-2 antibodies
WO2010085682A3 (en) * 2009-01-23 2010-09-30 Biogen Idec Ma Inc. Stabilized fc polypeptides with reduced effector function and methods of use
US20100256338A1 (en) * 2009-04-02 2010-10-07 Ulrich Brinkmann Multispecific antibodies comprising full length antibodies and single chain fab fragments
WO2010112194A1 (en) 2009-04-02 2010-10-07 F. Hoffmann-La Roche Ag Antigen-binding polypeptides and multispecific antibodies comprising them
WO2010115553A1 (en) 2009-04-07 2010-10-14 Roche Glycart Ag Bispecific anti-erbb-2/anti-c-met antibodies
WO2010115589A1 (en) 2009-04-07 2010-10-14 Roche Glycart Ag Trivalent, bispecific antibodies
WO2010115552A1 (en) 2009-04-07 2010-10-14 Roche Glycart Ag Bispecific anti-erbb-3/anti-c-met antibodies
US8021661B2 (en) 2005-01-05 2011-09-20 Biogen Idec Ma Inc. Cripto binding molecules
WO2011117330A1 (en) 2010-03-26 2011-09-29 Roche Glycart Ag Bispecific antibodies
WO2011117329A1 (en) 2010-03-26 2011-09-29 F. Hoffmann-La Roche Ag Bispecific, bivalent anti-vegf/anti-ang-2 antibodies
WO2011119888A2 (en) 2010-03-24 2011-09-29 The Regents Of The University Of California N-cadherin: target for cancer diagnosis and therapy
WO2012025530A1 (en) 2010-08-24 2012-03-01 F. Hoffmann-La Roche Ag Bispecific antibodies comprising a disulfide stabilized - fv fragment
WO2012085111A1 (en) 2010-12-23 2012-06-28 F. Hoffmann-La Roche Ag Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery
US8409577B2 (en) 2006-06-12 2013-04-02 Emergent Product Development Seattle, Llc Single chain multivalent binding proteins with effector function
WO2013150043A1 (en) 2012-04-05 2013-10-10 F. Hoffmann-La Roche Ag Bispecific antibodies against human tweak and human il17 and uses thereof
WO2014001326A1 (en) 2012-06-27 2014-01-03 F. Hoffmann-La Roche Ag Method for the selection and production of tailor-made, selective and multi-specific therapeutic molecules comprising at least two different targeting entities and uses thereof
WO2014006124A1 (en) 2012-07-04 2014-01-09 F. Hoffmann-La Roche Ag Covalently linked antigen-antibody conjugates
AU2014200661A1 (en) * 2006-06-12 2014-03-13 Aptevo Research And Development Llc Single-chain multivalent binding proteins with effector function
EP2638073A4 (en) * 2010-11-09 2014-05-07 Altimab Therapeutics Inc PROTEIN COMPLEXES FOR ANTIGEN BINDING, AND METHODS OF USE
US8853366B2 (en) 2001-01-17 2014-10-07 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
WO2015101587A1 (en) 2014-01-03 2015-07-09 F. Hoffmann-La Roche Ag Covalently linked helicar-anti-helicar antibody conjugates and uses thereof
WO2015101586A1 (en) 2014-01-03 2015-07-09 F. Hoffmann-La Roche Ag Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles
WO2015101589A1 (en) 2014-01-03 2015-07-09 F. Hoffmann-La Roche Ag Covalently linked polypeptide toxin-antibody conjugates
US9101609B2 (en) 2008-04-11 2015-08-11 Emergent Product Development Seattle, Llc CD37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof
US9133524B2 (en) 2010-07-01 2015-09-15 The Regents Of The University Of California Protein kinase ck2 gene mutations, amplifications and polymorphisms in human cancers and methods of use
WO2015138920A1 (en) 2014-03-14 2015-09-17 Novartis Ag Antibody molecules to lag-3 and uses thereof
WO2015142675A2 (en) 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor
WO2016014553A1 (en) 2014-07-21 2016-01-28 Novartis Ag Sortase synthesized chimeric antigen receptors
WO2016014565A2 (en) 2014-07-21 2016-01-28 Novartis Ag Treatment of cancer using humanized anti-bcma chimeric antigen receptor
WO2016014530A1 (en) 2014-07-21 2016-01-28 Novartis Ag Combinations of low, immune enhancing. doses of mtor inhibitors and cars
WO2016025880A1 (en) 2014-08-14 2016-02-18 Novartis Ag Treatment of cancer using gfr alpha-4 chimeric antigen receptor
US9266967B2 (en) 2007-12-21 2016-02-23 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
WO2016044605A1 (en) 2014-09-17 2016-03-24 Beatty, Gregory Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
WO2016061142A1 (en) 2014-10-14 2016-04-21 Novartis Ag Antibody molecules to pd-l1 and uses thereof
WO2016090034A2 (en) 2014-12-03 2016-06-09 Novartis Ag Methods for b cell preconditioning in car therapy
WO2016164731A2 (en) 2015-04-08 2016-10-13 Novartis Ag Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car) - expressing cell
WO2016172583A1 (en) 2015-04-23 2016-10-27 Novartis Ag Treatment of cancer using chimeric antigen receptor and protein kinase a blocker
WO2017019897A1 (en) 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to tim-3
WO2017019894A1 (en) 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to lag-3
US9567403B2 (en) 2013-08-06 2017-02-14 Bio-Thera Solutions, Ltd. Bispecific antibodies which bind EGFR and VEGF
US9676845B2 (en) 2009-06-16 2017-06-13 Hoffmann-La Roche, Inc. Bispecific antigen binding proteins
WO2017106810A2 (en) 2015-12-17 2017-06-22 Novartis Ag Combination of c-met inhibitor with antibody molecule to pd-1 and uses thereof
WO2017106656A1 (en) 2015-12-17 2017-06-22 Novartis Ag Antibody molecules to pd-1 and uses thereof
US9688758B2 (en) 2012-02-10 2017-06-27 Genentech, Inc. Single-chain antibodies and other heteromultimers
WO2017125897A1 (en) 2016-01-21 2017-07-27 Novartis Ag Multispecific molecules targeting cll-1
WO2017149515A1 (en) 2016-03-04 2017-09-08 Novartis Ag Cells expressing multiple chimeric antigen receptor (car) molecules and uses therefore
US9765153B2 (en) 2012-07-04 2017-09-19 Hoffmann-La Roche Inc. Anti-biotin antibodies and methods of use
WO2017165683A1 (en) 2016-03-23 2017-09-28 Novartis Ag Cell secreted minibodies and uses thereof
WO2017181119A2 (en) 2016-04-15 2017-10-19 Novartis Ag Compositions and methods for selective protein expression
WO2017210617A2 (en) 2016-06-02 2017-12-07 Porter, David, L. Therapeutic regimens for chimeric antigen receptor (car)- expressing cells
WO2018013918A2 (en) 2016-07-15 2018-01-18 Novartis Ag Treatment and prevention of cytokine release syndrome using a chimeric antigen receptor in combination with a kinase inhibitor
WO2018023025A1 (en) 2016-07-28 2018-02-01 Novartis Ag Combination therapies of chimeric antigen receptors adn pd-1 inhibitors
WO2018026819A2 (en) 2016-08-01 2018-02-08 Novartis Ag Treatment of cancer using a chimeric antigen receptor in combination with an inhibitor of a pro-m2 macrophage molecule
US9925272B2 (en) 2012-07-04 2018-03-27 Hoffmann-La Roche Inc. Anti-theophylline antibodies and methods of use
WO2018067992A1 (en) 2016-10-07 2018-04-12 Novartis Ag Chimeric antigen receptors for the treatment of cancer
US9982036B2 (en) 2011-02-28 2018-05-29 Hoffmann-La Roche Inc. Dual FC antigen binding proteins
US9994646B2 (en) 2009-09-16 2018-06-12 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
EP3348277A1 (en) 2009-11-20 2018-07-18 The Regents of The University of California Epithelial membrane protein-2 (emp2) and proliferative vitreoretinopathy (pvr)
WO2018140725A1 (en) 2017-01-26 2018-08-02 Novartis Ag Cd28 compositions and methods for chimeric antigen receptor therapy
WO2018160731A1 (en) 2017-02-28 2018-09-07 Novartis Ag Shp inhibitor compositions and uses for chimeric antigen receptor therapy
US10071158B2 (en) 2005-04-26 2018-09-11 Lindis Biotech Gmbh Combination of the application of antibodies for immunostimulation together with glucocorticoids
US10106612B2 (en) 2012-06-27 2018-10-23 Hoffmann-La Roche Inc. Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof
WO2018201051A1 (en) 2017-04-28 2018-11-01 Novartis Ag Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor
WO2018201056A1 (en) 2017-04-28 2018-11-01 Novartis Ag Cells expressing a bcma-targeting chimeric antigen receptor, and combination therapy with a gamma secretase inhibitor
US10138293B2 (en) 2007-12-21 2018-11-27 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
US10143748B2 (en) 2005-07-25 2018-12-04 Aptevo Research And Development Llc B-cell reduction using CD37-specific and CD20-specific binding molecules
WO2018237157A1 (en) 2017-06-22 2018-12-27 Novartis Ag CD73 BINDING ANTIBODY MOLECULES AND USES THEREOF
WO2019006007A1 (en) 2017-06-27 2019-01-03 Novartis Ag POSOLOGICAL REGIMES FOR ANTI-TIM3 ANTIBODIES AND USES THEREOF
WO2019018730A1 (en) 2017-07-20 2019-01-24 Novartis Ag DOSAGE REGIMES FOR ANTI-LAG3 ANTIBODIES AND USES THEREOF
US10251952B2 (en) 2014-06-26 2019-04-09 Hoffmann-La Roche Inc. Humanized anti-tau(pS422) antibody brain shuttles and use thereof
WO2019089798A1 (en) 2017-10-31 2019-05-09 Novartis Ag Anti-car compositions and methods
WO2019099838A1 (en) 2017-11-16 2019-05-23 Novartis Ag Combination therapies
US10323099B2 (en) 2013-10-11 2019-06-18 Hoffmann-La Roche Inc. Multispecific domain exchanged common variable light chain antibodies
EP3514179A1 (en) 2014-01-24 2019-07-24 Dana-Farber Cancer Institute, Inc. Antibody molecules to pd-1 and uses thereof
WO2019152660A1 (en) 2018-01-31 2019-08-08 Novartis Ag Combination therapy using a chimeric antigen receptor
WO2019200229A1 (en) 2018-04-13 2019-10-17 Novartis Ag Dosage regimens for anti-pd-l1 antibodies and uses thereof
WO2019210153A1 (en) 2018-04-27 2019-10-31 Novartis Ag Car t cell therapies with enhanced efficacy
US10465000B2 (en) 2013-12-20 2019-11-05 Hoffmann-La Roche Inc. Humanized anti-Tau(pS422) antibodies and methods of use
WO2019226658A1 (en) 2018-05-21 2019-11-28 Compass Therapeutics Llc Multispecific antigen-binding compositions and methods of use
WO2019226617A1 (en) 2018-05-21 2019-11-28 Compass Therapeutics Llc Compositions and methods for enhancing the killing of target cells by nk cells
WO2019227003A1 (en) 2018-05-25 2019-11-28 Novartis Ag Combination therapy with chimeric antigen receptor (car) therapies
WO2019232244A2 (en) 2018-05-31 2019-12-05 Novartis Ag Antibody molecules to cd73 and uses thereof
WO2019241426A1 (en) 2018-06-13 2019-12-19 Novartis Ag Bcma chimeric antigen receptors and uses thereof
WO2019246293A2 (en) 2018-06-19 2019-12-26 Atarga, Llc Antibody molecules to complement component 5 and uses thereof
WO2020012337A1 (en) 2018-07-10 2020-01-16 Novartis Ag 3-(5-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and their use in the treatment of i karos family zinc finger 2 (ikzf2)-dependent diseases
US10538589B2 (en) * 2015-01-14 2020-01-21 Armagen Inc. Methods and compositions for increasing N-acetylglucosaminidase (NAGLU) activity in the CNS using a fusion antibody comprising an anti-human insulin receptor antibody and NAGLU
WO2020021465A1 (en) 2018-07-25 2020-01-30 Advanced Accelerator Applications (Italy) S.R.L. Method of treatment of neuroendocrine tumors
US10611825B2 (en) 2011-02-28 2020-04-07 Hoffmann La-Roche Inc. Monovalent antigen binding proteins
US10633457B2 (en) 2014-12-03 2020-04-28 Hoffmann-La Roche Inc. Multispecific antibodies
EP3660042A1 (en) 2014-07-31 2020-06-03 Novartis AG Subset-optimized chimeric antigen receptor-containing t-cells
US10676533B2 (en) 2016-09-21 2020-06-09 Aptevo Research And Development Llc Methods of treatment of CD123 overexpressing disorders
WO2020128972A1 (en) 2018-12-20 2020-06-25 Novartis Ag Dosing regimen and pharmaceutical combination comprising 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives
WO2020128898A1 (en) 2018-12-20 2020-06-25 Novartis Ag Pharmaceutical combinations
WO2020165833A1 (en) 2019-02-15 2020-08-20 Novartis Ag 3-(1-oxo-5-(piperidin-4-yl)isoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
WO2020165834A1 (en) 2019-02-15 2020-08-20 Novartis Ag Substituted 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
WO2020165868A1 (en) 2019-02-15 2020-08-20 Perkinelmer Cellular Technologies Germany Gmbh Low-power microscope-objective pre-scan and high-power microscope-objective scan in x,y and z-direction for imaging objects such as cells using a microscope
WO2020172553A1 (en) 2019-02-22 2020-08-27 Novartis Ag Combination therapies of egfrviii chimeric antigen receptors and pd-1 inhibitors
EP3712171A1 (en) 2014-08-19 2020-09-23 Novartis AG Treatment of cancer using a cd123 chimeric antigen receptor
WO2020205523A1 (en) 2019-03-29 2020-10-08 Atarga, Llc Anti fgf23 antibody
EP3722316A1 (en) 2014-07-21 2020-10-14 Novartis AG Treatment of cancer using a cd33 chimeric antigen receptor
US10822402B2 (en) 2015-06-24 2020-11-03 Hoffmann-La Roche Inc. Humanized anti-tau(pS422) antibodies and methods of use
WO2021079195A1 (en) 2019-10-21 2021-04-29 Novartis Ag Tim-3 inhibitors and uses thereof
WO2021079188A1 (en) 2019-10-21 2021-04-29 Novartis Ag Combination therapies with venetoclax and tim-3 inhibitors
WO2021108661A2 (en) 2019-11-26 2021-06-03 Novartis Ag Chimeric antigen receptors and uses thereof
US11028156B2 (en) 2009-10-09 2021-06-08 Armagen, Inc. Methods and compositions for increasing iduronate 2-sulfatase activity in the CNS
WO2021123996A1 (en) 2019-12-20 2021-06-24 Novartis Ag Uses of anti-tgf-beta antibodies and checkpoint inhibitors for the treatment of proliferative diseases
US11046769B2 (en) 2018-11-13 2021-06-29 Compass Therapeutics Llc Multispecific binding constructs against checkpoint molecules and uses thereof
WO2021146636A1 (en) 2020-01-17 2021-07-22 Becton, Dickinson And Company Methods and compositions for single cell secretomics
WO2021144657A1 (en) 2020-01-17 2021-07-22 Novartis Ag Combination comprising a tim-3 inhibitor and a hypomethylating agent for use in treating myelodysplastic syndrome or chronic myelomonocytic leukemia
WO2021173995A2 (en) 2020-02-27 2021-09-02 Novartis Ag Methods of making chimeric antigen receptor-expressing cells
US11155631B2 (en) 2006-08-18 2021-10-26 Armagen, Inc. Macromolecular compositions that cross the blood-brain barrier and methods of use thereof
WO2021260528A1 (en) 2020-06-23 2021-12-30 Novartis Ag Dosing regimen comprising 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives
WO2022013787A1 (en) 2020-07-16 2022-01-20 Novartis Ag Anti-betacellulin antibodies, fragments thereof, and multi-specific binding molecules
WO2022026592A2 (en) 2020-07-28 2022-02-03 Celltas Bio, Inc. Antibody molecules to coronavirus and uses thereof
WO2022029573A1 (en) 2020-08-03 2022-02-10 Novartis Ag Heteroaryl substituted 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
WO2022043558A1 (en) 2020-08-31 2022-03-03 Advanced Accelerator Applications International Sa Method of treating psma-expressing cancers
WO2022043557A1 (en) 2020-08-31 2022-03-03 Advanced Accelerator Applications International Sa Method of treating psma-expressing cancers
EP2850106B1 (en) * 2012-05-18 2022-03-23 Aptevo Research and Development LLC Bispecific scfv immunofusion (bif) binding to cd123 and cd3
WO2022097065A2 (en) 2020-11-06 2022-05-12 Novartis Ag ANTIBODY Fc VARIANTS
US11333642B2 (en) 2016-10-25 2022-05-17 Regeneran Pharmaceuticals, Inc. Methods and systems for chromatography data analysis
WO2022104061A1 (en) 2020-11-13 2022-05-19 Novartis Ag Combination therapies with chimeric antigen receptor (car)-expressing cells
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
US11369896B2 (en) 2016-08-16 2022-06-28 Regeneron Pharmaceuticals, Inc. Methods for quantitating individual antibodies from a mixture
WO2022162569A1 (en) 2021-01-29 2022-08-04 Novartis Ag Dosage regimes for anti-cd73 and anti-entpd2 antibodies and uses thereof
US11421022B2 (en) 2012-06-27 2022-08-23 Hoffmann-La Roche Inc. Method for making antibody Fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof
WO2022215011A1 (en) 2021-04-07 2022-10-13 Novartis Ag USES OF ANTI-TGFβ ANTIBODIES AND OTHER THERAPEUTIC AGENTS FOR THE TREATMENT OF PROLIFERATIVE DISEASES
WO2022243846A1 (en) 2021-05-18 2022-11-24 Novartis Ag Combination therapies
US11512145B2 (en) 2007-07-27 2022-11-29 Armagen, Inc. Methods and compositions for increasing alpha-L-iduronidase activity in the CNS
WO2023044483A2 (en) 2021-09-20 2023-03-23 Voyager Therapeutics, Inc. Compositions and methods for the treatment of her2 positive cancer
WO2023092004A1 (en) 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Compositions and methods for the treatment of tau-related disorders
US11718679B2 (en) 2017-10-31 2023-08-08 Compass Therapeutics Llc CD137 antibodies and PD-1 antagonists and uses thereof
WO2023150778A1 (en) 2022-02-07 2023-08-10 Visterra, Inc. Anti-idiotype antibody molecules and uses thereof
US11752207B2 (en) 2017-07-11 2023-09-12 Compass Therapeutics Llc Agonist antibodies that bind human CD137 and uses thereof
WO2023220695A2 (en) 2022-05-13 2023-11-16 Voyager Therapeutics, Inc. Compositions and methods for the treatment of her2 positive cancer
US11851497B2 (en) 2017-11-20 2023-12-26 Compass Therapeutics Llc CD137 antibodies and tumor antigen-targeting antibodies and uses thereof
US11884698B2 (en) 2018-07-02 2024-01-30 Regeneron Pharmaceuticals, Inc. Systems and methods for preparing a polypeptide from a mixture
WO2024030976A2 (en) 2022-08-03 2024-02-08 Voyager Therapeutics, Inc. Compositions and methods for crossing the blood brain barrier
EP4324518A2 (en) 2014-01-31 2024-02-21 Novartis AG Antibody molecules to tim-3 and uses thereof
EP4378957A2 (en) 2015-07-29 2024-06-05 Novartis AG Combination therapies comprising antibody molecules to pd-1
US12042538B2 (en) 2017-09-19 2024-07-23 Regeneron Pharmaceuticals, Inc. Methods of reducing particle formation and compositions formed thereby
WO2024168061A2 (en) 2023-02-07 2024-08-15 Ayan Therapeutics Inc. Antibody molecules binding to sars-cov-2
US12070502B2 (en) 2014-10-09 2024-08-27 Regeneron Pharmaceuticals, Inc. Process for reducing subvisible particles in a pharmaceutical formulation
US12098215B2 (en) 2016-06-06 2024-09-24 Northwestern University Fusion protein construct
US12152073B2 (en) 2018-03-14 2024-11-26 Marengo Therapeutics, Inc. Multifunctional molecules that bind to calreticulin and uses thereof
US12247060B2 (en) 2018-01-09 2025-03-11 Marengo Therapeutics, Inc. Calreticulin binding constructs and engineered T cells for the treatment of diseases
US12286477B2 (en) 2018-07-03 2025-04-29 Marengo Therapeutics, Inc. Anti-TCR antibody molecules and uses thereof
WO2025122634A1 (en) 2023-12-05 2025-06-12 Voyager Therapeutics, Inc. Compositions and methods for the treatment of tau-related disorders
WO2025129010A1 (en) 2023-12-14 2025-06-19 Genentech, Inc. Methods of structure determination using antibodies
US12358982B2 (en) 2019-02-21 2025-07-15 Marengo Therapeutics, Inc. Multifunctional molecules that bind to T cell related cancer cells and uses thereof
US12384842B2 (en) 2019-02-21 2025-08-12 Marengo Therapeutics, Inc. Antibody molecules that bind to NKP30 and uses thereof
EP4643874A2 (en) 2015-12-22 2025-11-05 Novartis AG Mesothelin chimeric antigen receptor (car) and antibody against pd-l1 inhibitor for combined use in anticancer therapy
US12486326B2 (en) 2020-01-03 2025-12-02 Marengo Therapeutics, Inc. Anti-TCR antibody molecules and uses thereof

Families Citing this family (102)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136311A (en) 1996-05-06 2000-10-24 Cornell Research Foundation, Inc. Treatment and diagnosis of cancer
US7662925B2 (en) 2002-03-01 2010-02-16 Xencor, Inc. Optimized Fc variants and methods for their generation
US8188231B2 (en) 2002-09-27 2012-05-29 Xencor, Inc. Optimized FC variants
US20040132101A1 (en) 2002-09-27 2004-07-08 Xencor Optimized Fc variants and methods for their generation
US7317091B2 (en) 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
US8084582B2 (en) 2003-03-03 2011-12-27 Xencor, Inc. Optimized anti-CD20 monoclonal antibodies having Fc variants
US8388955B2 (en) 2003-03-03 2013-03-05 Xencor, Inc. Fc variants
US20090010920A1 (en) 2003-03-03 2009-01-08 Xencor, Inc. Fc Variants Having Decreased Affinity for FcyRIIb
US9051373B2 (en) 2003-05-02 2015-06-09 Xencor, Inc. Optimized Fc variants
US8101720B2 (en) 2004-10-21 2012-01-24 Xencor, Inc. Immunoglobulin insertions, deletions and substitutions
US9714282B2 (en) 2003-09-26 2017-07-25 Xencor, Inc. Optimized Fc variants and methods for their generation
US7276585B2 (en) 2004-03-24 2007-10-02 Xencor, Inc. Immunoglobulin variants outside the Fc region
US20150010550A1 (en) 2004-07-15 2015-01-08 Xencor, Inc. OPTIMIZED Fc VARIANTS
EP1817340B1 (en) 2004-11-12 2012-05-16 Xencor, Inc. Fc variants with altered binding to fcrn
US8802820B2 (en) 2004-11-12 2014-08-12 Xencor, Inc. Fc variants with altered binding to FcRn
US8367805B2 (en) 2004-11-12 2013-02-05 Xencor, Inc. Fc variants with altered binding to FcRn
US8546543B2 (en) 2004-11-12 2013-10-01 Xencor, Inc. Fc variants that extend antibody half-life
WO2006074399A2 (en) * 2005-01-05 2006-07-13 Biogen Idec Ma Inc. Multispecific binding molecules comprising connecting peptides
CA2606102C (en) 2005-04-26 2014-09-30 Medimmune, Inc. Modulation of antibody effector function by hinge domain engineering
WO2007005612A2 (en) 2005-07-01 2007-01-11 Medimmune, Inc. An integrated approach for generating multidomain protein therapeutics
EP1931709B1 (en) 2005-10-03 2016-12-07 Xencor, Inc. Fc variants with optimized fc receptor binding properties
JP4860703B2 (ja) 2005-10-06 2012-01-25 ゼンコー・インコーポレイテッド 最適化された抗ｃｄ３０抗体
WO2007047578A2 (en) 2005-10-14 2007-04-26 Medimmune, Inc. Cell display of antibody libraries
AR056142A1 (es) * 2005-10-21 2007-09-19 Amgen Inc Metodos para generar el anticuerpo igg monovalente
US8940871B2 (en) 2006-03-20 2015-01-27 The Regents Of The University Of California Engineered anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting
DK2059536T3 (da) 2006-08-14 2014-04-14 Xencor Inc Optimerede antistoffer, der er rettet mod cd19
WO2008036688A2 (en) 2006-09-18 2008-03-27 Xencor, Inc. Optimized antibodies that target hm1.24
US9212230B2 (en) * 2007-03-29 2015-12-15 Genmab A/S Bispecific antibodies and methods for production thereof
JP6126773B2 (ja) 2007-09-04 2017-05-10 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 癌のターゲッティングおよび検出のための高親和性抗前立腺幹細胞抗原（ｐｓｃａ）抗体
US20100226925A1 (en) * 2007-09-14 2010-09-09 Amgen Inc. Homogeneous Antibody Populations
ES2667729T3 (es) 2007-09-26 2018-05-14 Ucb Biopharma Sprl Fusiones de anticuerpos con doble especificidad
WO2009043051A2 (en) * 2007-09-27 2009-04-02 Biogen Idec Ma Inc. Cd23 binding molecules and methods of use thereof
US20100291113A1 (en) * 2007-10-03 2010-11-18 Cornell University Treatment of Proliferative Disorders Using Antibodies to PSMA
CA2703997C (en) 2007-12-26 2017-04-04 Xencor, Inc. Fc variants with altered binding to fcrn
US12492253B1 (en) 2008-02-25 2025-12-09 Xencor, Inc. Anti-human C5 antibodies
US20100069616A1 (en) * 2008-08-06 2010-03-18 The Regents Of The University Of California Engineered antibody-nanoparticle conjugates
WO2010035012A1 (en) 2008-09-26 2010-04-01 Ucb Pharma S.A. Biological products
WO2010079149A1 (de) * 2009-01-09 2010-07-15 Ipk Gatersleben Fusionsantikörper
CA2752510C (en) * 2009-02-17 2024-01-23 Neil Bander Methods and kits for diagnosis of cancer and prediction of therapeutic value
US9238878B2 (en) 2009-02-17 2016-01-19 Redwood Bioscience, Inc. Aldehyde-tagged protein-based drug carriers and methods of use
WO2011028952A1 (en) 2009-09-02 2011-03-10 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens
TW201120210A (en) * 2009-11-05 2011-06-16 Hoffmann La Roche Glycosylated repeat-motif-molecule conjugates
RU2673908C2 (ru) 2009-12-02 2018-12-03 Имэджинэб, Инк. Мини-антитела j591 и цис-диатела для направленной доставки простата-специфичного мембранного антигена (psma) человека и способы их применения
WO2011091078A2 (en) 2010-01-19 2011-07-28 Xencor, Inc. Antibody fc variants with enhanced complement activity
GB201005064D0 (en) 2010-03-25 2010-05-12 Ucb Pharma Sa Biological products
FI2560993T3 (fi) 2010-04-20 2024-09-23 Genmab As Heterodimeeristä vasta-ainetta FC sisältävät proteiinit ja menetelmät niiden tuottamiseksi
RU2606016C2 (ru) 2011-01-14 2017-01-10 Редвуд Байосайнс, Инк. Меченые альдегидом полипептиды иммуноглобулина и способы их применения
EP2686418A4 (en) 2011-03-17 2015-04-22 Minerva Biotechnologies Corp METHOD FOR THE PRODUCTION OF PLURIPOTENTAL STEM CELLS
CA2835453A1 (en) * 2011-05-09 2012-11-15 Minerva Biotechnologies Corporation Genetically engineered growth factor variants
SI2771364T1 (sl) 2011-10-27 2019-10-30 Genmab As Produkcija heterodimernih proteinov
ES2773107T3 (es) 2012-10-05 2020-07-09 Kyowa Kirin Co Ltd Composición de proteína heterodimérica
TWI635098B (zh) 2013-02-01 2018-09-11 再生元醫藥公司 含嵌合恆定區之抗體
MY173295A (en) * 2013-04-16 2020-01-14 Genentech Inc Pertuzumab variants and evaluation thereof
TWI596107B (zh) 2013-06-25 2017-08-21 卡地拉保健有限公司 單株抗體之新穎純化方法
GB2519786A (en) * 2013-10-30 2015-05-06 Sergej Michailovic Kiprijanov Multivalent antigen-binding protein molecules
JPWO2015079977A1 (ja) * 2013-11-26 2017-03-16 シャープ株式会社 抗体分離方法、抗体評価方法、医薬の評価方法、及び、抗体の２次元電気泳動用キット
TWI754319B (zh) 2014-03-19 2022-02-01 美商再生元醫藥公司 用於腫瘤治療之方法及抗體組成物
ES2785551T3 (es) 2014-06-30 2020-10-07 Glykos Finland Oy Derivado de sacárido de una carga útil tóxica y sus conjugados con anticuerpos
RU2016151645A (ru) 2014-07-01 2018-08-03 Пфайзер Инк. Биспецифические гетеродимерные диантитела и их применение
DK3221359T3 (da) 2014-11-17 2020-06-22 Regeneron Pharma Fremgangsmåder til tumorbehandling ved anvendelse af CD3XCD20-bispecifikt antistof
WO2016097300A1 (en) 2014-12-19 2016-06-23 Genmab A/S Rodent bispecific heterodimeric proteins
WO2016126950A1 (en) 2015-02-05 2016-08-11 Molecular Templates, Inc. Multivalent cd20-binding molecules comprising shiga toxin a subunit effector regions and enriched compositions thereof
WO2016161010A2 (en) 2015-03-30 2016-10-06 Regeneron Pharmaceuticals, Inc. Heavy chain constant regions with reduced binding to fc gamma receptors
US20180179274A1 (en) 2015-06-26 2018-06-28 Institute For Research In Biomedicine Proteins comprising a mutated lair-1 fragment and uses thereof
EP4137158A1 (en) 2015-08-07 2023-02-22 Imaginab, Inc. Antigen binding constructs to target molecules
CN108290956B (zh) * 2015-10-06 2022-07-05 希望之城公司 靶向psca的嵌合抗原受体
WO2017059878A1 (en) 2015-10-07 2017-04-13 Humabs Biomed Sa Antibodies that potently neutralize hepatitis b virus and uses thereof
EP3365366B1 (en) 2015-10-25 2021-07-14 Sanofi Trispecific and/or trivalent binding proteins for prevention or treatment of hiv infection
RS64771B1 (sr) 2016-04-13 2023-11-30 Sanofi Sa Trispecifični i/ili trovalentni vezujući proteini
US10882922B2 (en) 2016-04-13 2021-01-05 Sanofi Trispecific and/or trivalent binding proteins
CN109071634A (zh) 2016-04-26 2018-12-21 R.P.谢勒技术有限责任公司 抗体偶联物及其制备和使用方法
WO2018010789A1 (en) 2016-07-13 2018-01-18 Humabs Biomed Sa Novel antibodies specifically binding to zika virus epitopes and uses thereof
US20200024312A1 (en) 2017-01-25 2020-01-23 Molecular Templates, Inc. Cell-targeting molecules comprising de-immunized, shiga toxin a subunit effectors and cd8+ t-cell epitopes
WO2018147960A1 (en) 2017-02-08 2018-08-16 Imaginab, Inc. Extension sequences for diabodies
JP7278259B2 (ja) 2017-04-19 2023-05-19 インスティテュート・フォー・リサーチ・イン・バイオメディシン ワクチン及び標的としてのプラスモジウムスポロゾイトｎｐｄｐペプチド、新規マラリアワクチン、及びそれに結合する抗体
WO2019024979A1 (en) 2017-07-31 2019-02-07 Institute For Research In Biomedicine FUNCTIONAL DOMAIN ANTIBODIES IN THE ELBOW REGION
WO2019042555A1 (en) 2017-08-31 2019-03-07 Humabs Biomed Sa MULTISPECIFIC ANTIBODIES SPECIFICALLY BINDING TO ZIKA VIRUS EPITOPES AND USES THEREOF
ES2986579T3 (es) * 2017-10-10 2024-11-12 Sanofi Sa Anticuerpos anti-CD38 y combinaciones con anticuerpos anti-CD3 y anti-CD28
CN111246885B (zh) 2017-10-20 2024-06-11 豪夫迈·罗氏有限公司 从单特异性抗体生成多特异性抗体的方法
WO2019086362A1 (en) 2017-10-30 2019-05-09 F. Hoffmann-La Roche Ag Method for in vivo generation of multispecific antibodies from monospecific antibodies
GB2611498B (en) 2018-04-18 2023-07-19 Ucl Business Ltd Composition comprising an engineered regulatory T cell (Treg)
JP7440489B2 (ja) 2018-08-01 2024-02-28 シティ・オブ・ホープ Ｔａｇ７２陽性腫瘍治療のためのｔａｇ７２標的化キメラ抗原受容体修飾ｔ細胞
AU2019331024B2 (en) 2018-08-31 2026-01-15 Regeneron Pharmaceuticals, Inc. Dosing strategy that mitigates cytokine release syndrome for CD3/C20 bispecific antibodies
KR20210075129A (ko) 2018-10-09 2021-06-22 사노피 삼중특이적 항-cd38, 항-cd28, 및 항-cd3 결합 단백질 및 바이러스 감염을 치료하기 위한 사용 방법
WO2020078568A1 (en) 2018-10-19 2020-04-23 Humabs Biomed Sa Antibodies and methods for treatment of lyssavirus infection
TWI874341B (zh) 2018-12-18 2025-03-01 美商健生生物科技公司 產生異二聚體抗體之方法
WO2020132091A2 (en) 2018-12-19 2020-06-25 Humabs Biomed Sa Antibodies that neutralize hepatitis b virus and uses thereof
EA202191736A1 (ru) 2018-12-20 2021-10-01 Вир Байотекнолоджи, Инк. Комбинированная терапия hbv
EP3924387A4 (en) * 2019-02-15 2024-10-02 University Of Southern California LYM-1 AND LYM-2 ANTIBODY COMPOSITIONS AND CONSTRUCTS THEREOF
US11613576B2 (en) 2019-04-09 2023-03-28 Sanofi Trispecific binding proteins, methods, and uses thereof
WO2020221451A1 (en) 2019-04-30 2020-11-05 Humabs Biomed Sa Antibodies binding to plasmodium circumsporozoite protein and uses thereof
BR112021024417A2 (pt) * 2019-06-13 2022-01-18 Regeneron Pharma Métodos para remoção de componentes indesejados durante processos cromatográficos de múltiplos estágios
EP3757127A1 (en) 2019-06-28 2020-12-30 Institute for Research in Biomedicine Deimmunized antibodies binding to alpha-4 integrin and uses thereof
KR20220063188A (ko) 2019-08-29 2022-05-17 비르 바이오테크놀로지, 인코포레이티드 B형 간염 바이러스 감염을 치료하기 위한 항체 조성물 및 방법
CN113416258B (zh) 2019-10-24 2023-08-29 北京免疫方舟医药科技有限公司 一种多特异性抗体及其制备方法和用途
US20210155671A1 (en) 2019-11-24 2021-05-27 Molecular Templates, Inc. Uses of cd20-binding molecules and additional therapeutic agents
GB202007842D0 (en) 2020-05-26 2020-07-08 Quell Therapeutics Ltd Polypeptide useful in adoptive cell therapy
EP4171747A1 (en) 2020-06-24 2023-05-03 VIR Biotechnology, Inc. Engineered hepatitis b virus neutralizing antibodies and uses thereof
GB202013477D0 (en) 2020-08-27 2020-10-14 Quell Therapeutics Ltd Nucleic acid constructs for expressing polypeptides in cells
US20240034772A1 (en) 2020-12-08 2024-02-01 Humabs Biomed Sa Antibodies binding to f-protein of metapneumovirus and uses thereof
JP2024504167A (ja) 2021-01-26 2024-01-30 ヴィア・バイオテクノロジー・インコーポレイテッド Ｂ型肝炎ウイルス感染を処置するための抗体組成物および方法
WO2023036774A1 (en) 2021-09-07 2023-03-16 Institute For Research In Biomedicine Antibodies binding to tetanus toxin and uses thereof

Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5166322A (en) * 1989-04-21 1992-11-24 Genetics Institute Cysteine added variants of interleukin-3 and chemical modifications thereof
US5206344A (en) * 1985-06-26 1993-04-27 Cetus Oncology Corporation Interleukin-2 muteins and polymer conjugation thereof
US5208020A (en) * 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
US5219996A (en) * 1987-09-04 1993-06-15 Celltech Limited Recombinant antibodies and methods for their production in which surface residues are altered to cysteine residues for attachment of effector or receptor molecules
US5318876A (en) * 1991-04-08 1994-06-07 Basf Aktiengesellschaft Radiation-sensitive mixture containing acid-labile groups and production of relief patterns
US5348876A (en) * 1989-07-18 1994-09-20 Dynal As IgG3 antibodies with shortened hinge region and a complement activation test
US5624821A (en) * 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5641870A (en) * 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5677425A (en) * 1987-09-04 1997-10-14 Celltech Therapeutics Limited Recombinant antibody
US5766897A (en) * 1990-06-21 1998-06-16 Incyte Pharmaceuticals, Inc. Cysteine-pegylated proteins
US5821333A (en) * 1995-03-01 1998-10-13 Genetech, Inc. Method for making heteromultimeric polypeptides
US5837821A (en) * 1992-11-04 1998-11-17 City Of Hope Antibody construct
US5877291A (en) * 1992-12-11 1999-03-02 The Dow Chemical Company Multivalent single chain antibodies
US5976845A (en) * 1990-04-19 1999-11-02 The Dow Chemical Company Composite antibodies of human subgroup IV light chain capable of binding to TAG-72
US6011138A (en) * 1997-02-20 2000-01-04 Idec Pharmaceuticals Corporation Gamma-1 anti-human CD23 monoclonal antibodies
US6063905A (en) * 1997-01-07 2000-05-16 Board Of Regents, The University Of Texas System Recombinant human IGA-J. chain dimer
US6071515A (en) * 1992-08-21 2000-06-06 The Dow Chemical Company Dimer and multimer forms of single chain polypeptides
US6207815B1 (en) * 1988-10-19 2001-03-27 The Dow Chemical Company Family of high affinity, modified antibodies for cancer treatment
US6218149B1 (en) * 1988-09-15 2001-04-17 The Trustees Of Columbus University In The City Of New York Antibodies having modified carbohydrate content and methods of preparation and use
US6284536B1 (en) * 1998-04-20 2001-09-04 The Regents Of The University Of California Modified immunoglobin molecules and methods for use thereof
US20020009446A1 (en) * 1998-08-31 2002-01-24 Daniel Magilavy Method of modulating memory effector T-cells and compositions
US6348581B1 (en) * 1996-10-31 2002-02-19 The Dow Chemical Company High affinity humanized anti-TAG-72 monoclonalantibodies
US20020062010A1 (en) * 1997-05-02 2002-05-23 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
US6495137B1 (en) * 1990-04-19 2002-12-17 The Dow Chemical Company Humanized anti-tag-72 monoclonal antibodies using human subgroup 4 kappa light chains
US20030060444A1 (en) * 1997-06-25 2003-03-27 Celltech Therapeutics, Ltd. Cell activation process and reagents therefor
US6552170B1 (en) * 1990-04-06 2003-04-22 Amgen Inc. PEGylation reagents and compounds formed therewith
US6576746B2 (en) * 1998-10-13 2003-06-10 Immunomedics, Inc. Site-specific labeling of disulfide-containing targeting vectors
US20030207346A1 (en) * 1997-05-02 2003-11-06 William R. Arathoon Method for making multispecific antibodies having heteromultimeric and common components
US6737056B1 (en) * 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US20040101905A1 (en) * 2000-12-01 2004-05-27 Brekke Ole Henrik Andre Product
US6759518B1 (en) * 1998-04-09 2004-07-06 Vectron Therapeutics Ag Single-chain multiple antigen-binding molecule, its preparation and use
US20050097625A1 (en) * 2002-11-27 2005-05-05 Meade Harry M. Modified antibodies stably produced in milk and methods of producing same

Family Cites Families (85)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4485045A (en) * 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4816567A (en) * 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4544545A (en) * 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
NZ212207A (en) 1984-05-31 1991-07-26 Genentech Inc Recombinant lymphotoxin
US5618920A (en) * 1985-11-01 1997-04-08 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
GB8529014D0 (en) * 1985-11-25 1986-01-02 Biogen Nv Enhanced secretion of heterologous proteins
EP0284898A3 (de) * 1987-04-02 1990-06-27 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Hinge-Peptid, Verfahren zu seiner Herstellung und seine Verwendung zur Herstellung synthetischer Immunogene
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
JP3095168B2 (ja) 1988-02-05 2000-10-03 エル． モリソン，シェリー ドメイン‐変性不変部を有する抗体
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5714149A (en) * 1989-02-10 1998-02-03 Celltech Therapeutics Limited Crosslinked antibodies and processes for their preparation
US5225538A (en) * 1989-02-23 1993-07-06 Genentech, Inc. Lymphocyte homing receptor/immunoglobulin fusion proteins
US5859205A (en) * 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
GB8928874D0 (en) 1989-12-21 1990-02-28 Celltech Ltd Humanised antibodies
GB9009106D0 (en) * 1990-04-23 1990-06-20 3I Res Expl Ltd Processes and intermediates for synthetic antibody derivatives
US5670149A (en) * 1990-06-27 1997-09-23 Biogen, Inc. Lymphotoxin-β, Lymphotoxin-β complexes, pharmaceutical preparations and therapeutic uses thereof
US7030080B2 (en) * 1990-06-27 2006-04-18 Biogen, Inc. Lymphotoxin-β, lymphotoxin-β complexes, pharmaceutical preparations and therapeutic uses thereof
AU8228891A (en) * 1990-06-27 1992-01-23 Biogen, Inc. Surface complexed lymphotoxin
EP0519596B1 (en) 1991-05-17 2005-02-23 Merck & Co. Inc. A method for reducing the immunogenicity of antibody variable domains
DE4118120A1 (de) * 1991-06-03 1992-12-10 Behringwerke Ag Tetravalente bispezifische rezeptoren, ihre herstellung und verwendung
WO1994004679A1 (en) 1991-06-14 1994-03-03 Genentech, Inc. Method for making humanized antibodies
US20020037558A1 (en) * 1991-10-23 2002-03-28 Kin-Ming Lo E.coli produced immunoglobulin constructs
EP0672143B1 (en) 1992-12-04 2008-06-04 Biogen Idec MA Inc. Lymphotoxin-beta, lymphotoxin-beta complexes, pharmaceutical preparations and therapeutic uses thereof
IL108776A (en) 1993-03-11 1999-11-30 Genentech Inc A method of dissolving a cell or inhibiting cell growth, a bispecific molecule that binds FNT-1R and an SAF antibody, a medical preparation that contains it and its use
JPH08511420A (ja) 1993-06-16 1996-12-03 セルテック・セラピューテイクス・リミテッド 抗 体
WO1995009917A1 (en) 1993-10-07 1995-04-13 The Regents Of The University Of California Genetically engineered bispecific tetravalent antibodies
US5429746A (en) 1994-02-22 1995-07-04 Smith Kline Beecham Corporation Antibody purification
ES2220972T3 (es) 1995-01-26 2004-12-16 Biogen, Inc. Complejos de linfotoxinas-alfa/beta y anticuerpos de los receptores de anti-linfotoxina-beta como agentes antitumorales.
US6312691B1 (en) * 1996-01-26 2001-11-06 Jeffrey L. Browning Lymphotoxin-α/β complexes and anti-lympotoxin-β receptor antibodies as anti-tumor agents
US5925351A (en) * 1995-07-21 1999-07-20 Biogen, Inc. Soluble lymphotoxin-β receptors and anti-lymphotoxin receptor and ligand antibodies as therapeutic agents for the treatment of immunological disease
WO1997011370A1 (en) * 1995-09-22 1997-03-27 Immunomedics, Inc. Recombinant proteins having multiple disulfide bonds and thiol-substituted conjugates thereof
US5834597A (en) 1996-05-20 1998-11-10 Protein Design Labs, Inc. Mutated nonactivating IgG2 domains and anti CD3 antibodies incorporating the same
WO1998005787A1 (en) 1996-08-02 1998-02-12 Bristol-Myers Squibb Company A method for inhibiting immunoglobulin-induced toxicity resulting from the use of immunoglobulins in therapy and in vivo diagnosis
US7247302B1 (en) * 1996-08-02 2007-07-24 Bristol-Myers Squibb Company Method for inhibiting immunoglobulin-induced toxicity resulting from the use of immunoglobulins in therapy and in vivo diagnosis
US7255854B1 (en) * 1996-10-25 2007-08-14 Biogen, Inc. Use of lymphotoxin-β receptor blocking agents for the treatment of antibody mediated immunological diseases
WO1998023289A1 (en) 1996-11-27 1998-06-04 The General Hospital Corporation MODULATION OF IgG BINDING TO FcRn
RO120148B1 (ro) * 1997-03-14 2005-09-30 Idec Pharmaceuticals Corporation Sistem vector şi procedeu de inserţie a unui fragment adn, în genomul celulelor de mamifere
EP1724282B1 (en) 1997-05-21 2013-05-15 Merck Patent GmbH Method for the production of non-immunogenic proteins
US6165476A (en) * 1997-07-10 2000-12-26 Beth Israel Deaconess Medical Center Fusion proteins with an immunoglobulin hinge region linker
AUPO930697A0 (en) 1997-09-19 1997-10-09 Walter And Eliza Hall Institute Of Medical Research, The Catalytic antibodies and a method of producing same
GB9720054D0 (en) * 1997-09-19 1997-11-19 Celltech Therapeutics Ltd Biological products
US7026446B1 (en) * 1997-12-24 2006-04-11 Diatech Pty Ltd. Bifunctional molecules
NZ506529A (en) 1998-01-30 2002-12-20 Biogen Inc Treatment of follicular lymphomas using inhibitors of the lymphotoxin (LT) pathway
US7060667B1 (en) * 1998-01-30 2006-06-13 Biogen Idec Ma, Inc. Treatment of follicular lymphomas using inhibitors of the LT pathway
GB9809951D0 (en) 1998-05-08 1998-07-08 Univ Cambridge Tech Binding molecules
GB9809839D0 (en) 1998-05-09 1998-07-08 Glaxo Group Ltd Antibody
HUP0103773A3 (en) * 1998-10-09 2003-10-28 Biogen Idec Ma Inc Cambridge Reversal of viral-induced systemic schock and respiratory distress by blockade of the lymphotoxin beta pathway
DE69934967T2 (de) 1998-12-08 2007-12-06 Biovation Ltd. Verfahren zur verminderung der immunogenität von proteinen
AU3316600A (en) * 1999-02-22 2000-09-21 Torben F. Orntoft Gene expression in bladder tumors
AU778199B2 (en) 1999-10-29 2004-11-25 Genentech Inc. Anti-prostate stem cell antigen (PSCA) antibody compositions and methods of use
CN100455599C (zh) * 2000-03-03 2009-01-28 协和发酵工业株式会社 基因重组抗体及其抗体片段
EP1294904A1 (en) 2000-06-30 2003-03-26 Vlaams Interuniversitair Instituut voor Biotechnologie vzw. Heterodimeric fusion proteins
GB0020953D0 (en) 2000-08-24 2000-10-11 Smithkline Beecham Biolog Vaccine
DE10045591A1 (de) * 2000-09-15 2002-04-04 Klaus Pfizenmaier Ortsspezifische, antikörpervermittelte Aktivierung proapoptotischer Zytokine - AMAIZe (Antibody-Mediated Apoptosis Inducing Zytokine)
AR035352A1 (es) 2000-10-13 2004-05-12 Biogen Inc Anticuerpos anti-lt-beta-r humanizados
WO2002096948A2 (en) 2001-01-29 2002-12-05 Idec Pharmaceuticals Corporation Engineered tetravalent antibodies and methods of use
CA2436092A1 (en) 2001-01-29 2002-08-08 Idec Pharmaceutical Corporation Modified antibodies and methods of use
CA2438682A1 (en) 2001-02-20 2002-08-29 Zymogenetics, Inc. Antibodies that bind both bcma and taci
AUPR395801A0 (en) * 2001-03-26 2001-04-26 Austin Research Institute, The Antibodies against cancer
US20020155109A1 (en) 2001-04-20 2002-10-24 Lynch David H. Bispecific antibodies that bind TRAIL-R1 and TRAIL-R2
SK14432003A3 (sk) 2001-04-26 2004-07-07 Biogen, Inc. Protilátka špecifická pre Cripto, kompozícia obsahujúca takúto protilátku a jej použitie
ATE486092T1 (de) 2001-09-18 2010-11-15 Genentech Inc Zusammensetzungen und verfahren für die diagnose von tumoren
US20040014690A1 (en) * 2002-02-13 2004-01-22 Zhenkun Ma Macrolides with activity against methicillin-resistant staphylococcus aureus
US20040132101A1 (en) 2002-09-27 2004-07-08 Xencor Optimized Fc variants and methods for their generation
US7317091B2 (en) * 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
MY139983A (en) * 2002-03-12 2009-11-30 Janssen Alzheimer Immunotherap Humanized antibodies that recognize beta amyloid peptide
JP2005520566A (ja) 2002-03-22 2005-07-14 バイオジェン・アイデック・エムエイ・インコーポレイテッド Ｃｒｉｐｔｏ特異的抗体
DE60305919T2 (de) 2002-06-28 2007-01-18 Domantis Limited, Cambridge Dual-specifische liganden mit erhöhter halbwertszeit
AU2003248782A1 (en) * 2002-07-01 2004-01-19 Biogen Idec Ma Inc. Humanized anti-lymphotoxin beta receptor antibodies
JP2006508062A (ja) * 2002-09-17 2006-03-09 ジーティーシー バイオセラピューティックス インコーポレイテッド 重鎖間ジスルフィド結合を欠く免疫グロブリン分子の単離
DK1553975T3 (da) 2002-09-27 2012-05-07 Xencor Inc Optimerede Fc-varianter og fremgangsmåder til generering heraf.
AU2003287345A1 (en) 2002-10-31 2004-06-07 Genentech, Inc. Methods and compositions for increasing antibody production
CN100473664C (zh) * 2002-12-20 2009-04-01 拜奥根Idec马萨诸塞公司 多价淋巴毒素β受体激动剂及其治疗用途
RS20050481A (sr) * 2002-12-20 2007-08-03 Biogen Idec Ma Inc., Agensi limfotoksin beta receptora u kombinaciji sa hemoterapeutskim agensima
WO2004074434A2 (en) 2003-01-31 2004-09-02 Immunomedics, Inc. Methods and compositions for administering therapeutic and diagnostic agents
US20070041972A1 (en) 2003-05-30 2007-02-22 Alexion Pharmaceuticals, Inc. Antibodies and fusion proteins that include engineered constant regions
JP5068072B2 (ja) * 2003-06-27 2012-11-07 バイオジェン・アイデック・エムエイ・インコーポレイテッド 連結ペプチドを含む改変された結合分子
US20050152894A1 (en) * 2003-09-05 2005-07-14 Genentech, Inc. Antibodies with altered effector functions
ATE437950T1 (de) * 2003-09-15 2009-08-15 Res Dev Foundation Cripto-antagonismus von activin und tgf-b signalisierung
CA2560742A1 (en) * 2004-03-23 2005-10-06 Biogen Idec Ma Inc. Receptor coupling agents and therapeutic uses thereof
US7276585B2 (en) * 2004-03-24 2007-10-02 Xencor, Inc. Immunoglobulin variants outside the Fc region
WO2006074399A2 (en) * 2005-01-05 2006-07-13 Biogen Idec Ma Inc. Multispecific binding molecules comprising connecting peptides
WO2006074397A2 (en) 2005-01-05 2006-07-13 Biogen Idec Ma Inc. Cripto binding molecules
GB0514779D0 (en) 2005-07-19 2005-08-24 Celltech R&D Ltd Biological products
JP2009535033A (ja) 2006-04-28 2009-10-01 バイオジェン・アイデック・エムエイ・インコーポレイテッド Ｃｒｉｐｔｏ−３の検出のための組成物および方法

Patent Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5206344A (en) * 1985-06-26 1993-04-27 Cetus Oncology Corporation Interleukin-2 muteins and polymer conjugation thereof
US5624821A (en) * 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5219996A (en) * 1987-09-04 1993-06-15 Celltech Limited Recombinant antibodies and methods for their production in which surface residues are altered to cysteine residues for attachment of effector or receptor molecules
US5677425A (en) * 1987-09-04 1997-10-14 Celltech Therapeutics Limited Recombinant antibody
US20020028486A1 (en) * 1988-09-15 2002-03-07 The Trustees Of Columbia University In The City Of New York Antibodies having modified carbohydrate content and methods of preparation and use
US6218149B1 (en) * 1988-09-15 2001-04-17 The Trustees Of Columbus University In The City Of New York Antibodies having modified carbohydrate content and methods of preparation and use
US6207815B1 (en) * 1988-10-19 2001-03-27 The Dow Chemical Company Family of high affinity, modified antibodies for cancer treatment
US5166322A (en) * 1989-04-21 1992-11-24 Genetics Institute Cysteine added variants of interleukin-3 and chemical modifications thereof
US5348876A (en) * 1989-07-18 1994-09-20 Dynal As IgG3 antibodies with shortened hinge region and a complement activation test
US5208020A (en) * 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
US6552170B1 (en) * 1990-04-06 2003-04-22 Amgen Inc. PEGylation reagents and compounds formed therewith
US5976845A (en) * 1990-04-19 1999-11-02 The Dow Chemical Company Composite antibodies of human subgroup IV light chain capable of binding to TAG-72
US6495137B1 (en) * 1990-04-19 2002-12-17 The Dow Chemical Company Humanized anti-tag-72 monoclonal antibodies using human subgroup 4 kappa light chains
US5766897A (en) * 1990-06-21 1998-06-16 Incyte Pharmaceuticals, Inc. Cysteine-pegylated proteins
US5318876A (en) * 1991-04-08 1994-06-07 Basf Aktiengesellschaft Radiation-sensitive mixture containing acid-labile groups and production of relief patterns
US6329507B1 (en) * 1992-08-21 2001-12-11 The Dow Chemical Company Dimer and multimer forms of single chain polypeptides
US6071515A (en) * 1992-08-21 2000-06-06 The Dow Chemical Company Dimer and multimer forms of single chain polypeptides
US5837821A (en) * 1992-11-04 1998-11-17 City Of Hope Antibody construct
US5877291A (en) * 1992-12-11 1999-03-02 The Dow Chemical Company Multivalent single chain antibodies
US5821333A (en) * 1995-03-01 1998-10-13 Genetech, Inc. Method for making heteromultimeric polypeptides
US5641870A (en) * 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US6066719A (en) * 1995-04-20 2000-05-23 Genetech, Inc. Antibody fragments
US20020002271A1 (en) * 1995-04-20 2002-01-03 Genentech, Inc. Antibody purification
US6214984B1 (en) * 1995-04-20 2001-04-10 Genentech, Inc. Isolated nucleic acid encoding, and methods for preparing, antibody fragments
US6348581B1 (en) * 1996-10-31 2002-02-19 The Dow Chemical Company High affinity humanized anti-TAG-72 monoclonalantibodies
US6063905A (en) * 1997-01-07 2000-05-16 Board Of Regents, The University Of Texas System Recombinant human IGA-J. chain dimer
US6011138A (en) * 1997-02-20 2000-01-04 Idec Pharmaceuticals Corporation Gamma-1 anti-human CD23 monoclonal antibodies
US20020062010A1 (en) * 1997-05-02 2002-05-23 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
US20030207346A1 (en) * 1997-05-02 2003-11-06 William R. Arathoon Method for making multispecific antibodies having heteromultimeric and common components
US20030078385A1 (en) * 1997-05-02 2003-04-24 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
US20030060444A1 (en) * 1997-06-25 2003-03-27 Celltech Therapeutics, Ltd. Cell activation process and reagents therefor
US6759518B1 (en) * 1998-04-09 2004-07-06 Vectron Therapeutics Ag Single-chain multiple antigen-binding molecule, its preparation and use
US6284536B1 (en) * 1998-04-20 2001-09-04 The Regents Of The University Of California Modified immunoglobin molecules and methods for use thereof
US20020009446A1 (en) * 1998-08-31 2002-01-24 Daniel Magilavy Method of modulating memory effector T-cells and compositions
US6576746B2 (en) * 1998-10-13 2003-06-10 Immunomedics, Inc. Site-specific labeling of disulfide-containing targeting vectors
US6737056B1 (en) * 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US20040101905A1 (en) * 2000-12-01 2004-05-27 Brekke Ole Henrik Andre Product
US20050097625A1 (en) * 2002-11-27 2005-05-05 Meade Harry M. Modified antibodies stably produced in milk and methods of producing same

Cited By (211)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8853366B2 (en) 2001-01-17 2014-10-07 Emergent Product Development Seattle, Llc Binding domain-immunoglobulin fusion proteins
US8603473B2 (en) * 2003-06-27 2013-12-10 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
US20090041758A1 (en) * 2003-06-27 2009-02-12 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
US8021661B2 (en) 2005-01-05 2011-09-20 Biogen Idec Ma Inc. Cripto binding molecules
US10071158B2 (en) 2005-04-26 2018-09-11 Lindis Biotech Gmbh Combination of the application of antibodies for immunostimulation together with glucocorticoids
US10576149B2 (en) 2005-04-26 2020-03-03 Lindis Biotech Gmbh Combination of the application of antibodies for immunostimulation together with glucocorticoids
US10143748B2 (en) 2005-07-25 2018-12-04 Aptevo Research And Development Llc B-cell reduction using CD37-specific and CD20-specific binding molecules
US10307481B2 (en) 2005-07-25 2019-06-04 Aptevo Research And Development Llc CD37 immunotherapeutics and uses thereof
US7960142B2 (en) 2006-03-17 2011-06-14 Biogen Idec Ma Inc. Stabilized polypeptide compositions
US20090048122A1 (en) * 2006-03-17 2009-02-19 Biogen Idec Ma Inc. Stabilized polypeptide compositions
US8892364B2 (en) 2006-03-17 2014-11-18 Biogen Idec Ma Inc. Stabilized polypeptide compositions
US7951918B2 (en) 2006-03-17 2011-05-31 Biogen Idec Ma Inc. Stabilized polypeptide compositions
US20140154250A1 (en) * 2006-06-12 2014-06-05 Emergent Product Development Seattle, Llc Single-chain multivalent binding proteins with effector function
US8409577B2 (en) 2006-06-12 2013-04-02 Emergent Product Development Seattle, Llc Single chain multivalent binding proteins with effector function
US20140154252A1 (en) * 2006-06-12 2014-06-05 Emergent Product Development Seattle, Llc Single-chain multivalent binding proteins with effector function
AU2014200661B2 (en) * 2006-06-12 2016-06-30 Aptevo Research And Development Llc Single-chain multivalent binding proteins with effector function
AU2014200661A1 (en) * 2006-06-12 2014-03-13 Aptevo Research And Development Llc Single-chain multivalent binding proteins with effector function
US11155631B2 (en) 2006-08-18 2021-10-26 Armagen, Inc. Macromolecular compositions that cross the blood-brain barrier and methods of use thereof
US11512145B2 (en) 2007-07-27 2022-11-29 Armagen, Inc. Methods and compositions for increasing alpha-L-iduronidase activity in the CNS
US9266967B2 (en) 2007-12-21 2016-02-23 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
US10927163B2 (en) 2007-12-21 2021-02-23 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
US10138293B2 (en) 2007-12-21 2018-11-27 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
US9101609B2 (en) 2008-04-11 2015-08-11 Emergent Product Development Seattle, Llc CD37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof
EP2781526A1 (en) 2008-10-08 2014-09-24 F. Hoffmann-La Roche AG Bispecific anti-VEGF/anti-ANG-2 antibodies
EP2792687A1 (en) 2008-10-08 2014-10-22 F. Hoffmann-La Roche AG Bispecific anti-VEGF/anti-ANG-2 antibodies
WO2010040508A1 (en) 2008-10-08 2010-04-15 F. Hoffmann-La Roche Ag Bispecific anti-vegf/anti-ang-2 antibodies
WO2010085682A3 (en) * 2009-01-23 2010-09-30 Biogen Idec Ma Inc. Stabilized fc polypeptides with reduced effector function and methods of use
WO2010112193A1 (en) 2009-04-02 2010-10-07 Roche Glycart Ag Multispecific antibodies comprising full length antibodies and single chain fab fragments
US20100256338A1 (en) * 2009-04-02 2010-10-07 Ulrich Brinkmann Multispecific antibodies comprising full length antibodies and single chain fab fragments
US9382323B2 (en) 2009-04-02 2016-07-05 Roche Glycart Ag Multispecific antibodies comprising full length antibodies and single chain fab fragments
WO2010112194A1 (en) 2009-04-02 2010-10-07 F. Hoffmann-La Roche Ag Antigen-binding polypeptides and multispecific antibodies comprising them
WO2010115553A1 (en) 2009-04-07 2010-10-14 Roche Glycart Ag Bispecific anti-erbb-2/anti-c-met antibodies
WO2010115589A1 (en) 2009-04-07 2010-10-14 Roche Glycart Ag Trivalent, bispecific antibodies
WO2010115552A1 (en) 2009-04-07 2010-10-14 Roche Glycart Ag Bispecific anti-erbb-3/anti-c-met antibodies
US11993642B2 (en) 2009-04-07 2024-05-28 Hoffmann-La Roche Inc. Trivalent, bispecific antibodies
WO2010115551A1 (en) 2009-04-07 2010-10-14 Roche Glycart Ag Bispecific anti-erbb-1/anti-c-met antibodies
US9890204B2 (en) 2009-04-07 2018-02-13 Hoffmann-La Roche Inc. Trivalent, bispecific antibodies
US11673945B2 (en) 2009-06-16 2023-06-13 Hoffmann-La Roche Inc. Bispecific antigen binding proteins
US9676845B2 (en) 2009-06-16 2017-06-13 Hoffmann-La Roche, Inc. Bispecific antigen binding proteins
US10640555B2 (en) 2009-06-16 2020-05-05 Hoffmann-La Roche Inc. Bispecific antigen binding proteins
US9994646B2 (en) 2009-09-16 2018-06-12 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
US11028156B2 (en) 2009-10-09 2021-06-08 Armagen, Inc. Methods and compositions for increasing iduronate 2-sulfatase activity in the CNS
US12043661B2 (en) 2009-10-09 2024-07-23 Armagen, Inc. Methods and compositions for increasing iduronate 2-sulfatase activity in the CNS
EP3348277A1 (en) 2009-11-20 2018-07-18 The Regents of The University of California Epithelial membrane protein-2 (emp2) and proliferative vitreoretinopathy (pvr)
WO2011119888A2 (en) 2010-03-24 2011-09-29 The Regents Of The University Of California N-cadherin: target for cancer diagnosis and therapy
US10106600B2 (en) 2010-03-26 2018-10-23 Roche Glycart Ag Bispecific antibodies
WO2011117329A1 (en) 2010-03-26 2011-09-29 F. Hoffmann-La Roche Ag Bispecific, bivalent anti-vegf/anti-ang-2 antibodies
WO2011117330A1 (en) 2010-03-26 2011-09-29 Roche Glycart Ag Bispecific antibodies
US9133524B2 (en) 2010-07-01 2015-09-15 The Regents Of The University Of California Protein kinase ck2 gene mutations, amplifications and polymorphisms in human cancers and methods of use
WO2012025530A1 (en) 2010-08-24 2012-03-01 F. Hoffmann-La Roche Ag Bispecific antibodies comprising a disulfide stabilized - fv fragment
US9879095B2 (en) 2010-08-24 2018-01-30 Hoffman-La Roche Inc. Bispecific antibodies comprising a disulfide stabilized-Fv fragment
EP2638073A4 (en) * 2010-11-09 2014-05-07 Altimab Therapeutics Inc PROTEIN COMPLEXES FOR ANTIGEN BINDING, AND METHODS OF USE
US9518132B2 (en) 2010-11-09 2016-12-13 Altimab Therapeutics, Inc. Protein complexes for antigen binding and methods of use
WO2012085111A1 (en) 2010-12-23 2012-06-28 F. Hoffmann-La Roche Ag Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery
US11618790B2 (en) 2010-12-23 2023-04-04 Hoffmann-La Roche Inc. Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery
US9982036B2 (en) 2011-02-28 2018-05-29 Hoffmann-La Roche Inc. Dual FC antigen binding proteins
US10611825B2 (en) 2011-02-28 2020-04-07 Hoffmann La-Roche Inc. Monovalent antigen binding proteins
US10793621B2 (en) 2011-02-28 2020-10-06 Hoffmann-La Roche Inc. Nucleic acid encoding dual Fc antigen binding proteins
US9688758B2 (en) 2012-02-10 2017-06-27 Genentech, Inc. Single-chain antibodies and other heteromultimers
WO2013150043A1 (en) 2012-04-05 2013-10-10 F. Hoffmann-La Roche Ag Bispecific antibodies against human tweak and human il17 and uses thereof
US9714292B2 (en) 2012-04-05 2017-07-25 Hoffmann-La Roche Inc. Bispecific antibodies against human TWEAK and human IL17 and uses thereof
EP4053162A1 (en) * 2012-05-18 2022-09-07 Aptevo Research and Development LLC Bispecific scfv immunofusion (bif) binding to cd123 and cd3
EP2850106B1 (en) * 2012-05-18 2022-03-23 Aptevo Research and Development LLC Bispecific scfv immunofusion (bif) binding to cd123 and cd3
WO2014001326A1 (en) 2012-06-27 2014-01-03 F. Hoffmann-La Roche Ag Method for the selection and production of tailor-made, selective and multi-specific therapeutic molecules comprising at least two different targeting entities and uses thereof
US11407836B2 (en) 2012-06-27 2022-08-09 Hoffmann-La Roche Inc. Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof
US10106612B2 (en) 2012-06-27 2018-10-23 Hoffmann-La Roche Inc. Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof
US11421022B2 (en) 2012-06-27 2022-08-23 Hoffmann-La Roche Inc. Method for making antibody Fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof
US10517945B2 (en) 2012-07-04 2019-12-31 Hoffman-La Roche Inc. Covalently linked antigen-antibody conjugates
WO2014006124A1 (en) 2012-07-04 2014-01-09 F. Hoffmann-La Roche Ag Covalently linked antigen-antibody conjugates
US12023378B2 (en) 2012-07-04 2024-07-02 Hoffmann-La Roche Inc. Covalently linked antigen-antibody conjugates
US9925272B2 (en) 2012-07-04 2018-03-27 Hoffmann-La Roche Inc. Anti-theophylline antibodies and methods of use
US9765153B2 (en) 2012-07-04 2017-09-19 Hoffmann-La Roche Inc. Anti-biotin antibodies and methods of use
US9567403B2 (en) 2013-08-06 2017-02-14 Bio-Thera Solutions, Ltd. Bispecific antibodies which bind EGFR and VEGF
US10323099B2 (en) 2013-10-11 2019-06-18 Hoffmann-La Roche Inc. Multispecific domain exchanged common variable light chain antibodies
US10465000B2 (en) 2013-12-20 2019-11-05 Hoffmann-La Roche Inc. Humanized anti-Tau(pS422) antibodies and methods of use
US10519249B2 (en) 2014-01-03 2019-12-31 Hoffmann-La Roche Inc. Covalently linked polypeptide toxin-antibody conjugates
US10407511B2 (en) 2014-01-03 2019-09-10 Hoffmann-La Roche Inc. Covalently linked helicar-anti-helicar antibody conjugates and uses thereof
WO2015101589A1 (en) 2014-01-03 2015-07-09 F. Hoffmann-La Roche Ag Covalently linked polypeptide toxin-antibody conjugates
WO2015101586A1 (en) 2014-01-03 2015-07-09 F. Hoffmann-La Roche Ag Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles
WO2015101587A1 (en) 2014-01-03 2015-07-09 F. Hoffmann-La Roche Ag Covalently linked helicar-anti-helicar antibody conjugates and uses thereof
US10561737B2 (en) 2014-01-03 2020-02-18 Hoffmann-La Roche Inc. Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles
EP3514179A1 (en) 2014-01-24 2019-07-24 Dana-Farber Cancer Institute, Inc. Antibody molecules to pd-1 and uses thereof
EP4324518A2 (en) 2014-01-31 2024-02-21 Novartis AG Antibody molecules to tim-3 and uses thereof
WO2015138920A1 (en) 2014-03-14 2015-09-17 Novartis Ag Antibody molecules to lag-3 and uses thereof
EP3660050A1 (en) 2014-03-14 2020-06-03 Novartis AG Antibody molecules to lag-3 and uses thereof
WO2015142675A2 (en) 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor
US10251952B2 (en) 2014-06-26 2019-04-09 Hoffmann-La Roche Inc. Humanized anti-tau(pS422) antibody brain shuttles and use thereof
EP3722316A1 (en) 2014-07-21 2020-10-14 Novartis AG Treatment of cancer using a cd33 chimeric antigen receptor
WO2016014553A1 (en) 2014-07-21 2016-01-28 Novartis Ag Sortase synthesized chimeric antigen receptors
WO2016014565A2 (en) 2014-07-21 2016-01-28 Novartis Ag Treatment of cancer using humanized anti-bcma chimeric antigen receptor
WO2016014530A1 (en) 2014-07-21 2016-01-28 Novartis Ag Combinations of low, immune enhancing. doses of mtor inhibitors and cars
EP3660042A1 (en) 2014-07-31 2020-06-03 Novartis AG Subset-optimized chimeric antigen receptor-containing t-cells
EP4205749A1 (en) 2014-07-31 2023-07-05 Novartis AG Subset-optimized chimeric antigen receptor-containing cells
WO2016025880A1 (en) 2014-08-14 2016-02-18 Novartis Ag Treatment of cancer using gfr alpha-4 chimeric antigen receptor
EP3712171A1 (en) 2014-08-19 2020-09-23 Novartis AG Treatment of cancer using a cd123 chimeric antigen receptor
WO2016044605A1 (en) 2014-09-17 2016-03-24 Beatty, Gregory Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
EP3967709A1 (en) 2014-09-17 2022-03-16 Novartis AG Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
US12070502B2 (en) 2014-10-09 2024-08-27 Regeneron Pharmaceuticals, Inc. Process for reducing subvisible particles in a pharmaceutical formulation
EP4245376A2 (en) 2014-10-14 2023-09-20 Novartis AG Antibody molecules to pd-l1 and uses thereof
WO2016061142A1 (en) 2014-10-14 2016-04-21 Novartis Ag Antibody molecules to pd-l1 and uses thereof
US10633457B2 (en) 2014-12-03 2020-04-28 Hoffmann-La Roche Inc. Multispecific antibodies
US11999801B2 (en) 2014-12-03 2024-06-04 Hoffman-La Roche Inc. Multispecific antibodies
WO2016090034A2 (en) 2014-12-03 2016-06-09 Novartis Ag Methods for b cell preconditioning in car therapy
US10538589B2 (en) * 2015-01-14 2020-01-21 Armagen Inc. Methods and compositions for increasing N-acetylglucosaminidase (NAGLU) activity in the CNS using a fusion antibody comprising an anti-human insulin receptor antibody and NAGLU
EP4056588A1 (en) 2015-04-08 2022-09-14 Novartis AG Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car)- expressing cell
EP4491715A2 (en) 2015-04-08 2025-01-15 Novartis AG Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car)- expressing cell
WO2016164731A2 (en) 2015-04-08 2016-10-13 Novartis Ag Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car) - expressing cell
WO2016172583A1 (en) 2015-04-23 2016-10-27 Novartis Ag Treatment of cancer using chimeric antigen receptor and protein kinase a blocker
US10822402B2 (en) 2015-06-24 2020-11-03 Hoffmann-La Roche Inc. Humanized anti-tau(pS422) antibodies and methods of use
WO2017019894A1 (en) 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to lag-3
EP3964528A1 (en) 2015-07-29 2022-03-09 Novartis AG Combination therapies comprising antibody molecules to lag-3
EP4378957A2 (en) 2015-07-29 2024-06-05 Novartis AG Combination therapies comprising antibody molecules to pd-1
EP3878465A1 (en) 2015-07-29 2021-09-15 Novartis AG Combination therapies comprising antibody molecules to tim-3
WO2017019897A1 (en) 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to tim-3
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
EP4424322A2 (en) 2015-12-17 2024-09-04 Novartis AG Antibody molecules to pd-1 and uses thereof
WO2017106656A1 (en) 2015-12-17 2017-06-22 Novartis Ag Antibody molecules to pd-1 and uses thereof
WO2017106810A2 (en) 2015-12-17 2017-06-22 Novartis Ag Combination of c-met inhibitor with antibody molecule to pd-1 and uses thereof
EP4643874A2 (en) 2015-12-22 2025-11-05 Novartis AG Mesothelin chimeric antigen receptor (car) and antibody against pd-l1 inhibitor for combined use in anticancer therapy
EP3851457A1 (en) 2016-01-21 2021-07-21 Novartis AG Multispecific molecules targeting cll-1
WO2017125897A1 (en) 2016-01-21 2017-07-27 Novartis Ag Multispecific molecules targeting cll-1
WO2017149515A1 (en) 2016-03-04 2017-09-08 Novartis Ag Cells expressing multiple chimeric antigen receptor (car) molecules and uses therefore
WO2017165683A1 (en) 2016-03-23 2017-09-28 Novartis Ag Cell secreted minibodies and uses thereof
EP4219721A2 (en) 2016-04-15 2023-08-02 Novartis AG Compositions and methods for selective protein expression
WO2017181119A2 (en) 2016-04-15 2017-10-19 Novartis Ag Compositions and methods for selective protein expression
WO2017210617A2 (en) 2016-06-02 2017-12-07 Porter, David, L. Therapeutic regimens for chimeric antigen receptor (car)- expressing cells
US12098215B2 (en) 2016-06-06 2024-09-24 Northwestern University Fusion protein construct
WO2018013918A2 (en) 2016-07-15 2018-01-18 Novartis Ag Treatment and prevention of cytokine release syndrome using a chimeric antigen receptor in combination with a kinase inhibitor
WO2018023025A1 (en) 2016-07-28 2018-02-01 Novartis Ag Combination therapies of chimeric antigen receptors adn pd-1 inhibitors
WO2018026819A2 (en) 2016-08-01 2018-02-08 Novartis Ag Treatment of cancer using a chimeric antigen receptor in combination with an inhibitor of a pro-m2 macrophage molecule
US11369896B2 (en) 2016-08-16 2022-06-28 Regeneron Pharmaceuticals, Inc. Methods for quantitating individual antibodies from a mixture
US11571636B2 (en) 2016-08-16 2023-02-07 Regeneron Pharmaceuticals, Inc. Methods for quantitating individual antibodies from a mixture
US11850535B2 (en) 2016-08-16 2023-12-26 Regeneron Pharmaceuticals, Inc. Methods for quantitating individual antibodies from a mixture
US12441798B2 (en) 2016-09-21 2025-10-14 Aptevo Research And Development Llc CD123 binding proteins and related compositions and methods
US10676533B2 (en) 2016-09-21 2020-06-09 Aptevo Research And Development Llc Methods of treatment of CD123 overexpressing disorders
US11939392B2 (en) 2016-09-21 2024-03-26 Aptevo Research And Development Llc CD123 binding proteins and related compositions and methods
US11242400B2 (en) 2016-09-21 2022-02-08 Aptevo Research And Development Llc CD123 binding proteins and related compositions and methods
WO2018067992A1 (en) 2016-10-07 2018-04-12 Novartis Ag Chimeric antigen receptors for the treatment of cancer
US11680930B2 (en) 2016-10-25 2023-06-20 Regeneron Pharmaceuticals, Inc. Methods and systems for chromatography data analysis
US11333642B2 (en) 2016-10-25 2022-05-17 Regeneran Pharmaceuticals, Inc. Methods and systems for chromatography data analysis
EP4043485A1 (en) 2017-01-26 2022-08-17 Novartis AG Cd28 compositions and methods for chimeric antigen receptor therapy
WO2018140725A1 (en) 2017-01-26 2018-08-02 Novartis Ag Cd28 compositions and methods for chimeric antigen receptor therapy
WO2018160731A1 (en) 2017-02-28 2018-09-07 Novartis Ag Shp inhibitor compositions and uses for chimeric antigen receptor therapy
WO2018201051A1 (en) 2017-04-28 2018-11-01 Novartis Ag Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor
WO2018201056A1 (en) 2017-04-28 2018-11-01 Novartis Ag Cells expressing a bcma-targeting chimeric antigen receptor, and combination therapy with a gamma secretase inhibitor
WO2018237157A1 (en) 2017-06-22 2018-12-27 Novartis Ag CD73 BINDING ANTIBODY MOLECULES AND USES THEREOF
WO2019006007A1 (en) 2017-06-27 2019-01-03 Novartis Ag POSOLOGICAL REGIMES FOR ANTI-TIM3 ANTIBODIES AND USES THEREOF
US11752207B2 (en) 2017-07-11 2023-09-12 Compass Therapeutics Llc Agonist antibodies that bind human CD137 and uses thereof
WO2019018730A1 (en) 2017-07-20 2019-01-24 Novartis Ag DOSAGE REGIMES FOR ANTI-LAG3 ANTIBODIES AND USES THEREOF
US12042538B2 (en) 2017-09-19 2024-07-23 Regeneron Pharmaceuticals, Inc. Methods of reducing particle formation and compositions formed thereby
WO2019089798A1 (en) 2017-10-31 2019-05-09 Novartis Ag Anti-car compositions and methods
US11718679B2 (en) 2017-10-31 2023-08-08 Compass Therapeutics Llc CD137 antibodies and PD-1 antagonists and uses thereof
US12286483B2 (en) 2017-10-31 2025-04-29 Compass Therapeutics Llc Method of treating cancer using CD137 antibodies and PD-1 antagonists
WO2019099838A1 (en) 2017-11-16 2019-05-23 Novartis Ag Combination therapies
US11851497B2 (en) 2017-11-20 2023-12-26 Compass Therapeutics Llc CD137 antibodies and tumor antigen-targeting antibodies and uses thereof
US12247060B2 (en) 2018-01-09 2025-03-11 Marengo Therapeutics, Inc. Calreticulin binding constructs and engineered T cells for the treatment of diseases
WO2019152660A1 (en) 2018-01-31 2019-08-08 Novartis Ag Combination therapy using a chimeric antigen receptor
US12152073B2 (en) 2018-03-14 2024-11-26 Marengo Therapeutics, Inc. Multifunctional molecules that bind to calreticulin and uses thereof
WO2019200229A1 (en) 2018-04-13 2019-10-17 Novartis Ag Dosage regimens for anti-pd-l1 antibodies and uses thereof
WO2019210153A1 (en) 2018-04-27 2019-10-31 Novartis Ag Car t cell therapies with enhanced efficacy
WO2019226617A1 (en) 2018-05-21 2019-11-28 Compass Therapeutics Llc Compositions and methods for enhancing the killing of target cells by nk cells
WO2019226658A1 (en) 2018-05-21 2019-11-28 Compass Therapeutics Llc Multispecific antigen-binding compositions and methods of use
WO2019227003A1 (en) 2018-05-25 2019-11-28 Novartis Ag Combination therapy with chimeric antigen receptor (car) therapies
WO2019232244A2 (en) 2018-05-31 2019-12-05 Novartis Ag Antibody molecules to cd73 and uses thereof
WO2019241426A1 (en) 2018-06-13 2019-12-19 Novartis Ag Bcma chimeric antigen receptors and uses thereof
WO2019246293A2 (en) 2018-06-19 2019-12-26 Atarga, Llc Antibody molecules to complement component 5 and uses thereof
US11884698B2 (en) 2018-07-02 2024-01-30 Regeneron Pharmaceuticals, Inc. Systems and methods for preparing a polypeptide from a mixture
US12286477B2 (en) 2018-07-03 2025-04-29 Marengo Therapeutics, Inc. Anti-TCR antibody molecules and uses thereof
US12351632B2 (en) 2018-07-03 2025-07-08 Marengo Therapeutics, Inc. Anti-TCR antibody molecules and uses thereof
WO2020012337A1 (en) 2018-07-10 2020-01-16 Novartis Ag 3-(5-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and their use in the treatment of i karos family zinc finger 2 (ikzf2)-dependent diseases
WO2020021465A1 (en) 2018-07-25 2020-01-30 Advanced Accelerator Applications (Italy) S.R.L. Method of treatment of neuroendocrine tumors
US11970538B2 (en) 2018-11-13 2024-04-30 Compass Therapeutics Llc Multispecific binding constructs against checkpoint molecules and uses thereof
US11046769B2 (en) 2018-11-13 2021-06-29 Compass Therapeutics Llc Multispecific binding constructs against checkpoint molecules and uses thereof
US12503511B2 (en) 2018-11-13 2025-12-23 Compass Therapeutics, LLC Multispecific binding constructs against checkpoint molecules and uses thereof
WO2020128898A1 (en) 2018-12-20 2020-06-25 Novartis Ag Pharmaceutical combinations
WO2020128972A1 (en) 2018-12-20 2020-06-25 Novartis Ag Dosing regimen and pharmaceutical combination comprising 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives
WO2020165868A1 (en) 2019-02-15 2020-08-20 Perkinelmer Cellular Technologies Germany Gmbh Low-power microscope-objective pre-scan and high-power microscope-objective scan in x,y and z-direction for imaging objects such as cells using a microscope
WO2020165833A1 (en) 2019-02-15 2020-08-20 Novartis Ag 3-(1-oxo-5-(piperidin-4-yl)isoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
WO2020165834A1 (en) 2019-02-15 2020-08-20 Novartis Ag Substituted 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
US12358982B2 (en) 2019-02-21 2025-07-15 Marengo Therapeutics, Inc. Multifunctional molecules that bind to T cell related cancer cells and uses thereof
US12384842B2 (en) 2019-02-21 2025-08-12 Marengo Therapeutics, Inc. Antibody molecules that bind to NKP30 and uses thereof
WO2020172553A1 (en) 2019-02-22 2020-08-27 Novartis Ag Combination therapies of egfrviii chimeric antigen receptors and pd-1 inhibitors
WO2020205523A1 (en) 2019-03-29 2020-10-08 Atarga, Llc Anti fgf23 antibody
WO2021079188A1 (en) 2019-10-21 2021-04-29 Novartis Ag Combination therapies with venetoclax and tim-3 inhibitors
WO2021079195A1 (en) 2019-10-21 2021-04-29 Novartis Ag Tim-3 inhibitors and uses thereof
WO2021108661A2 (en) 2019-11-26 2021-06-03 Novartis Ag Chimeric antigen receptors and uses thereof
WO2021123996A1 (en) 2019-12-20 2021-06-24 Novartis Ag Uses of anti-tgf-beta antibodies and checkpoint inhibitors for the treatment of proliferative diseases
WO2021123902A1 (en) 2019-12-20 2021-06-24 Novartis Ag Combination of anti tim-3 antibody mbg453 and anti tgf-beta antibody nis793, with or without decitabine or the anti pd-1 antibody spartalizumab, for treating myelofibrosis and myelodysplastic syndrome
US12486326B2 (en) 2020-01-03 2025-12-02 Marengo Therapeutics, Inc. Anti-TCR antibody molecules and uses thereof
WO2021144657A1 (en) 2020-01-17 2021-07-22 Novartis Ag Combination comprising a tim-3 inhibitor and a hypomethylating agent for use in treating myelodysplastic syndrome or chronic myelomonocytic leukemia
WO2021146636A1 (en) 2020-01-17 2021-07-22 Becton, Dickinson And Company Methods and compositions for single cell secretomics
WO2021173995A2 (en) 2020-02-27 2021-09-02 Novartis Ag Methods of making chimeric antigen receptor-expressing cells
WO2021260528A1 (en) 2020-06-23 2021-12-30 Novartis Ag Dosing regimen comprising 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives
WO2022013787A1 (en) 2020-07-16 2022-01-20 Novartis Ag Anti-betacellulin antibodies, fragments thereof, and multi-specific binding molecules
WO2022026592A2 (en) 2020-07-28 2022-02-03 Celltas Bio, Inc. Antibody molecules to coronavirus and uses thereof
WO2022029573A1 (en) 2020-08-03 2022-02-10 Novartis Ag Heteroaryl substituted 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
WO2022043558A1 (en) 2020-08-31 2022-03-03 Advanced Accelerator Applications International Sa Method of treating psma-expressing cancers
WO2022043557A1 (en) 2020-08-31 2022-03-03 Advanced Accelerator Applications International Sa Method of treating psma-expressing cancers
WO2022097065A2 (en) 2020-11-06 2022-05-12 Novartis Ag ANTIBODY Fc VARIANTS
WO2022104061A1 (en) 2020-11-13 2022-05-19 Novartis Ag Combination therapies with chimeric antigen receptor (car)-expressing cells
WO2022162569A1 (en) 2021-01-29 2022-08-04 Novartis Ag Dosage regimes for anti-cd73 and anti-entpd2 antibodies and uses thereof
WO2022215011A1 (en) 2021-04-07 2022-10-13 Novartis Ag USES OF ANTI-TGFβ ANTIBODIES AND OTHER THERAPEUTIC AGENTS FOR THE TREATMENT OF PROLIFERATIVE DISEASES
WO2022243846A1 (en) 2021-05-18 2022-11-24 Novartis Ag Combination therapies
WO2023044483A2 (en) 2021-09-20 2023-03-23 Voyager Therapeutics, Inc. Compositions and methods for the treatment of her2 positive cancer
WO2023092004A1 (en) 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Compositions and methods for the treatment of tau-related disorders
WO2023150778A1 (en) 2022-02-07 2023-08-10 Visterra, Inc. Anti-idiotype antibody molecules and uses thereof
WO2023220695A2 (en) 2022-05-13 2023-11-16 Voyager Therapeutics, Inc. Compositions and methods for the treatment of her2 positive cancer
WO2024030976A2 (en) 2022-08-03 2024-02-08 Voyager Therapeutics, Inc. Compositions and methods for crossing the blood brain barrier
WO2024168061A2 (en) 2023-02-07 2024-08-15 Ayan Therapeutics Inc. Antibody molecules binding to sars-cov-2
WO2025122634A1 (en) 2023-12-05 2025-06-12 Voyager Therapeutics, Inc. Compositions and methods for the treatment of tau-related disorders
WO2025129010A1 (en) 2023-12-14 2025-06-19 Genentech, Inc. Methods of structure determination using antibodies

Also Published As

Publication number Publication date
AU2004252171A1 (en) 2005-01-06
US20090041758A1 (en) 2009-02-12
US20050163783A1 (en) 2005-07-28
WO2005000899A2 (en) 2005-01-06
AU2004252171B2 (en) 2011-04-21
JP2007535297A (ja) 2007-12-06
US7700097B2 (en) 2010-04-20
US8603473B2 (en) 2013-12-10
CA2529945A1 (en) 2005-01-06
EP1641827A2 (en) 2006-04-05
NZ544924A (en) 2009-03-31
JP2010273686A (ja) 2010-12-09
AU2004252170B2 (en) 2011-01-27
AU2011201880A1 (en) 2011-05-19
NZ544923A (en) 2009-02-28
EP1641826A2 (en) 2006-04-05
WO2005000898A2 (en) 2005-01-06
JP2007535296A (ja) 2007-12-06
JP5091476B2 (ja) 2012-12-05
WO2005000898A3 (en) 2005-09-09
AU2004252170A1 (en) 2005-01-06
JP5068072B2 (ja) 2012-11-07
WO2005000899A3 (en) 2005-08-11
JP2010280683A (ja) 2010-12-16
CA2530388A1 (en) 2005-01-06

Similar Documents

Publication Publication Date Title
US8603473B2 (en) Modified binding molecules comprising connecting peptides
US8084026B2 (en) Multispecific binding molecules comprising connecting peptides
JP2007535297A5 (enExample)
US9738707B2 (en) Heterodimeric Fc regions, binding molecules comprising same, and methods relating thereto
US8618252B2 (en) Neonatal Fc receptor (FcRn)-binding polypeptide variants, dimeric Fc binding proteins and methods related thereto
US8021661B2 (en) Cripto binding molecules
JP2007535296A5 (enExample)
US20090258420A1 (en) Altered polypeptides, immunoconjugates thereof, and methods related thereto
CN1842538B (zh) 包含连接肽的修饰的结合分子

Legal Events

Date Code Title Description
AS Assignment

Owner name: BIOGEN IDEC MA INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GLASER, SCOTT;REFF, MITCHELL;YANG, TZUNG-HORNG;AND OTHERS;REEL/FRAME:016395/0885;SIGNING DATES FROM 20050315 TO 20050316

AS Assignment

Owner name: BIOGEN IDEC MA INC., MASSACHUSETTS

Free format text: CORRECTIVE COVERSHEET TO CORRECT ATTORNEY DOCKET NUMBER IDV-005 THAT WAS PREVIOUSLY RECORDED ON REEL 016395 FRAME 0885.;ASSIGNORS:GLASER, SCOTT;REFF, MITCHELL;YANG, TZUNG-HORNG;AND OTHERS;REEL/FRAME:018119/0149;SIGNING DATES FROM 20050315 TO 20050316

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION