US20050106667A1 - Binding polypeptides with restricted diversity sequences - Google Patents

Binding polypeptides with restricted diversity sequences Download PDF

Info

Publication number
US20050106667A1
US20050106667A1 US10/901,011 US90101104A US2005106667A1 US 20050106667 A1 US20050106667 A1 US 20050106667A1 US 90101104 A US90101104 A US 90101104A US 2005106667 A1 US2005106667 A1 US 2005106667A1
Authority
US
United States
Prior art keywords
polypeptide
amino acid
variant
antibody
sequence
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
US10/901,011
Other languages
English (en)
Inventor
Frederic Fellouse
Sachdev Sidhu
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.)
Genentech Inc
Original Assignee
Genentech 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 Genentech Inc filed Critical Genentech Inc
Priority to US10/901,011 priority Critical patent/US20050106667A1/en
Assigned to GENENTECH, INC. reassignment GENENTECH, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FELLOUSE, FREDERIC, SIDHU, SACHDEV
Publication of US20050106667A1 publication Critical patent/US20050106667A1/en
Priority to US12/015,451 priority patent/US20090023602A1/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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • 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
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • 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
    • 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
    • A61P17/06—Antipsoriatics
    • 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
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention generally relates to variant CDRs diversified using highly limited amino acid repertoires, and libraries comprising a plurality of such sequences.
  • the invention also relates to fusion polypeptides comprising these variant CDRs.
  • the invention also relates to methods and compositions useful for identifying novel binding polypeptides that can be used therapeutically or as reagents.
  • Phage display technology has provided a powerful tool for generating and selecting novel proteins that bind to a ligand, such as an antigen. Using the techniques of phage display allows the generation of large libraries of protein variants that can be rapidly sorted for those sequences that bind to a target antigen with high affinity. Nucleic acids encoding variant polypeptides are fused to a nucleic acid sequence encoding a viral coat protein, such as the gene III protein or the gene VIII protein. Monovalent phage display systems where the nucleic acid sequence encoding the protein or polypeptide is fused to a nucleic acid sequence encoding a portion of the gene III protein have been developed.
  • Phage display technology has several advantages over conventional hybridoma and recombinant methods for preparing antibodies with the desired characteristics. This technology allows the development of large libraries of antibodies with diverse sequences in less time and without the use of animals. Preparation of hybridomas or preparation of humanized antibodies can easily require several months of preparation. In addition, since no immunization is required, phage antibody libraries can be generated for antigens which are toxic or have low antigenicity (Hogenboom, Immunotechniques (1988), 4:1-20). Phage antibody libraries can also be used to generate and identify novel human antibodies.
  • Antibodies have become very useful as therapeutic agents for a wide variety of conditions.
  • humanized antibodies to HER-2, a tumor antigen are useful in the diagnosis and treatment of cancer.
  • Other antibodies, such as anti-INF- ⁇ antibody are useful in treating inflammatory conditions such as Crohn's disease.
  • Phage display libraries have been used to generate human antibodies from immunized, non-immunized humans, germ line sequences, or na ⁇ ve B cell Ig repertories (Barbas & Burton, Trends Biotech (1996), 14:230; Griffiths et al., EMBO J . (1994), 13:3245; Vaughan et al., Nat. Biotech . (1996), 14:309; Winter EP 0368 684 B 1).
  • Na ⁇ ve, or nonimmune, antigen binding libraries have been generated using a variety of lymphoidal tissues. Some of these libraries are commercially available, such as those developed by Cambridge Antibody Technology and Morphosys (Vaughan et al., Nature Biotech 14:309 (1996); Knappik et al., J. Mol. Biol. 296:57 (1999)). However, many of these libraries have limited diversity.
  • the ability to identify and isolate high affinity antibodies from a phage display library is important in isolating novel human antibodies for therapeutic use. Isolation of high affinity antibodies from a library is traditionally thought to be dependent, at least in part, on the size of the library, the efficiency of production in bacterial cells and the diversity of the library. See, for e.g., Knappik et al., J. Mol. Biol . (1999), 296:57.
  • the size of the library is decreased by inefficiency of production due to improper folding of the antibody or antigen binding protein and the presence of stop codons. Expression in bacterial cells can be inhibited if the antibody or antigen binding domain is not properly folded.
  • Expression can be improved by mutating residues in turns at the surface of the variable/constant interface, or at selected CDR residues.
  • CDR3 regions are of interest in part because they often are found to participate in antigen binding. CDR3 regions on the heavy chain vary greatly in size, sequence and structural conformation.
  • the present invention provides simplified and flexible methods of generating polypeptides comprising variant CDRs that comprise sequences with restricted diversity yet retain target antigen binding capability. Unlike conventional methods that are based on the proposition that adequate diversity of target binders can be generated only if a particular CDR(s), or all CDRs are diversified, and unlike conventional notions that adequate diversity is dependent upon the broadest range of amino acid substitutions (generally by substitution using all or most of the 20 amino acids), the invention provides methods capable of generating high quality target binders that are not necessarily dependent upon diversifying a particular CDR(s) or a particular number of CDRs of a reference polypeptide or source antibody.
  • the invention is based, at least in part, on the surprising and unexpected finding that highly diverse libraries of high quality comprising functional polypeptides capable of binding target antigens can be generated by diversifying a minimal number of amino acid positions with a highly restricted number of amino acid residues.
  • Methods of the invention are rapid, convenient and flexible, based on using restricted codon sets that encode a low number of amino acids.
  • the restricted sequence diversity, and thus generally smaller size of the populations (for e.g., libraries) of polypeptides generated by methods of the invention allows for further diversification of these populations, where necessary or desired. This is an advantage generally not provided by conventional methods.
  • Candidate binder polypeptides generated by the invention possess high-quality target binding characteristics and have structural characteristics that provide for high yield of production in cell culture.
  • the invention provides methods for generating these binder polypeptides, methods for using these polypeptides, and compositions comprising the same.
  • the invention provides fusion polypeptides comprising diversified CDR(s) and a heterologous polypeptide sequence (preferably that of at least a portion of a viral polypeptide), as single polypeptides and as a member of a plurality of unique individual polypeptides that are candidate binders to targets of interest.
  • Compositions (such as libraries) comprising such polypeptides find use in a variety of applications, for e.g., as pools of candidate immunoglobulin polypeptides (for e.g., antibodies and antibody fragments) that bind to targets of interest.
  • Such polypeptides may also be generated using non-immunoglobulin scaffolds (for e.g., proteins, such as human growth hormone, etc.).
  • the invention encompasses various aspects, including polynucleotides and polypeptides generated according to methods of the invention, and systems, kits and articles of manufacture for practicing methods of the invention, and/or using polypeptides/polynucleotides and/or compositions of the invention.
  • the invention provides a method of generating a polypeptide comprising at least one, two, three, four, five or all of variant CDRs selected from the group consisting of H1, H2, H3, L1, L2 and L3, wherein said polypeptide is capable of binding a target antigen of interest, said method comprising identifying at least one (or any number up to all) solvent accessible and highly diverse amino acid position in a reference CDR corresponding to the variant CDR; and (ii) varying the amino acid at the solvent accessible and high diverse position by generating variant copies of the CDR using a restricted codon set (the definition of “restricted codon set” as provided below).
  • a restricted codon set the definition of “restricted codon set” as provided below.
  • the invention provides a method of generating a composition comprising a plurality of polypeptides, each polypeptide comprising at least one, two, three, four, five or all of variant CDRs selected from the group consisting of H1, H2, H3, L1, L2 and L3, wherein said polypeptide is capable of binding a target antigen of interest, said method comprising identifying at least one (or any number up to all) solvent accessible and highly diverse amino acid position in a reference CDR corresponding to the variant CDR; and (ii) varying the amino acid at the solvent accessible and high diverse position by generating variant copies of the CDR using a restricted codon set; wherein a plurality of polypeptides are generated by amplifying a template polynucleotide with a set of oligon
  • the invention provides a method comprising: constructing an expression vector comprising a polynucleotide sequence which encodes a light chain, a heavy chain, or both the light chain and the heavy chain variable domains of a source antibody comprising at least one, two, three, four, five or all CDRs selected from the group consisting of CDR L1, L2, L3, H1, H2 and H3; and mutating at least one, two, three, four, five or all CDRs of the source antibody at at least one (or any number up to all) solvent accessible and highly diverse amino acid position using a restricted codon set.
  • the invention provides a method comprising: constructing a library of phage or phagemid particles displaying a plurality of polypeptides of the invention; contacting the library of particles with a target antigen under conditions suitable for binding of the particles to the target antigen; and separating the particles that bind from those that do not bind to the target antigen.
  • a solvent accessible and/or highly diverse amino acid position can be any that meet the criteria as described herein, in particular any combination of the positions as described herein, for example any combination of the positions described for the polypeptides of the invention (as described in greater detail herein).
  • Suitable variant amino acids can be any that meet the criteria as described herein, for example variant amino acids in polypeptides of the invention as described in greater detail below.
  • Designing diversity in CDRs may involve designing diversity in the length and/or in sequence of the CDR.
  • CDRH3 may be diversified in length to be, for e.g., 7 to 19 amino acids in length, and/or in its sequence, for e.g. by varying highly diverse and/or solvent accessible positions with amino acids encoded by a restricted codon set.
  • a portion of CDRH3 has a length ranging from 5 to 22, 7 to 20, 9 to 15, or 11 to 13 amino acids, and has a variant amino acid at one or more positions encoded by a restricted codon set that encodes a limited number of amino acids such as codon sets encoding no more than 10, 8, 6, 4 or 2 amino acids.
  • the C terminal end has an amino acid sequence AM or AMDY.
  • polypeptides of the invention can be in a variety of forms as long as the target binding function of the polypeptides is retained.
  • a polypeptide of the invention is a fusion polypeptide (ie. a fusion of two or more sequences from heterologous polypeptides).
  • Polypeptides with diversified CDRs according to the invention can be prepared as fusion polypeptides to at least a portion of a viral coat protein, for e.g., for use in phage display.
  • Viral coat proteins that can be used for display of the polypeptides of the invention comprise protein p III, major coat protein pVIII, Soc (T4 phage), Hoc (T4 phage), gpD (lambda phage), pVI, or variants or fragments thereof.
  • the fusion polypeptide is fused to at least a portion of a viral coat protein, such as a viral coat protein selected from the group consisting of pIII, pVIII, Soc, Hoc, gpD, pVI, and variants or fragments thereof.
  • the antibody variable domains can be displayed on the surface of the virus in a variety of formats including ScFv, Fab, ScFv 2 , F(ab′) 2 and F(ab) 2 .
  • the fusion protein preferably includes a dimerization domain.
  • the dimerization domain can comprise a dimerization sequence and/or a sequence comprising one or more cysteine residues.
  • the dimerization domain is preferably linked, directly or indirectly, to the C-terminal end of a heavy chain variable or constant domain (e.g., CH1).
  • the structure of the dimerization domain can be varied depending on whether the antibody variable domain is produced as a fusion protein component with the viral coat protein component (without an amber stop codon after dimerization domain) or whether the antibody variable domain is produced predominantly without viral coat protein component (eg. with an amber stop codon after dimerization domain).
  • the antibody variable domain is produced predominantly as a fusion protein with viral coat protein component, one or more disulfide bonds and/or a single dimerization sequence provides for bivalent display.
  • a dimerization domain comprising both a cysteine residue and a dimerization sequence.
  • a fusion polypeptide can comprise a tag that may be useful in purification, detection and/or screening such as FLAG, poly-his, gD tag, c-myc, fluorescence protein or B-galactosidase.
  • a fusion polypeptide comprises a light chain variable or constant domain fused to a polypeptide tag.
  • a polypeptide such as an antibody variable domain is obtained from a single source or template molecule.
  • the source or template molecule is preferably selected or designed for characteristics such as good yield and stability when produced in prokaryotic or eukaryotic cell culture, and/or to accommodate CDRH3 regions of varying lengths.
  • the sequence of the template molecule can be altered to improve folding and/or display of the variable domain when presented as a fusion protein with a phage coat protein component.
  • a source antibody may comprise the amino acid sequence of the variable domains of humanized antibody 4D5 (light chain variable domain ( FIG. 15 ; SEQ ID NO: 1)); (heavy chain variable domain ( FIG. 15 ; SEQ ID NO: 2)).
  • framework region residues can be modified or altered from the source or template molecule to improve folding, yield, display or affinity of the antibody variable domain.
  • framework residues are selected to be modified from the source or template molecule when the amino acid in the framework position of the source molecule is different from the amino acid or amino acids commonly found at that position in naturally occurring antibodies or in a subgroup consensus sequence. The amino acids at those positions can be changed to the amino acids most commonly found in the naturally occurring antibodies or in a subgroup consensus sequence at that position.
  • framework residue 71 of the heavy chain may be R, V or A.
  • framework residue 93 of the heavy chain may be S or A.
  • framework residue 94 may be R, K or T or encoded by MRT.
  • framework residue 49 in the heavy chain may be alanine or glycine.
  • Framework residues in the light chain may also be changed.
  • the amino acid at position 66 may be arginine or glycine.
  • Methods of the invention are capable of generating a large variety of polypeptides comprising a diverse set of CDR sequences.
  • the invention provides a polypeptide comprising a variant CDRH3 region that comprises an amino acid sequence:
  • the invention provides a polypeptide comprising a variant CDRH2 that comprises an amino acid sequence:
  • the invention provides a polypeptide comprising a variant CDRH1 that comprises an amino acid sequence:
  • the invention provides a polypeptide comprising a variant CDRL3 that comprises an amino acid sequence:
  • the invention provides a polypeptide comprising a variant CDRL2 that comprises an amino acid sequence:
  • the invention provides a polypeptide comprising a variant CDRL1 that comprises an amino acid sequence:
  • amino acid X can be any of the amino acids encoded by a particular restricted codon set.
  • the 4 X1 amino acids in the variant CDRH3 can be, for e.g., AADY, AAAY, DSYA, SAYY, AAAA, SAAY, AAAY, AYDS, or any combination of one or more of the four amino acids encoded by the restricted codon set.
  • a restricted codon set encodes from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or only 2 amino acids. In some embodiments, a restricted codon set encodes at least 2 but 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer amino acids. In one embodiment, a restricted codon set is a tetranomial codon set. In another embodiment, a restricted codon set is a binomial codon set.
  • the invention provides a polypeptide comprising a variant CDRH1, H2, H3, L1, L2 and/or L3, wherein the variant CDR has a variant amino acid in at least one solvent accessible and highly diverse amino acid position, wherein the variant amino acid is encoded by a restricted codon set.
  • the restricted codon set is TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC, MRT, WMT, or a combination thereof.
  • the codon set is TMT and/or KMT.
  • a variant CDR comprises an amino acid sequence as set forth above.
  • the invention provides a polypeptide comprising a variant CDRH3 comprising a variant amino acid in at least one (or any number up to all) of positions 95, 96, 97, 98, 99, 100 and 100a, numbering of positions according to the Kabat system.
  • the C terminal residues of CDRH3 are kept constant as AMDY (although some changes can be made as long as the desired polypeptide characteristics (such as target antigen binding) are substantially retained).
  • all positions between 100 and A in the AMDY region comprise variant amino acids.
  • at least one position between 100 and A in the AMDY region comprises a variant amino acid.
  • a polypeptide comprises a variant CDRH3 comprising a variant amino acid in at least one of positions 95, 96, 97, 98, 99, 100, and at least one position between 100 and C-terminal sequence AMDY.
  • the variant CDRH3 comprises an insertion of one or more residues/positions, wherein said one or more positions comprises an amino acid encoded by a restricted codon set.
  • said insertion comprises 1-15, 3-13, 5-11, or 7-9 residues/positions.
  • said insertion comprises at least 1, at least 3, at least 5, at least 7, at least 9, at least 11, at least 13 residues/positions.
  • said insertion comprises 15 or fewer, 13 or fewer, 111 or fewer, 9 or fewer, 7 or fewer, or 5 or fewer residues/positions.
  • the invention provides a polypeptide comprising a variant CDRH2 comprising a variant amino acid in at least one (or any number up to all) of positions 50, 52, 53, 54, 56 and 58, numbering of positions according to the Kabat system.
  • the invention provides a polypeptide comprising a variant CDRH1 comprising a variant amino acid in at least one (or any number up to all) of positions 28, 30, 31, 32 and 33, numbering of positions according to the Kabat system.
  • the invention provides a polypeptide comprising a variant CDRL3 comprising a variant amino acid in at least one (or any number up to all) of positions 91, 92, 93, 94 and 96, numbering of positions according to the Kabat system.
  • the invention provides a polypeptide comprising a variant CDRL2 comprising a variant amino acid in at least one or both of positions 50 and 53, numbering of positions according to the Kabat system.
  • the invention provides a polypeptide comprising a variant CDRL1 comprising a variant amino acid in at least one (or any number up to all) of positions 28, 29, 30, 31 and 32, numbering of positions according to the Kabat system.
  • the invention provides a polypeptide comprising a variant CDR as described above, wherein the polypeptide further comprises at least one, two, three, four or five additional variant CDRs selected from the group consisting of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 or CDRL3, wherein a variant amino acid is encoded by a restricted codon set.
  • the restricted codon set is TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC, MRT, WMT, or a combination thereof.
  • a restricted codon set encodes at least Y and/or S.
  • a restricted codon set does not encode alanine.
  • the restricted codon set encodes 4 or fewer amino acids. In one embodiment, the restricted codon set encodes only 2 amino acids, which in one embodiment are Y and S. In one embodiment of the invention, a restricted codon set encodes from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or only 2 amino acids. In some embodiments, a restricted codon set encodes at least 2 but 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer amino acids. In one embodiment, a restricted codon set is a tetranomial codon set. In another embodiment, a restricted codon set is a binomial codon set.
  • a polypeptide of the invention comprises a variant CDRH3, and at least one additional variant CDR which is CDRH1 and/or CDRH2.
  • the polypeptide further comprises at least one variant light chain CDR.
  • a variant light chain CDR is CDRL3.
  • a polypeptide of the invention further comprises a variant CDRL1 and/or CDRL2 (in some instances, in combination with a variant CDRL3).
  • a polypeptide of the invention comprises at least one, or both, of heavy chain and light chain antibody variable domains, wherein the antibody variable domain comprises one, two or three variant CDRs as described herein (for e.g., as described in the foregoing).
  • a polypeptide of the invention (in particular those comprising an antibody variable domain) further comprises an antibody framework sequence, for e.g., FR1, FR2, FR3 and/or FR4 for an antibody variable domain corresponding to the variant CDR, the FR sequences obtained from a single antibody template.
  • the FR sequences are obtained from a human antibody.
  • the FR sequences are obtained from a human consensus sequence (e.g., subgroup III consensus sequence).
  • the framework sequences comprise a modified consensus sequence as described herein (e.g., comprising modifications at position 49, 71, 93 and/or 94 in the heavy chain, and/or position 66 in the light chain).
  • each of the FR has the sequence of antibody 4D5 (SEQ ID NO: 1).
  • the invention provides methods of generating compositions comprising polypeptides and/or polynucleotides of the invention. Accordingly, in one aspect, the invention provides a method of generating a composition comprising a plurality of polypeptides comprising:
  • a method of the invention also comprises generating a plurality of polypeptides comprising a variant CDRL1, CDRL2 or CDRL3 or mixtures thereof, wherein the variant CDRs are formed with at least one variant amino acid in a solvent accessible and highly diverse position; wherein the variant amino acid is encoded by a restricted codon set.
  • polypeptides comprising variant CDRL3 comprise an amino acid sequence:
  • the invention provides a polypeptide comprising at least one, two, three, four, five or all of variant CDRs selected from the group consisting of CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 and CDR H3, wherein the variant CDR is as described above.
  • a polypeptide of the invention comprises a light chain and a heavy chain antibody variable domain, wherein the light chain variable domain comprises at least 1, 2 or 3 variant CDRs selected from the group consisting of CDR L1, L2 and L3, and the heavy chain variable domain comprises at least 1, 2 or 3 variant CDRs selected from the group consisting of CDR H1, H2 and H3.
  • a polypeptide of the invention is an ScFv. In some embodiments, it is a Fab fragment. In some embodiments, it is a F(ab) 2 or F(ab′) 2 Accordingly, in some embodiments, a polypeptide of the invention further comprises a dimerization domain. In some embodiments, the dimerization domain is located between an antibody heavy chain or light chain variable domain and at least a portion of a viral coat protein. The dimerization domain can comprise a dimerization sequence, and/or sequence comprising one or more cysteine residues. The dimerization domain is preferably linked, directly or indirectly, to the C-terminal end of a heavy chain variable or constant domain.
  • the structure of the dimerization domain can be varied depending on whether the antibody variable domain is produced as a fusion protein component with the viral coat protein component (without an amber stop codon after dimerization domain) or whether the antibody variable domain is produced predominantly without viral coat protein component (eg. with an amber stop codon after dimerization domain).
  • the antibody variable domain is produced predominantly as a fusion protein with viral coat protein component, one or more disulfide bond and/or a single dimerization sequence provides for bivalent display.
  • a dimerization domain comprising both a cysteine residue and a dimerization sequence.
  • heavy chains of the F(ab) 2 dimerize at a dimerization domain not including a hinge region.
  • the dimerization domain may comprise a leucine zipper sequence (for example, a GCN4 sequence such as GRMKQLEDKVEELLSKNYHLENEVARLKKLVGERG (SEQ ID NO: 3)).
  • a polypeptide of the invention further comprises a light chain constant domain fused to a light chain variable domain, which in some embodiments comprises at least one, two or three variant CDRs.
  • the polypeptide comprises a heavy chain constant domain fused to a heavy chain variable domain, which in some embodiments comprises at least one, two or three variant CDRs.
  • framework residue 71 of the heavy chain may be amino acid R, V or A.
  • framework residue 93 of the heavy chain may be amino acid S or A.
  • framework residue 94 of the heavy chain may be amino acid R, K or T or encoded by MRT.
  • framework residue 49 of the heavy chain may be amino acid A or G.
  • Framework residues in the light chain may also be mutated.
  • framework residue 66 in the light chain may be amino acid R or G.
  • a variant CDR refers to a CDR with a sequence variance as compared to the corresponding CDR of a single reference polypeptide/source antibody. Accordingly, the CDRs of a single polypeptide of the invention preferably correspond to the set of CDRs of a single reference polypeptide or source antibody.
  • Polypeptides of the invention may comprise any one or combinations of variant CDRs.
  • a polypeptide of the invention may comprise a variant CDRH1 and variant CDRH2.
  • a polypeptide of the invention may comprise a variant CDRH1, variant CDRH2 and a variant CDRH3.
  • a polypeptide of the invention may comprise a variant CDRH1, variant CDRH2, variant CDRH3 and variant CDRL3.
  • a polypeptide of the invention comprises a variant CDRL1, variant CDRL2 and variant CDRL3.
  • Any polypeptide of the invention may further comprise a variant CDRL3.
  • Any polypeptide of the invention may further comprise a variant CDRH3.
  • a polypeptide of the invention comprises one or more variant CDR sequences as depicted in FIG. 5 .
  • Polypeptides of the invention may be in a complex with one another.
  • the invention provides a polypeptide complex comprising two polypeptides, wherein each polypeptide is a polypeptide of the invention, and wherein one of said polypeptides comprises at least one, two or all of variant CDRs H1, H2 and H3, and the other polypeptide comprises a variant light chain CDR (e.g., CDR L3).
  • a polypeptide complex may comprise a first and a second polypeptide (wherein the first and second polypeptides are polypeptides of the invention), wherein the first polypeptide comprises at least one, two or three variant light chain CDRs, and the second polypeptide comprises at least one, two or three variant heavy chain CDRs.
  • the invention also provides complexes of polypeptides that comprise the same variant CDR sequences.
  • Complexing can be mediated by any suitable technique, including by dimerization/multimerization at a dimerization/multimerization domain such as those described herein or covalent interactions (such as through a disulfide linkage) (which in some contexts is part of a dimerization domain, for e.g. a dimerization domain may contain a leucine zipper sequence and a cysteine).
  • the invention provides compositions comprising polypeptides and/or polynucleotides of the invention.
  • the invention provides a composition comprising a plurality of any of the polypeptides of the invention described herein. Said plurality may comprise polypeptides encoded by a plurality of polynucleotides generated using a set of oligonucleotides comprising degeneracy in the sequence encoding a variant amino acid, wherein said degeneracy is that of the multiple codon sequences of the restricted codon set encoding the variant amino acid.
  • a composition comprising a polynucleotide or polypeptide or library of the invention may be in the form of a kit or an article of manufacture (optionally packaged with instructions, buffers, etc.).
  • the invention provides a polynucleotide encoding a polypeptide of the invention as described herein.
  • the invention provides a vector comprising a sequence encoding a polypeptide of the invention.
  • the vector can be, for e.g., a replicable expression vector (for e.g., the replicable expression vector can be M13, f1, fd, Pf3 phage or a derivative thereof, or a lambdoid phage, such as lambda, 21, phi80, phi81, 82, 424, 434, etc., or a derivative thereof).
  • the vector can comprise a promoter region linked to the sequence encoding a polypeptide of the invention.
  • the promoter can be any suitable for expression of the polypeptide, for e.g., the lac Z promoter system, the alkaline phosphatase pho A promoter (Ap), the bacteriophage l PL promoter (a temperature sensitive promoter), the tac promoter, the tryptophan promoter, and the bacteriophage T7 promoter.
  • the invention also provides a vector comprising a promoter selected from the group consisting of the foregoing promoter systems.
  • Polypeptides of the invention can be displayed in any suitable form in accordance with the need and desire of the practitioner.
  • a polypeptide of the invention can be displayed on a viral surface, for e.g., a phage or phagemid viral particle.
  • the invention provides viral particles comprising a polypeptide of the invention and/or polynucleotide encoding a polypeptide of the invention.
  • the invention provides a population comprising a plurality of polypeptide or polynucleotide of the invention, wherein each type of polypeptide or polynucleotide is a polypeptide or polynucleotide of the invention as described herein.
  • polypeptides and/or polynucleotides are provided as a library, for e.g., a library comprising a plurality of at least about 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 distinct polypeptide and/or polynucleotide sequences of the invention.
  • the invention also provides a library comprising a plurality of the viruses or viral particles of the invention, each virus or virus particle displaying a polypeptide of the invention.
  • a library of the invention may comprise viruses or viral particles displaying any number of distinct polypeptides (sequences), for e.g., at least about 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 distinct polypeptides.
  • the invention provides host cells comprising a polynucleotide or vector comprising a sequence encoding a polypeptide of the invention.
  • the invention provides methods for selecting for high affinity binders to specific target antigens such as growth hormone, bovine growth hormone, insulin like growth factors, human growth hormone including n-methionyl human growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, amylin, an apoptosis protein, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), leutinizing hormone (LH), hemapoietic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factors, hepatocyte growth factor, hepatocyte growth factor receptor (c-met), mullerian inhibiting substance, mouse gonadotropin-associated polypeptide, inhibin, activin, vascular endothelial growth factors, integrin, nerve growth factors such as NGF-beta, insulin-like growth factor-I and II, erythropoietin, osteoinductive factors, interferons, colony stimulating
  • the methods of the invention provide populations of polypeptides (for e.g., libraries of polypeptides (eg. antibody variable domains)) with one or more diversified CDR regions. These libraries are sorted (selected) and/or screened to identify high affinity binders to a target antigen.
  • polypeptide binders from the library are selected for binding to target antigens, and for affinity.
  • the polypeptide binders selected using one or more of these selection strategies, may then be screened for affinity and/or for specificity (binding only to target antigen and not to non-target antigens).
  • a method of the invention comprises generating a plurality of polypeptides with one or more diversified CDR regions, sorting the plurality of polypeptides for binders to a target antigen by contacting the plurality of polypeptides with a target antigen under conditions suitable for binding; separating the binders to the target antigen from those that do not bind; isolating the binders; and identifying the high affinity binders (or any binders having a desired binding affinity).
  • the affinity of the binders that bind to the target antigen can be determined using a variety of techniques known in the art, for e.g., competition ELISA such as described herein.
  • the polypeptides can be fused to a polypeptide tag, such as gD, poly his or FLAG, which can be used to sort binders in combination with sorting for the target antigen.
  • Another embodiment provides a method of isolating or selecting for an antibody variable domain that binds to a target antigen from a library of antibody variable domains, said method comprising: a) contacting a population comprising a plurality of polypeptides of the invention with an immobilized target antigen under conditions suitable for binding to isolate target antigen polypeptide binders; b) separating the polypeptide binders from nonbinders, and eluting the binders from the target antigen; c) optionally, repeating steps a-b at least once (in some embodiments, at least twice).
  • a method may further comprise: d) incubating the polypeptide binders with a concentration of labelled target antigen in the range of 0.1 nM to 1000 nM under conditions suitable for binding to form a mixture; e) contacting the mixture with an immobilized agent that binds to the label on the target antigen; f) eluting the polypeptide binders from the labelled target antigen; g) optionally, repeating steps d) to f) at least once (in some embodiments, at least twice), using a successively lower concentration of labelled target antigen each time.
  • the method may comprise adding an excess of unlabelled target antigen to the mixture and incubating for a period of time sufficient to elute low affinity binders from the labelled target antigen.
  • Another aspect of the invention provides a method of isolating or selecting for high affinity binders (or binders having a desired binding affinity) to a target antigen.
  • said method comprises: a) contacting a population comprising a plurality of polypeptides of the invention with a target antigen, wherein the antigen is provided at a concentration in the range of about 0.1 nM to 1000 nM to isolate polypeptide binders to the target antigen; b) separating the polypeptide binders from the target antigen; c) optionally, repeating steps a-b at least once (in some embodiments, at least twice), each time with a successively lower concentration of target antigen to isolate polypeptide binders that bind to lowest concentration of target antigen; d) selecting the polypeptide binder that binds to the lowest concentration of the target antigen for high affinity (or any desired affinity) by incubating the polypeptide binders with several different dilutions of the target antigen and determining the
  • Another embodiment provides an assay for isolating or selecting polypeptide binders comprising (a) contacting a population comprising a plurality of polypeptides of the invention with a labelled target antigen, wherein the labeled target antigen is provided at a concentration in a range of 0.1 nM to 1000 nM, under conditions suitable for binding to form a complex of a polypeptide binder and the labelled target antigen; b) isolating the complexes and separating the polypeptide binder from the labelled target antigen; c) optionally, repeating steps a-b at least once, each time using a lower concentration of target antigen.
  • the method may further comprise contacting the complex of polypeptide binder and target antigen with an excess of unlabelled target antigen.
  • the steps of the method are repeated twice and the concentration of target in a first round of selection is in the range of about 100 nM to 250 nM, and, in a second round of selection (if performed) is in the range of about 25 nM to 100 nM, and in the third round of selection (if performed) is in the range of about 0.1 nM to 25 nM.
  • the invention also includes a method of screening a population comprising a plurality of polypeptides of the invention, said method comprising: a) incubating a first sample of the population of polypeptides with a target antigen under conditions suitable for binding of the polypeptides to the target antigen; b) subjecting a second sample of the population of polypeptides to a similar incubation but in the absence of the target antigen; (c) contacting each of the first and second sample with immobilized target antigen under conditions suitable for binding of the polypeptides to the immobilized target antigen; d) detecting amount of polypeptides bound to immobilized target antigen for each sample; e) determining affinity of a particular polypeptide for the target antigen by calculating the ratio of the amount of the particular polypeptide that is bound in the first sample over the amount of the particular polypeptide that is bound in the second sample.
  • the invention provides a method of screening for a polypeptide, such as an antibody variable domain of the invention, that binds to a specific target antigen from a library of antibody variable domains, said method comprising: a) generating a population comprising a plurality of polypeptides of the invention; b) contacting the population of polypeptides with a target antigen under conditions suitable for binding; c) separating a binder polypeptide in the library from nonbinder polypeptides; d) identifying a target antigen-specific binder polypeptide by determining whether the binder polypeptide binds to a non-target antigen; and e) isolating a target antigen-specific binder polypeptide.
  • step (e) comprises eluting the binder polypeptide from the target antigen, and amplifying a replicable expression vector en
  • polypeptide binders are first selected for binding to an immobilized target antigen.
  • Polypeptide binders that bind to the immobilized target antigen can then be screened for binding to the target antigen and for lack of binding to nontarget antigens.
  • Polypeptide binders that bind specifically to the target antigen can be amplified as necessary.
  • polypeptide binders can be selected for higher affinity by contact with a concentration of a labelled target antigen to form a complex, wherein the concentration range of labelled target antigen is from about 0.1 nM to about 1000 nM, and the complexes are isolated by contact with an agent that binds to the label on the target antigen.
  • a polypeptide binder can then be eluted from the labeled target antigen and optionally, the rounds of selection are repeated, each time a lower concentration of labelled target antigen is used.
  • the binder polypeptides that can be isolated using this selection method can then be screened for high affinity using for example, the solution phase ELISA assay as described in Example 8 or other conventional methods known in the art.
  • polypeptides of the invention used in methods of the invention can be provided in any form suitable for the selection/screening steps.
  • the polypeptides can be in free soluble form, attached to a matrix, or present at the surface of a viral particle such as phage or phagemid particle.
  • the plurality of polypeptides are encoded by a plurality of replicable vectors provided in the form of a library.
  • vectors encoding a binder polypeptide may be further amplified to provide sufficient quantities of the polypeptide for use in repetitions of the selection/screening steps (which, as indicated above, are optional in methods of the invention).
  • the invention provides a method of selecting for a polypeptide that binds to a target antigen comprising:
  • the invention provides a method of selecting for an antigen binding variable domain that binds to a target antigen from a library of antibody variable domains comprising:
  • the concentration of target antigen is about 100 to 250 nM, or about 25 to 100 nM.
  • the invention provides a method of selecting for a polypeptide that binds to a target antigen from a library of polypeptides comprising:
  • methods of the invention further comprise the steps of:
  • these methods further comprise adding an excess of unlabelled target antigen to the mixture and incubating for a period of time sufficient to elute low affinity binders from the labelled target antigen.
  • the invention provides a method of isolating high affinity binders to a target antigen comprising:
  • the invention provides an assay for selecting polypeptide binders from a library comprising a plurality of polypeptides of the invention (as described herein) comprising:
  • the method further comprises adding an excess of unlabelled target antigen to the complex of the polypeptide binder and target antigen.
  • the steps set forth above are repeated at least once (in some embodiments, at least twice) and the concentration of target in the first round of selection is about 100 nM to 250 nM, and in the second round of selection is about 25 nM to 100 nM, and in the third round of selection is about 0.1 nM to 25 nM.
  • the invention provides a method of screening a library comprising a plurality of polypeptides of the invention, said method comprising:
  • a polypeptide in the population used in methods of the invention comprises variant CDRH2 comprising an amino acid sequence:
  • a polypeptide in the population used in methods of the invention comprises variant CDRH1 comprising an amino acid sequence:
  • a polypeptide in the population used in methods of the invention comprises variant CDRL3 comprising an amino acid sequence:
  • a polypeptide in the population used in methods of the invention comprises variant CDRL2 comprising an amino acid sequence:
  • a polypeptide in the population used in methods of the invention comprises variant CDRL1 comprising an amino acid sequence:
  • the invention provides a method for determining the presence of a protein of interest comprising exposing a sample suspected of containing the protein to a binder polypeptide of the invention and determining binding of the binder polypeptide to the sample.
  • the invention provides a kit comprising the binder polypeptide and instructions for using the binder polypeptide to detect the protein.
  • the invention further provides: isolated nucleic acid encoding the binder polypeptide; a vector comprising the nucleic acid, optionally, operably linked to control sequences recognized by a host cell transformed with the vector; a host cell transformed with the vector; a process for producing the binder polypeptide comprising culturing this host cell so that the nucleic acid is expressed and, optionally, recovering the binder polypeptide from the host cell culture (e.g. from the host cell culture medium).
  • the invention also provides a composition
  • a composition comprising a binder polypeptide of the invention and a carrier (e.g., a pharmaceutically acceptable carrier) or diluent.
  • a carrier e.g., a pharmaceutically acceptable carrier
  • This composition for therapeutic use is sterile and may be lyophilized.
  • a binder polypeptide of this invention in the manufacture of a medicament for treating an indication described herein.
  • the composition can further comprise a second thereapeutic agent such as a chemotherapeutic agent, a cytotoxic agent or an anti-angiogenic agent.
  • the invention further provides a method for treating a mammal, comprising administering an effective amount of a binder polypeptide of the invention to the mammal.
  • the mammal to be treated in the method may be a nonhuman mammal, e.g. a primate suitable for gathering preclinical data or a rodent (e.g., mouse or rat or rabbit).
  • the nonhuman mammal may be healthy (e.g. in toxicology studies) or may be suffering from a disorder to be treated with the binder polypeptide of interest.
  • the mammal is suffering from or is at risk of developing abnormal angiogenesis (e.g., pathological angiogenesis).
  • the disorder is a cancer selected from the group consisting of colorectal cancer, renal cell carcinoma, ovarian cancer, lung cancer, non-small-cell lung cancer (NSCLC), bronchoalveolar carcinoma and pancreatic cancer.
  • the disorder is a disease caused by ocular neovascularisation, e.g., diabetic blindness, retinopathies, primarily diabetic retinopathy, age-induced macular degeneration and rubeosis.
  • the mammal to be treated is suffering from or is at risk of developing an edema (e.g., an edema associated with brain tumors, an edema associated with stroke, or a cerebral edema).
  • the mammal is suffering from or at risk of developing a disorder or illness selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, refractory ascites, psoriasis, sarcoidosis, arterial arteriosclerosis, sepsis, burns and pancreatitis.
  • the mammal is provided from or is at risk of developing a genitourinary illness selected from the group consisting of polycystic ovarian disease (POD), endometriosis and uterine fibroids.
  • POD polycystic ovarian disease
  • the disorder is a disease caused by dysregulation of cell survival (e.g., abnormal amount of cell death), including but not limited to cancer, disorders of the immune system, disorders of the nervous system and disorders of the vascular system.
  • the amount of binder polypeptide of the invention that is administered will be a therapeutically effective amount to treat the disorder. In dose escalation studies, a variety of doses of the binder polypeptide may be administered to the mammal. In another embodiment, a therapeutically effective amount of the binder polypeptide is administered to a human patient to treat a disorder in that patient.
  • binder polypeptides of this invention useful for treating inflammatory or immune diseases described herein are Fab or scFv antibodies. Accordingly, such binder polypeptides can be used in the manufacture of a medicament for treating an inflammatory or immune disease.
  • a mammal that is suffering from or is at risk for developing a disorder or illness described herein can be treated by administering, a second therapeutic agent, simultaneously, sequentially or in combination with, a polypeptide (e.g., an antibody) of this invention.
  • a polypeptide e.g., an antibody
  • polypeptides can be used to understand the role of host stromal cell collaboration in the growth of implanted non-host tumors, such as in mouse models wherein human tumors have been implanted. These polypeptides can be used in methods of identifying human tumors that can escape therapeutic treatment by observing or monitoring the growth of the tumor implanted into a rodent or rabbit after treatment with a polypeptide of this invention.
  • the polypeptides of this invention can also be used to study and evaluate combination therapies with a polypeptide of this invention and other therapeutic agents.
  • the polypeptides of this invention can be used to study the role of a target molecule of interest in other diseases by administering the polypeptides to an animal suffering from the disease or a similar disease and determining whether one or more symptoms of the disease are alleviated.
  • FIG. 1 illustrates CDR positions diversified in a library based on a binomial codon set that encodes only Y and S. CDR positions shown are numbered according to the Kabat nomenclature.
  • FIG. 3 shows enrichment ratios for libraries YADS-A and YADS-B following 5 rounds of selection against various target antigens.
  • FIG. 4 shows results of sorting of YS-A and YS-B libraries. Number of specific binders obtained is shown. Numbers are shown as X/Y, with X representing the number of specific clones (i.e., those binding to the target antigen at least 10 times higher (based on ELISA signal read at 450 nm) than the binding of bovine serum albumin (BSA), and Y representing the number of clones screened for a given library, round and target antigen.
  • BSA bovine serum albumin
  • FIG. 5 shows sequences of binders obtained from selection of library YS-A and YS-B. Note: Asterisks correspond to absence of an amino acid normally found in the corresponding position in a template sequence.
  • FIG. 6 shows an illustrative set of restricted codon sets.
  • the codon sets shown are tetranomial, i.e., they each encode only 4 amino acids.
  • FIG. 7 shows the number of specific binders assessed by phage ELISAs. Numbers are shown as X/Y, with X being the number of specific binders, and Y being the number of clones screened.
  • FIG. 8 shows the number of unique clones obtained from individual restricted diversity libraries for each target antigen.
  • FIG. 9 shows mutagenic oligonucleotides used in the construction of libraries YADS-A and YADS-B, which are based on tetranomial codon sets that encode only 4 amino acids.
  • WMT encodes S, Y, T and N.
  • KMT encodes Y, A, D and S. Codon sets are represented in the IUB code.
  • FIG. 10 shows the number of specific binders assessed by phage ELISAs for libraries YADS-A and YADS-B. Numbers are shown as X[Y, with X being the number of specific binders, and Y being the number of clones screened.
  • FIG. 11 shows values of IC50 of clones YS1-AP, YS2-AP and YS3-AP with respect to its corresponding human target antigen and cyno target antigen, measured by competitive phage ELISA
  • FIG. 12 shows light chain CDR positions that were diversified in a library based on a tetranomial codon set (YADS).
  • the library is referred to as the YADS-II library.
  • CDR positions are numbered according to the Kabat nomenclature.
  • FIG. 13 shows mutagenic oligonucleotides used in the construction of library YADS-II.
  • KMT encodes Y, A, D and S. Codon sets are represented in the IUB code.
  • FIG. 14 shows the results of screening YADS-II hVEGF selectants.
  • the figure shows clone number, BSA binding (measured by phage ELISA—numbers lower than 0.200 were considered to be below background and are indicated in bold character), and percent inhibition of binding by 100 nM of human VEGF (numbers showing inhibition greater than 75% are indicated in bold character).
  • FIG. 15 depicts the sequences of 4D5 light chain and heavy chain variable domain (SED ID NO:1 & 2, respectively).
  • FIG. 16 graphically depicts results of phage ELISA of 3 binders obtained from a YADS library on plates coated with different target antigens, shown for increasing amounts of phage.
  • FIG. 17 shows values of association (k a ), dissociation rate (k d ) and affinity (K d ) of 3 binders for human VEGF and murine VEGF.
  • FIG. 18 shows the DNA sequence of Ptac promoter driven cassette for display of Fab-zip (SEQ ID NO: 4). Two open reading frames are indicated. The first open reading frame encodes a malE secretion signal, humanized 4D5 light chain variable and constant domain. The second open reading frame encodes a stII secretion signal, humanized 4D5 heavy chain variable domain, humanized 4D5 heavy chain first constant domain (CH1), zipper sequence, and C-terminal of p3 (cP3).
  • the first open reading frame encodes a malE secretion signal, humanized 4D5 light chain variable and constant domain.
  • the second open reading frame encodes a stII secretion signal, humanized 4D5 heavy chain variable domain, humanized 4D5 heavy chain first constant domain (CH1), zipper sequence, and C-terminal of p3 (cP3).
  • FIG. 19 illustrates a bicistronic vector allowing expression of separate transcripts for display of F(ab) 2 .
  • a suitable promoter drives expression of the first and second cistron.
  • the first cistron encodes a secretion signal sequence (malE or stII), a light chain variable and constant domain and a gD tag.
  • the second cistron encodes a secretion signal, a sequence encoding heavy chain variable domain and constant domain 1 (CH1) and dimerization domain and at least a portion of the viral coat protein.
  • FIG. 20 shows a 3-D modeled structure of humanized 4D5 showing CDR residues that form contiguous patches.
  • Contiguous patches are formed by amino acid residues 28, 29, 30, 31 and 32 in CDRL1; amino acids residues 50 and 53 of CDRL2; amino acid residues 91, 92, 93, 94 and 96 of CDRL3; amino acid residues 28, 30, 31, 32, 33 in CDRH1; and amino acid residues 50, 52, 53, 54, 56, and 58 in CDRH2.
  • FIG. 21 shows the frequency of amino acids (identified by single letter code) in human antibody light chain CDR sequences from the Kabat database. The frequency of each amino acid at a particular amino acid position is shown starting with the most frequent amino acid at that position at the left and continuing on to the right to the least frequent amino acid. The number below the amino acid represents the number of naturally occurring sequences in the Kabat database that have that amino acid in that position.
  • FIG. 22 shows the frequency of amino acids (identified by single letter code) in human antibody heavy chain CDR sequences from the Kabat database. The frequency of each amino acid at a particular amino acid position is shown starting with the most frequent amino acid at that position at the left and continuing on to the right to the least frequent amino acid. The number below the amino acid represents the number of naturally occurring sequences in the Kabat database that have that amino acid in that position. Framework amino acid positions 71, 93 and 94 are also shown.
  • FIG. 23 shows values of association (k a ), dissociation rate (k d ) and affinity (K d ) of two anti-VEGF binders obtained from YS libraries (as described in Example 2) for human VEGF and murine VEGF.
  • FIGS. 24 A-F show the DNA (SEQ ID NO: 5) and amino acid (SEQ ID NOs: 6 & 7, for light and heavy chain, respectively) sequence of vector pV-0350-4, which is a vector that comprises a dimerization domain between heavy chain constant CH1 domain and p3 sequences.
  • the invention provides novel, unconventional, greatly simplified and flexible methods for diversifying CDR sequences (including antibody variable domain sequences), and libraries comprising a multiplicity, generally a great multiplicity of diversified CDRs (including antibody variable domain sequences).
  • Such libraries provide combinatorial libraries useful for, for example, selecting and/or screening for synthetic antibody clones with desirable activities such as binding affinities and avidities.
  • These libraries are useful for identifying immunoglobulin polypeptide sequences that are capable of interacting with any of a wide variety of target antigens.
  • libraries comprising diversified immunoglobulin polypeptides of the invention expressed as phage displays are particularly useful for, and provide a high throughput, efficient and automatable systems of, selecting and/or screening for antigen binding molecules of interest.
  • the methods of the invention are designed to provide high affinity binders to target antigens with minimal changes to a source or template molecule and provide for good production yields when the antibody or antigens binding fragments are produced in cell culture.
  • Methods and compositions of the invention provide numerous additional advantages. For example, relatively simple variant CDR sequences can be generated, using codon sets encoding a restricted number of amino acids (as opposed to the conventional approach of using codon sets encoding the maximal number of amino acids), while retaining sufficient diversity of unique target binding sequences.
  • the simplified nature (and generally relatively smaller size) of sequence populations generated according to the invention permits further diversification once a population, or sub-population thereof, has been identified to possess the desired characteristics.
  • sequences of target antigen binders obtained by methods of the invention leaves significantly greater room for individualized further sequence modifications to achieve the desired results.
  • sequence modifications are routinely performed in affinity maturation, humanization, etc.
  • restricted codon sets that encode only a limited number of amino acids
  • it would be possible to target different epitopes using different restricted codon sets thus providing the practitioner greater control of the diversification approach as compared with randomization based on a maximal number of amino acids.
  • An added advantage of using restricted codon sets is that undesirable amino acids can be eliminated from the process, for e.g., methionine or stop codons, thus improving the overall quality and productivity of a library.
  • variant CDRs, and binders comprising such variant CDRs can be generated that contain sequences that have a predominance of tyrosine residues.
  • Amino acids are represented herein as either a single letter code or as the three letter code or both.
  • affinity purification means the purification of a molecule based on a specific attraction or binding of the molecule to a chemical or binding partner to form a combination or complex which allows the molecule to be separated from impurities while remaining bound or attracted to the partner moiety.
  • antibody is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, affinity matured antibodies, humanized antibodies, chimeric antibodies, as well as antigen binding fragments (e.g., Fab, F(ab′) 2 , scFv and Fv), so long as they exhibit the desired biological activity.
  • antibody also includes human antibodies.
  • antibody variable domain refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2, and CDR3), and Framework Regions (FRs).
  • CDRs Complementarity Determining Regions
  • FRs Framework Regions
  • V H refers to the variable domain of the heavy chain.
  • V L refers to the variable domain of the light chain.
  • the amino acid positions assigned to CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). Amino acid numbering of antibodies or antigen binding fragments is also according to that of Kabat.
  • CDRs Complementarity Determining Regions
  • Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.
  • Each complementarity determining region may comprise amino acid residues from a “complementarity determining region” as defined by Kabat (i.e.
  • a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.
  • the CDRH1 of the heavy chain of antibody 4D5 includes amino acids 26 to 35.
  • FR Framework regions
  • Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.
  • the CDRs are defined according to Kabat, the light chain FR residues are positioned at about residues 1-23 (LCFR1), 3549 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are positioned about at residues 1-30 (HCFR1), 3649 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residues.
  • the light chain FR residues are positioned about at residues 1-25 (LCFR1), 3349 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain and the heavy chain FR residues are positioned about at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain residues.
  • the FR residues can be adjusted accordingly.
  • CDRH1 includes amino acids H26-H35
  • the heavy chain FR1 residues are at positions 1-25 and the FR2 residues are at positions 36-49.
  • codon set refers to a set of different nucleotide triplet sequences used to encode desired variant amino acids.
  • a set of oligonucleotides can be synthesized, for example, by solid phase synthesis, including sequences that represent all possible combinations of nucleotide triplets provided by the codon set and that will encode the desired group of amino acids.
  • a standard form of codon designation is that of the IUB code, which is known in the art and described herein.
  • a codon set typically is represented by 3 capital letters in italics, eg. NNK, NNS, XYZ, DVK and the like.
  • oligonucleotides with selected nucleotide “degeneracy” at certain positions is well known in that art, for example the TRIM approach (Knappek et al.; J. Mol. Biol. (1999), 296:57-86); Garrard & Henner, Gene (1993), 128:103).
  • Such sets of oligonucleotides having certain codon sets can be synthesized using commercial nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, Calif.), or can be obtained commercially (for example, from Life Technologies, Rockville, Md.).
  • a set of oligonucleotides synthesized having a particular codon set will typically include a plurality of oligonucleotides with different sequences, the differences established by the codon set within the overall sequence.
  • Oligonucleotides, as used according to the invention have sequences that allow for hybridization to a variable domain nucleic acid template and also can, but does not necessarily, include restriction enzyme sites useful for, for example, cloning purposes.
  • restricted codon set refers to a codon set that encodes a much more limited number of amino acids than the codon sets typically utilized in art methods of generating sequence diversity.
  • restricted codon sets used for sequence diversification encode from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or only 2 amino acids.
  • a restricted codon set used for sequence diversification encodes at least 2 but 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer amino acids.
  • a tetranomial codon set is used. Examples of tetranomial codon sets include those listed in FIG.
  • a binomial codon set is used.
  • binomial codon sets include TMT, KAT, YAC, WAC, TWC, TYT, YTC, WTC, KTT, YCT, MCG, SCG, MGC, SGT, GRT, GKT and GYT.
  • Determination of suitable restricted codons, and the identification of specific amino acids encoded by a particular restricted codon is well known and would be evident to one skilled in the art. Determination of suitable amino acid sets to be used for diversification of a CDR sequence can be empirical and/or guided by criteria known in the art (for e.g., inclusion of a combination of hydrophobic and hydrophilic amino acid types, etc.)
  • an “Fv” fragment is an antibody fragment which contains a complete antigen recognition and binding site.
  • This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H -V L dimer.
  • the six CDRs or a subset thereof confer antigen binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • the “Fab” fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain.
  • F(ab′) 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.
  • Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the scFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H and V L ).
  • V H heavy chain variable domain
  • V L light chain variable domain
  • linear antibodies refers to the antibodies described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H 1-V H -C H 1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
  • Cell Cell
  • cell line cell culture
  • progeny include all progeny of a cell or cell line.
  • terms like “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • Control sequences when referring to expression means DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • coat protein means a protein, at least a portion of which is present on the surface of the virus particle. From a functional perspective, a coat protein is any protein which associates with a virus particle during the viral assembly process in a host cell, and remains associated with the assembled virus until it infects another host cell.
  • the coat protein may be the major coat protein or may be a minor coat protein.
  • a “major” coat protein is generally a coat protein which is present in the viral coat at preferably at least about 5, more preferably at least about 7, even more preferably at least about 10 copies of the protein or more.
  • a major coat protein may be present in tens, hundreds or even thousands of copies per virion.
  • An example of a major coat protein is the p8 protein of filamentous phage.
  • the “detection limit” for a chemical entity in a particular assay is the minimum concentration of that entity which can be detected above the background level for that assay.
  • the “detection limit” for a particular phage displaying a particular antigen binding fragment is the phage concentration at which the particular phage produces an ELISA signal above that produced by a control phage not displaying the antigen binding fragment.
  • a “fusion protein” and a “fusion polypeptide” refers to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property.
  • the property may be a biological property, such as activity in vitro or in vivo.
  • the property may also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc.
  • the two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each other.
  • the two portions of the polypeptide are obtained from heterologous or different polypeptides.
  • Heterologous DNA is any DNA that is introduced into a host cell.
  • the DNA may be derived from a variety of sources including genomic DNA, cDNA, synthetic DNA and fusions or combinations of these.
  • the DNA may include DNA from the same cell or cell type as the host or recipient cell or DNA from a different cell type, for example, from a mammal or plant.
  • the DNA may, optionally, include marker or selection genes, for example, antibiotic resistance genes, temperature resistance genes, etc.
  • highly diverse position refers to a position of an amino acid located in the variable regions of the light and heavy chains that have a number of different amino acid represented at the position when the amino acid sequences of known and/or naturally occurring antibodies or antigen binding fragments are compared.
  • the highly diverse positions are typically in the CDR regions.
  • the ability to determine highly diverse positions in known and/or naturally occurring antibodies is facilitated by the data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991).
  • an amino acid position is highly diverse if it has preferably from about 2 to about 11, preferably from about 4 to about 9, and preferably from about 5 to about 7 different possible amino acid residue variations at that position.
  • an amino acid position is highly diverse if it has preferably at least about 2, preferably at least about 4, preferably at least about 6, and preferably at least about 8 different possible amino acid residue variations at that position.
  • library refers to a plurality of antibody or antibody fragment sequences (for example, polypeptides of the invention), or the nucleic acids that encode these sequences, the sequences being different in the combination of variant amino acids that are introduced into these sequences according to the methods of the invention.
  • “Ligation” is the process of forming phosphodiester bonds between two nucleic acid fragments.
  • the ends of the fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary first to convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation.
  • the DNA is treated in a suitable buffer for at least 15 minutes at 15° C. with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of the four deoxyribonucleotide triphosphates.
  • the DNA is then purified by phenol-chloroform extraction and ethanol precipitation or by silica purification.
  • the DNA fragments that are to be ligated together are put in solution in about equimolar amounts.
  • the solution will also contain ATP, ligase buffer, and a ligase such as T4 DNA ligase at about 10 units per 0.5 ⁇ g of DNA.
  • the vector is first linearized by digestion with the appropriate restriction endonuclease(s).
  • the linearized fragment is then treated with bacterial alkaline phosphatase or calf intestinal phosphatase to prevent self-ligation during the ligation step.
  • a “mutation” is a deletion, insertion, or substitution of a nucleotide(s) relative to a reference nucleotide sequence, such as a wild type sequence.
  • natural or “naturally occurring” antibodies refers to antibodies identified from a nonsynthetic source, for example, from a differentiated antigen-specific B cell obtained ex vivo, or its corresponding hybridoma cell line, or from antibodies obtained from the serum of an animal. These antibodies can include antibodies generated in any type of immune response, either natural or otherwise induced. Natural antibodies include the amino acid sequences, and the nucleotide sequences that constitute or encode these antibodies, for example, as identified in the Kabat database.
  • natural antibodies are different than “synthetic antibodies”, synthetic antibodies referring to antibody sequences that have been changed from a source or template sequence, for example, by the replacement, deletion, or addition, of an amino acid, or more than one amino acid, at a certain position with a different amino acid, the different amino acid providing an antibody sequence different from the source antibody sequence.
  • “Operably linked” when referring to nucleic acids means that the nucleic acids are placed in a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promotor or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contingent and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accord with conventional practice.
  • “Phage display” is a technique by which variant polypeptides are displayed as fusion proteins to at least a portion of coat protein on the surface of phage, e.g., filamentous phage, particles.
  • a utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and efficiently sorted for those sequences that bind to a target antigen with high affinity. Display of peptide and protein libraries on phage has been used for screening millions of polypeptides for ones with specific binding properties. Polyvalent phage display methods have been used for displaying small random peptides and small proteins through fusions to either gene III or gene VIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct.
  • phagemid vectors are used, which simplify DNA manipulations. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).
  • a “phagemid” is a plasmid vector having a bacterial origin of replication, e.g., Co1E1, and a copy of an intergenic region of a bacteriophage.
  • the phagemid may be used on any known bacteriophage, including filamentous bacteriophage and lambdoid bacteriophage.
  • the plasmid will also generally contain a selectable marker for antibiotic resistance. Segments of DNA cloned into these vectors can be propagated as plasmids. When cells harboring these vectors are provided with all genes necessary for the production of phage particles, the mode of replication of the plasmid changes to rolling circle replication to generate copies of one strand of the plasmid DNA and package phage particles.
  • the phagemid may form infectious or non-infectious phage particles.
  • This term includes phagemids which contain a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene as a gene fusion such that the heterologous polypeptide is displayed on the surface of the phage particle.
  • phage vector means a double stranded replicative form of a bacteriophage containing a heterologous gene and capable of replication.
  • the phage vector has a phage origin of replication allowing phage replication and phage particle formation.
  • the phage is preferably a filamentous bacteriophage, such as an M13, f1, fd, Pf3 phage or a derivative thereof, or a lambdoid phage, such as lambda, 21, phi80, phi81, 82, 424, 434, etc., or a derivative thereof.
  • “Oligonucleotides” are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (such as phosphotriester, phosphite, or phosphoramidite chemistry, using solid-phase techniques such as described in EP 266,032 published 4 May 1988, or via deoxynucloside H-phosphonate intermediates as described by Froeshler et al., Nucl. Acids, Res., 14:5399-5407 (1986)). Further methods include the polymerase chain reaction defined below and other autoprimer methods and oligonucleotide syntheses on solid supports. All of these methods are described in Engels et al., Agnew. Chem.
  • oligonucleotides can be purified on polyacrylamide gels or molecular sizing columns or by precipitation.
  • DNA is “purified” when the DNA is separated from non-nucleic acid impurities.
  • the impurities may be polar, non-polar, ionic, etc.
  • a “source antibody”, as used herein, refers to an antibody or antigen binding fragment whose antigen binding sequence serves as the template sequence upon which diversification according to the criteria described herein is performed.
  • An antigen binding sequence generally includes an antibody variable region, preferably at least one CDR, preferably including framework regions.
  • solvent accessible position refers to a position of an amino acid residue in the variable regions of the heavy and light chains of a source antibody or antigen binding fragment that is determined, based on structure, ensemble of structures and/or modeled structure of the antibody or antigen binding fragment, as potentially available for solvent access and/or contact with a molecule, such as an antibody-specific antigen. These positions are typically found in the CDRs and on the exterior of the protein.
  • the solvent accessible positions of an antibody or antigen binding fragment, as defined herein, can be determined using any of a number of algorithms known in the art.
  • solvent accessible positions are determined using coordinates from a 3-dimensional model of an antibody (or portion thereof, for e.g., an antibody variable domain, or CDR segment(s)), preferably using a computer program such as the InsightII program (Accelrys, San Diego, Calif.).
  • Solvent accessible positions can also be determined using algorithms known in the art (e.g., Lee and Richards, J. Mol. Biol. 55, 379 (1971) and Connolly, J. Appl. Cryst. 16, 548 (1983)). Determination of solvent accessible positions can be performed using software suitable for protein modeling and 3-dimensional structural information obtained from an antibody (or portion thereof). Software that can be utilized for these purposes includes SYBYL Biopolymer Module software (Tripos Associates).
  • the “size” of a probe which is used in the calculation is set at about 1.4 Angstrom or smaller in radius.
  • Pacios ((1994) “ARVOMOL/CONTOUR: molecular surface areas and volumes on Personal Computers.” Comput. Chem. 18(4): 377-386; and (1995). “Variations of Surface Areas and Volumes in Distinct Molecular Surfaces of Biomolecules.” J. Mol. Model. 1: 46-53.)
  • a “transcription regulatory element” will contain one or more of the following components: an enhancer element, a promoter, an operator sequence, a repressor gene, and a transcription termination sequence. These components are well known in the art. U.S. Pat. No. 5,667,780.
  • a “transformant” is a cell which has taken up and maintained DNA as evidenced by the expression of a phenotype associated with the DNA (e.g., antibiotic resistance conferred by a protein encoded by the DNA).
  • Transformation means a process whereby a cell takes up DNA and becomes a “transformant”.
  • the DNA uptake may be permanent or transient.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • affinity matured antibody is one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).
  • Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by V H and V L domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al.
  • blocking antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it bind.
  • Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
  • an “agonist antibody”, as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.
  • a salvage receptor binding epitope to the antibody (especially an antibody fragment), as described, e.g., in U.S. Pat. No. 5,739,277.
  • a nucleic acid molecule encoding the salvage receptor binding epitope can be linked in frame to a nucleic acid encoding a polypeptide sequence of this invention so that the fusion protein expressed by the engineered nucleic acid molecule comprises the salvage receptor binding epitope and a polypeptide sequence of this invention.
  • the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG 1 , IgG 2 , IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule (e.g., Ghetie, V et al., (2000) Ann. Rev. Immunol. 18:739-766, Table 1). Antibodies with substitutions in an Fc region thereof and increased serum half-lives are also described in WO00/42072 (Presta, L.), WO 02/060919; Shields, R.
  • the serum half-life can also be increased, for example, by attaching other polypeptide sequences.
  • antibodies of this invention or other polypeptide containing the amino acid sequences of this invention can be attached to serum albumin or a portion of serum albumin that binds to the FcRn receptor or a serum albumin binding peptide so that serum albumin binds to the antibody or polypeptide, e.g., such polypeptide sequences are disclosed in WO01/45746.
  • the serum albumin peptide to be attached comprises an amino acid sequence of DICLPRWGCLW.
  • the half-life of a Fab according to this invention is increased by these methods. See also, Dennis, M. S., et al., (2002) JBC 277(38):35035-35043 for serum albumin binding peptide sequences.
  • a “disorder” is any condition that would benefit from treatment with a substance/molecule or method of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • disorders to be treated herein include malignant and benign tumors; non-leukemias and lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, immunologic and other angiogenesis-related disorders.
  • cell proliferative disorder and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation.
  • the cell proliferative disorder is cancer.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
  • examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • Dysregulation of angiogenesis can lead to many disorders that can be treated by compositions and methods of the invention. These disorders include both non-neoplastic and neoplastic conditions.
  • Neoplastics include but are not limited those described above.
  • Non-neoplastic disorders include but are not limited to undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, corneal graft rejection, retinal/choroidal neovascularization, neovascularization of the angle (rubeosis), ocular
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or disorder.
  • an “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a “therapeutically effective amount” of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents e.g.
  • methotrexate adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below.
  • a tumoricidal agent causes destruction of tumor cells.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topote
  • dynemicin including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubi
  • anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves.
  • SERMs selective estrogen receptor modulators
  • tamoxifen including NOLVADEX® tamoxifen
  • EVISTA® raloxifene droloxifene
  • 4-hydroxytamoxifen trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene
  • anti-progesterones anti-progesterones
  • estrogen receptor down-regulators ETDs
  • agents that function to suppress or shut down the ovaries for example, leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and
  • LHRH leutinizing
  • chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell whose growth is dependent upon activity of a target molecule of interest either in vitro or in vivo.
  • the growth inhibitory agent may be one which significantly reduces the percentage of target molecule-dependent cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
  • Doxorubicin is an anthracycline antibiotic.
  • the full chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy- ⁇ -L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,1-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.
  • a “variant” or “mutant” of a starting or reference polypeptide for e.g., a source antibody or its variable domain(s)/CDR(s)), such as a fusion protein (polypeptide) or a heterologous polypeptide (heterologous to a phage), is a polypeptide that 1) has an amino acid sequence different from that of the starting or reference polypeptide and 2) was derived from the starting or reference polypeptide through either natural or artificial (manmade) mutagenesis.
  • variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of the polypeptide of interest.
  • a fusion polypeptide of the invention generated using an oligonucleotide comprising a restricted codon set that encodes a sequence with a variant amino acid (with respect to the amino acid found at the corresponding position in a source antibody/antigen binding fragment) would be a variant polypeptide with respect to a source antibody and/or antigen binding fragment and/or CDR.
  • a variant CDR refers to a CDR comprising a variant sequence with respect to a starting or reference polypeptide sequence (such as that of a source antibody and/or antigen binding fragment and/or CDR).
  • a variant amino acid in this context, refers to an amino acid different from the amino acid at the corresponding position in a starting or reference polypeptide sequence (such as that of a source antibody and/or antigen binding fragment and/or CDR). Any combination of deletion, insertion, and substitution may be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics.
  • binder sequences contain point mutations such as deletions or additions.
  • a VEGF clone from the YADS library exhibits a missing Q in CDRL3 which was not the result of vector construction.
  • the Q in position 89 of the 4D5 CDRL3 was intentionally deleted in the construction of the vector.
  • amino acid changes also may alter post-translational processes of the polypeptide, such as changing the number or position of glycosylation sites.
  • Methods for generating amino acid sequence variants of polypeptides are described in U.S. Pat. No. 5,534,615, expressly incorporated herein by reference.
  • a “wild type” or “reference” sequence or the sequence of a “wild type” or “reference” protein/polypeptide, such as a coat protein, or a CDR or variable domain of a source antibody, maybe the reference sequence from which variant polypeptides are derived through the introduction of mutations.
  • the “wild type” sequence for a given protein is the sequence that is most common in nature.
  • a “wild type” gene sequence is the sequence for that gene which is most commonly found in nature. Mutations may be introduced into a “wild type” gene (and thus the protein it encodes) either through natural processes or through man induced means. The products of such processes are “variant” or “mutant” forms of the original “wild type” protein or gene.
  • a “plurality” of a substance such as a polypeptide or polynucleotide of the invention, as used herein, generally refers to a collection of two or more types or kinds of the substance. There are two or more types or kinds of a substance if two or more of the substances differ from each other with respect to a particular characteristic, such as the variant amino acid found at a particular amino acid position. For example, there is a plurality of polypeptides of the invention if there are two or more polypeptides of the invention that are substantially the same, preferably identical, in sequence except for the sequence of a variant CDR or except for the variant amino acid at a particular solvent accessible and highly diverse amino acid position.
  • polynucleotides of the invention there is a plurality of polynucleotides of the invention if there are two or more polynucleotides of the invention that are substantially the same, preferably identical, in sequence except for the sequence that encodes a variant CDR or except for the sequence that encodes a variant amino acid for a particular solvent accessible and highly diverse amino acid position.
  • the invention provides methods for generating and isolating novel target antigen binding polypeptides, such as antibodies or antigen binding fragments, that can have a high affinity for a selected antigen.
  • a plurality of different binder polypeptides are prepared by mutating (diversifying) one or more selected amino acid positions in a source antibody light chain variable domain and/or heavy chain variable domain with restricted codon sets to generate a library of with variant amino acids in at least one CDR sequence, wherein the number of types of variant amino acids is kept to a minimum (i.e., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, or only 2, but generally at least 2).
  • amino acid positions include those that are solvent accessible, for example as determined by analyzing the structure of a source antibody, and/or that are highly diverse among known and/or natural occurring immunoglobulin polypeptides.
  • a further advantage afforded by the limited nature of diversification of the invention is that additional amino acid positions other than those that are highly diverse and/or solvent accessible can also be diversified in accordance with the need or desire of the practitioner; examples of these embodiments are described herein.
  • amino acid positions that are solvent accessible and highly diverse are preferably those in the CDR regions of the antibody variable domains selected from the group consisting of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, CDRH3, and mixtures thereof.
  • Amino acid positions are each mutated using a restricted codon set encoding a limited number of amino acids, the choice of amino acids generally being independent of the commonly occurring amino acids at each position.
  • a codon set is selected that encodes preferably from 2 to 10, preferably from 2 to 8, preferably from 2 to 6, preferably from 2 to 4, preferably only 2 amino acids.
  • a codon set when a solvent accessible and highly diverse position in a CDR region is to be mutated, a codon set is selected that encodes preferably from 2 to 10, from 3 to 9, from 4 to 8, from 5 to 7 amino acids. In some embodiments, a codon set encodes at least 2, but 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer amino acids.
  • CDR sequences can also be diversified by varying the length, for e.g., for CDRH3, variant CDRH3 regions can be generated that have different lengths and/or are randomized at selected positions using restricted codon sets.
  • the diversity of the library of the polypeptides comprising variant CDRs is designed using codon sets that encode only a limited number of amino acids, such that a minimum but sufficient amount of sequence diversity is introduced into a CDR.
  • the number of positions mutated in the CDR is minimized and the variant amino acids at each position are designed to include a limited number of amino acids, independent of the amino acids that deemed to be commonly occurring at that position in known and/or naturally occurring CDRs.
  • a single antibody, including at least one CDR is used as the source antibody. It is surprising that a library of antibody variable domains having diversity in sequences and size can be generated using a single source antibody as a template and targeting diversity to particular positions using an unconventionally limited number of amino acid substitutions.
  • high quality libraries of antibody variable domains are generated.
  • the libraries have restricted diversity of different sequences of CDR sequences, for e.g., diversity of the antibody variable domains.
  • the libraries include high affinity binding antibody variable domains for one or more antigens, including, for example, neutravidin, an apoptosis protein (AP), maltose binding protein 2 (MBP2), erbin-GST, insulin, murine and human VEGF.
  • the diversity in the library is designed by selecting amino acid positions that are solvent accessible and highly diverse in a single source antibody and mutating those positions in at least one CDR using restricted codon sets.
  • the restricted codon set preferably encodes preferably fewer 10, 8, 6, 4 amino acids, or encodes only 2 amino acids.
  • a source antibody is humanized antibody 4D5, but the methods for diversification can be applied to other source antibodies whose sequence is known.
  • a source antibody can be a naturally occurring antibody, synthetic antibody, recombinant antibody, humanized antibody, germ line derived antibody, chimeric antibody, affinity matured antibody, or antigen binding fragment thereof.
  • the antibodies can be obtained from a variety of mammalian species including humans, mice and rats.
  • a source antibody is an antibody that is obtained after one or more initial affinity screening rounds, but prior to an affinity maturation step(s).
  • a source antibody may be selected or modified to provide for high yield and stability when produced in cell culture.
  • Antibody 4D5 is a humanized antibody specific for a cancer-associated antigen known as Her-2 (erbB2).
  • the antibody includes variable domains having consensus framework regions; a few positions were reverted to mouse sequence during the process of increasing affinity of the humanized antibody.
  • the sequence and crystal structure of humanized antibody 4D5 have been described in U.S. Pat. No. 6,054,297, Carter et al, PNAS 89:4285 (1992), the crystal structure is shown in J. Mol. Biol. 229:969 (1993) and online at www/ncbi/nih/gov/structure/mmdb(MMDB#s-990-992).
  • a criterion for generating diversity in antibody variable domains is to mutate residues at positions that are solvent accessible (as defined above). These positions are typically found in the CDRs, and are typically on the exterior of the protein.
  • solvent accessible positions are determined using coordinates from a 3-dimensional model of an antibody, using a computer program such as the InsightII program (Accelrys, San Diego, Calif.). Solvent accessible positions can also be determined using algorithms known in the art (e.g., Lee and Richards, J. Mol. Biol. 55, 379 (1971) and Connolly, J. Appl. Cryst. 16, 548 (1983)). Determination of solvent accessible positions can be performed using software suitable for protein modeling and 3-dimensional structural information obtained from an antibody.
  • selection of solvent accessible residues is further refined by choosing solvent accessible residues that collectively form a minimum contiguous patch, for example when the reference polypeptide or source antibody is in its 3-D folded structure.
  • a compact (minimum) contiguous patch is formed by residues selected for CDRH1/H2/H3/L1/L2/L3 of humanized 4D5.
  • a compact (minimum) contiguous patch may comprise only a subset (for example, 2-5 CDRs) of the full range of CDRs, for example, CDRH1/H2/H3/L3. Solvent accessible residues that do not contribute to formation of such a patch may optionally be excluded from diversification.
  • this selection criterion permits the practitioner to minimize, as desired, the number of residues to be diversified.
  • residue 28 in H1 can optionally be excluded in diversification since it is on the edge of the patch.
  • this selection criterion can also be used, where desired, to choose residues to be diversified that may not necessarily be deemed solvent accessible.
  • a residue that is not deemed solvent accessible, but forms a contiguous patch in the 3-D folded structure with other residues that are deemed solvent accessible may be selected for diversification.
  • An example of this is CDRL1-29. Selection of such residues would be evident to one skilled in the art, and its appropriateness can also be determined empirically and according to the needs and desires of the skilled practitioner.
  • positions to be mutated are those positions which show variability in amino acid sequence when the sequences of known and/or natural-antibodies are compared.
  • a highly diverse position refers to a position of an amino acid located in the variable regions of the light or heavy chains that have a number of different amino acids represented at the position when the amino acid sequences of known and/or natural antibodies/antigen binding fragments are compared.
  • the highly diverse positions are preferably in the CDR regions.
  • the positions of CDRH3 are all considered highly diverse.
  • amino acid residues are highly diverse if they have preferably from about 2 to about 11 (although the numbers can range as described herein) different possible amino acid residue variations at that position.
  • identification of highly diverse positions in known and/or naturally occurring antibodies is facilitated by the data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991).
  • An internet-based database located at http/immuno/bme/nwu/edu provides an extensive collection and alignment of human light and heavy chain sequences and facilitates determination of highly diverse positions in these sequences.
  • the diversity at the solvent accessible positions of humanized antibody 4D5 in known and/or naturally occurring light and heavy chains is shown in FIGS. 22 and 23 .
  • the highly diverse and solvent accessible residues in at least one, two, three, four, five or all of CDRs selected from the group consisting of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, CDRH3, and mixtures thereof are mutated (i.e., randomized using restricted codon sets as described herein).
  • a population of polypeptides may be generated by diversifying at least one solvent accessible and/or highly diverse residue in CDRL3 and CDRH3 using restricted codons.
  • the invention provides for a large number of novel antibody sequences formed by replacing at least one solvent accessible and highly diverse position of at least one CDR of the source antibody variable domain with variant amino acids encoded by a restricted codon.
  • a variant CDR or antibody variable domain can comprise a variant amino acid in one or more amino acid positions 28, 30, 31, 32 and/or 33 of CDRH1; and/or in one or more amino acid positions 50, 52, 53, 54, 56 and/or 58 of CDRH2; and/or in one or more amino acid positions 28, 29, 30 and/or 31 of CDRL1; and/or in one or more amino acid positions 50 and/or 53 in CDRL2; and/or in one or more amino acid positions 91, 92, 93, 94 and/or 96 in CDRL3.
  • the variant amino acids at these positions are encoded by restricted codon sets, as described herein.
  • a codon set is a set of different nucleotide triplet sequences which can be used to form a set of oligonucleotides used to encode the desired group of amino acids.
  • a set of oligonucleotides can be synthesized, for example, by solid phase synthesis, containing sequences that represent all possible combinations of nucleotide triplets provided by the codon set and that will encode the desired group of amino acids. Synthesis of oligonucleotides with selected nucleotide “degeneracy” at certain positions is well known in that art.
  • Such sets of nucleotides having certain codon sets can be synthesized using commercial nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, Calif.), or can be obtained commercially (for example, from Life Technologies, Rockville, Md.). Therefore, a set of oligonucleotides synthesized having a particular codon set will typically include a plurality of oligonucleotides with different sequences, the differences established by the codon set within the overall sequence. Oligonucleotides, as used according to the invention, have sequences that allow for hybridization to a variable domain nucleic acid template and also can include restriction enzyme sites for cloning purposes.
  • the restricted repertoire of amino acids intended to occupy one or more of the solvent accessible and highly diverse positions in CDRs of humanized antibody 4D5 are determined (based on the desire of the practitioner, which can be based on any of a number of criteria, including specific amino acids desired for particular positions, specific amino acid(s) desired to be absent from a particular position, size of library desired, characteristic of antigen binders sought, etc.).
  • CDRH3s Heavy chain CDR3s in known antibodies have diverse sequences, structural conformations, and lengths. CDRH3s are often found in the middle of the antigen binding pocket and often participate in antigen contact. The design of CDRH3 is thus preferably developed separately from that of the other CDRs because it can be difficult to predict the structural conformation of CDRH3 and the amino acid diversity in this region is especially diverse in known antibodies. In accordance with the present invention, CDRH3 is designed to generate diversity at specific positions within CDRH3, for e.g., positions 95, 96, 97, 98, 99, 100 and 100a (for e.g., according to Kabat numbering in 4D5).
  • diversity is also generated by varying CDRH3 length using restricted codon sets.
  • Length diversity can be of any range determined empirically to be suitable for generating a population of polypeptides containing substantial proportions of antigen binding proteins.
  • Illustrative embodiments of oligonucleotides that can be utilized to provide for variety in CDRH3 sequence length include those shown in FIG. 2 and FIG. 9 .
  • sequence diversity of libraries created by introduction of variant amino acids in a particular CDR can be increased by combining the variant CDR with other CDRs comprising variations in other regions of the antibody, specifically in other CDRs of either the light or heavy chain variable sequences.
  • nucleic acid sequences that encode members of this set can be further diversified by introduction of other variant amino acids in the CDRs of either the light or heavy chain sequences, via codon sets.
  • CDRH3 sequences from fusion polypeptides that bind a target antigen can be combined with diversified CDRL3, CDRH1, or CDRH2 sequences, or any combination of diversified CDRs.
  • framework residues may be varied relative to the sequence of a source antibody or antigen binding fragment, for example, to reflect a consensus sequence or to improve stability or display.
  • framework residues 49, 93, 94 or 71 in the heavy chain may be varied.
  • Heavy chain framework residue 93 may be serine or alanine (which is the human consensus sequence amino acid at that position.)
  • Heavy chain framework residue 94 may be changed to reflect framework consensus sequence from threonine to arginine or lysine.
  • Another example of a framework residue that may be altered is heavy chain framework residue 71, which is R in about 1970 polypeptides, V in about 627 polypeptides and A in about 527 polypeptides, as found in the Kabat database.
  • Heavy chain framework residue 49 may be alanine or glycine.
  • the 3 N-terminal amino acids of the heavy chain variable domain can be removed.
  • the arginine at amino acid position 66 can be changed to glycine.
  • the invention provides vector constructs for generating fusion polypeptides that bind with significant affinity to potential ligands.
  • These constructs comprise a dimerizable domain that when present in a fusion polypeptide provides for increased tendency for heavy chains to dimerize to form dimers of Fab or Fab′ antibody fragments/portions.
  • These dimerization domains may include, eg. a heavy chain hinge sequence (for e.g., a sequence comprising TCPPCPAPELLG (SEQ ID NO: 120) that may be present in the fusion polypeptide.
  • Dimerization domains in fusion phage polypeptides bring two sets of fusion polypeptides (LC/HC-phage protein/fragment (such as pIII)) together, thus allowing formation of suitable linkages (such as interheavy chain disulfide bridges) between the two sets of fusion polypeptide.
  • Vector constructs containing such dimerization domains can be used to achieve divalent display of antibody variable domains, for example the diversified fusion proteins described herein, on phage.
  • the intrinsic affinity of each monomeric antibody fragment (fusion polypeptide) is not significantly altered by fusion to the dimerization domain.
  • dimerization results in divalent phage display which provides increased avidity of phage binding, with significant decrease in off-rate, which can be determined by methods known in the art and as described herein.
  • Dimerization domain-containing vectors of the invention may or may not also include an amber stop codon after the dimerization domain.
  • Dimerization can be varied to achieve different display characteristics.
  • Dimerization domains can comprise a sequence comprising a cysteine residue, a hinge region from a full-length antibody, a dimerization sequence such as leucine zipper sequence or GCN4 zipper sequence or mixtures thereof.
  • Dimerization sequences are known in the art, and include, for example, the GCN4 zipper sequence (GRMKQLEDKVEELLSKNYHLENEVARLKKLVGERG) (SEQ ID NO: 3).
  • GCN4 zipper sequence GCN4 zipper sequence (GRMKQLEDKVEELLSKNYHLENEVARLKKLVGERG) (SEQ ID NO: 3).
  • GCN4 zipper sequence GCN4 zipper sequence
  • the dimerization domain is preferably located at the C-terminal end of the heavy chain variable or constant domain sequence and/or between the heavy chain variable or constant domain sequence and any viral coat protein component sequence.
  • An amber stop codon may also be present at or after the C-terminal end of the dimerization domain.
  • the dimerization domain encodes at least one cysteine and a dimerizing sequence such as leucine zipper. In another embodiment, wherein no amber stop codon is present, the dimerization domain may comprise a single cysteine residue.
  • polypeptides of the invention can also be fused to other types of polypeptides in order to provide for display of the variant polypeptides or to provide for purification, screening or sorting, and detection of the polypeptide.
  • the polypeptides of the invention are fused to all or a portion of a viral coat protein.
  • viral coat protein include protein PIII, major coat protein, pVIII, Soc, Hoc, gpD, pVI and variants thereof.
  • the variant polypeptides generated according to the methods of the invention can optionally be fused to a polypeptide marker or tag such as FLAG, polyhistidine, gD, c-myc, B-galactosidase and the like.
  • libraries can be created by targeting solvent accessible and/or highly diverse positions in at least one CDR region for amino acid substitution with variant amino acids using the Kunkel method. See, for e.g., Kunkel et al., Methods Enzymol. (1987), 154:367-382. Generation of randomized sequences is also described below in the Examples.
  • the sequence of oligonucleotides includes one or more of the designed restricted codon sets for different lengths of CDRH3 or for the solvent accessible and highly diverse positions in a CDR.
  • a codon set is a set of different nucleotide triplet sequences used to encode desired variant amino acids. Codon sets can be represented using symbols to designate particular nucleotides or equimolar mixtures of nucleotides as shown below according to the IUB code. Typically, a codon set is represented by three capital letters eg. KMT, TMT and the like.
  • TMT is the nucleotide thymine; and M can be A or C.
  • This codon set can present multiple codons and can encode only a limited number of amino acids, namely tyrosine and serine.
  • Oligonucleotide or primer sets can be synthesized using standard methods.
  • a set of oligonucleotides can be synthesized, for example, by solid phase synthesis, containing sequences that represent all possible combinations of nucleotide triplets provided by the restricted codon set and that will encode the desired restricted group of amino acids. Synthesis of oligonucleotides with selected nucleotide “degeneracy” at certain positions is well known in that art.
  • Such sets of oligonucleotides having certain codon sets can be synthesized using commercial nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, Calif.), or can be obtained commercially (for example, from Life Technologies, Rockville, Md.).
  • a set of oligonucleotides synthesized having a particular codon set will typically include a plurality of oligonucleotides with different sequences, the differences established by the codon set within the overall sequence.
  • Oligonucleotides, as used according to the invention have sequences that allow for hybridization to a CDR (for e.g., as contained within a variable domain) nucleic acid template and also can include restriction enzyme sites for cloning purposes.
  • nucleic acid sequences encoding variant amino acids can be created by oligonucleotide-mediated mutagenesis of a nucleic acid sequence encoding a source or template polypeptide such as the antibody variable domain of 4D5. This technique is well known in the art as described by Zoller et al. Nucleic Acids Res. 10:6487-6504(1987). Briefly, nucleic acid sequences encoding variant amino acids are created by hybridizing an oligonucleotide set encoding the desired restricted codon sets to a DNA template, where the template is the single-stranded form of the plasmid containing a variable region nucleic acid template sequence.
  • DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will contain the restricted codon sets as provided by the oligonucleotide set.
  • Nucleic acids encoding other source or template molecules are known or can be readily determined.
  • oligonucleotides of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have at least 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation(s). This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
  • the oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al., Proc. Natl. Acad. Sci. USA, 75:5765 (1978).
  • the DNA template is generated by those vectors that are either derived from bacteriophage M13 vectors (the commercially available M13mp18 and M13mp19 vectors are suitable), or those vectors that contain a single-stranded phage origin of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987).
  • the DNA that is to be mutated can be inserted into one of these vectors in order to generate single-stranded template. Production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al., above.
  • the oligonucleotide is hybridized to the single stranded template under suitable hybridization conditions.
  • a DNA polymerizing enzyme usually T7 DNA polymerase or the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis.
  • a heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of gene 1, and the other strand (the original template) encodes the native, unaltered sequence of gene 1.
  • This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JM101. After growing the cells, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabelled with a 32-Phosphate to identify the bacterial colonies that contain the mutated DNA.
  • the method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s).
  • the modifications are as follows:
  • the single stranded oligonucleotide is annealed to the single-stranded template as described above.
  • a mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTT) is combined with a modified thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from Amersham). This mixture is added to the template-oligonucleotide complex.
  • this new strand of DNA Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated.
  • this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion.
  • the template strand can be digested with ExoIII nuclease or another appropriate nuclease past the region that contains the site(s) to be mutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded.
  • a complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell.
  • the sequence of the oligonucleotide set is of sufficient length to hybridize to the template nucleic acid and may also, but does not necessarily, contain restriction sites.
  • the DNA template can be generated by those vectors that are either derived from bacteriophage M13 vectors or vectors that contain a single-stranded phage origin of replication as described by Viera et al. ((1987) Meth. Enzymol., 153:3). Thus, the DNA that is to be mutated must be inserted into one of these vectors in order to generate single-stranded template. Production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al., supra.
  • a library can be generated by providing upstream and downstream oligonucleotide sets, each set having a plurality of oligonucleotides with different sequences, the different sequences established by the codon sets provided within the sequence of the oligonucleotides.
  • the upstream and downstream oligonucleotide sets, along with a variable domain template nucleic acid sequence, can be used in a polymerase chain reaction to generate a “library” of PCR products.
  • the PCR products can be referred to as “nucleic acid cassettes”, as they can be fused with other related or unrelated nucleic acid sequences, for example, viral coat protein components and dimerization domains, using established molecular biology techniques.
  • the sequence of the PCR primers includes one or more of the designed codon sets for the solvent accessible and highly diverse positions in a CDR region.
  • a codon set is a set of different nucleotide triplet sequences used to encode desired variant amino acids.
  • Oligonucleotide sets can be used in a polymerase chain reaction using a variable region nucleic acid template sequence as the template to create nucleic acid cassettes.
  • the variable region nucleic acid template sequence can be any portion of the light or heavy immunoglobulin chains containing the target nucleic acid sequences (ie., nucleic acid sequences encoding amino acids targeted for substitution).
  • the variable region nucleic acid template sequence is a portion of a double stranded DNA molecule having a first nucleic acid strand and complementary second nucleic acid strand.
  • the variable region nucleic acid template sequence contains at least a portion of a variable domain and has at least one CDR. In some cases, the variable region nucleic acid template sequence contains more than one CDR.
  • An upstream portion and a downstream portion of the variable region nucleic acid template sequence can be targeted for hybridization with members of an upstream oligonucleotide set and a downstream oligonucleotide set.
  • a first oligonucleotide of the upstream primer set can hybridize to the first nucleic acid strand and a second oligonucleotide of the downstream primer set can hybridize to the second nucleic acid strand.
  • the oligonucleotide primers can include one or more codon sets and be designed to hybridize to a portion of the variable region nucleic acid template sequence. Use of these oligonucleotides can introduce two or more codon sets into the PCR product (ie., the nucleic acid cassette) following PCR.
  • the oligonucleotide primer that hybridizes to regions of the nucleic acid sequence encoding the antibody variable domain includes portions that encode CDR residues that are targeted for amino acid substitution.
  • the upstream and downstream oligonucleotide sets can also be synthesized to include restriction sites within the oligonucleotide sequence. These restriction sites can facilitate the insertion of the nucleic acid cassettes [ie., PCR reaction products] into an expression vector having additional antibody sequences. Preferably, the restriction sites are designed to facilitate the cloning of the nucleic acid cassettes without introducing extraneous nucleic acid sequences or removing original CDR or framework nucleic acid sequences.
  • Nucleic acid cassettes can be cloned into any suitable vector for expression of a portion or the entire light or heavy chain sequence containing the targeted amino acid substitutions generated. According to methods detailed in the invention, the nucleic acid cassette is cloned into a vector allowing production of a portion or the entire light or heavy chain sequence fused to all or a portion of a viral coat protein (ie., creating a fusion protein) and displayed on the surface of a particle or cell. While several types of vectors are available and may be used to practice this invention, phagemid vectors are the preferred vectors for use herein, as they may be constructed with relative ease, and can be readily amplified. Phagemid vectors generally contain a variety of components including promoters, signal sequences, phenotypic selection genes, origin of replication sites, and other necessary components as are known to those of ordinary skill in the art.
  • the nucleic acid cassette contains a sequence that is able to encode all or a portion of the heavy or light chain variable domain, and is able to encode the variant amino acid combinations.
  • the nucleic acid cassettes can be inserted into an expression vector containing additional antibody sequence, for example all or portions of the variable or constant domains of the light and heavy chain variable regions.
  • additional antibody sequences can also be fused to other nucleic acid sequences, such as sequences which encode viral coat protein components and therefore allow production of a fusion protein.
  • One aspect of the invention includes a replicable expression vector comprising a nucleic acid sequence encoding a gene fusion, wherein the gene fusion encodes a fusion protein comprising a CDR-containing polypeptide (such as an antibody variable domain), or an antibody variable domain and a constant domain, fused to all or a portion of a viral coat protein. Also included is a library of diverse replicable expression vectors comprising a plurality of gene fusions encoding a plurality of different fusion proteins including a plurality of the fusion polypeptides generated with diverse sequences as described above.
  • the vectors can include a variety of components and may be constructed to allow for movement of antibody variable domain between different vectors and/or to provide for display of the fusion proteins in different formats.
  • a phage vector generally has a phage origin of replication allowing phage replication and phage particle formation.
  • the phage is generally a filamentous bacteriophage, such as an M13, f1, fd, Pf3 phage or a derivative thereof, or a lambdoid phage, such as lambda, 21, phi80, phi81, 82, 424, 434, etc., or a derivative thereof.
  • viral coat proteins examples include infectivity protein PIII (sometimes also designated p3), major coat protein PVIII, Soc (T4), Hoc (T4), gpD (of bacteriophage lambda), minor bacteriophage coat protein 6 (pVI) (filamentous phage; J Immunol Methods. 1999 Dec. 10;231 (1-2):39-51), variants of the M13 bacteriophage major coat protein (P8) ( Protein Sci 2000 April; 9(4):647-54).
  • infectivity protein PIII sometimes also designated p3
  • major coat protein PVIII major coat protein
  • Soc T4
  • Hoc T4
  • gpD of bacteriophage lambda
  • pVI minor bacteriophage coat protein 6
  • P8 Protein Sci 2000 April; 9(4):647-54
  • the fusion protein can be displayed on the surface of a phage and suitable phage systems include M13KO7 helper phage, M13R408, M13-VCS, and Phi X 174, pJuFo phage system (J. Virol. 2001 August; 75(15):7107-13.v), hyperphage ( Nat Biotechnol. 2001 January; 19(1):75-8).
  • the preferred helper phage is M13KO7
  • the preferred coat protein is the M 13 Phage gene III coat protein.
  • the preferred host is E. coli , and protease deficient strains of E. coli .
  • Vectors, such as the fth 1 vector Nucleic Acids Res. 2001 May 15;29(10):E50-0
  • Vectors such as the fth 1 vector ( Nucleic Acids Res. 2001 May 15;29(10):E50-0) can be useful for the expression of the fusion protein.
  • the expression vector also can have a secretory signal sequence fused to the DNA encoding a CDR-containing fusion polypeptide (for e.g., each subunit of an antibody, or fragment thereof).
  • This sequence is typically located immediately 5′ to the gene encoding the fusion protein, and will thus be transcribed at the amino terminus of the fusion protein.
  • the signal sequence has been demonstrated to be located at positions other than 5′ to the gene encoding the protein to be secreted. This sequence targets the protein to which it is attached across the inner membrane of the bacterial cell.
  • the DNA encoding the signal sequence may be obtained as a restriction endonuclease fragment from any gene encoding a protein that has a signal sequence.
  • Suitable prokaryotic signal sequences may be obtained from genes encoding, for example, LamB or OmpF (Wong et al., Gene, 68:1931 (1983), MalE, PhoA and other genes.
  • a prokaryotic signal sequence for practicing this invention is the E. coli heat-stable enterotoxin II (STII) signal sequence as described by Chang et al., Gene 55:189 (1987), and/or malE.
  • STII E. coli heat-stable enterotoxin II
  • a vector also typically includes a promoter to drive expression of the fusion polypeptide.
  • Promoters most commonly used in prokaryotic vectors include the lac Z promoter system, the alkaline phosphatase pho A promoter (Ap), the bacteriophage l PL promoter (a temperature sensitive promoter), the tac promoter (a hybrid trp-lac promoter that is regulated by the lac repressor), the tryptophan promoter, and the bacteriophage T7 promoter.
  • the lac Z promoter system the alkaline phosphatase pho A promoter (Ap)
  • the bacteriophage l PL promoter a temperature sensitive promoter
  • the tac promoter a hybrid trp-lac promoter that is regulated by the lac repressor
  • tryptophan promoter a hybrid trp-lac promoter that is regulated by the lac repressor
  • the tryptophan promoter a hybrid trp-lac promoter that is
  • the vector can also include other nucleic acid sequences, for example, sequences encoding gD tags, c-Myc epitopes, poly-histidine tags, fluorescence proteins (eg., GFP), or beta-galactosidase protein which can be useful for detection or purification of the fusion protein expressed on the surface of the phage or cell.
  • Nucleic acid sequences encoding, for example, a gD tag also provide for positive or negative selection of cells or virus expressing the fusion protein.
  • the gD tag is preferably fused to an antibody variable domain which is not fused to the viral coat protein component.
  • Nucleic acid sequences encoding, for example, a polyhistidine tag are useful for identifying fusion proteins including antibody variable domains that bind to a specific antigen using immunohistochemistry. Tags useful for detection of antigen binding can be fused to either an antibody variable domain not fused to a viral coat protein component or an antibody variable domain fused to a viral coat protein component.
  • phenotypic selection genes are those encoding proteins that confer antibiotic resistance upon the host cell.
  • ampicillin resistance gene ampr
  • tetr tetracycline resistance gene
  • the vector can also include nucleic acid sequences containing unique restriction sites and suppressible stop codons.
  • the unique restriction sites are useful for moving antibody variable domains between different vectors and expression systems, especially useful for production of full-length antibodies or antigen binding fragments in cell cultures.
  • the suppressible stop codons are useful to control the level of expression of the fusion protein and to facilitate purification of soluble antibody fragments.
  • an amber stop codon can be read as Gln in a supE host to enable phage display, while in a non-supE host it is read as a stop codon to produce soluble antibody fragments without fusion to phage coat proteins.
  • vector systems that allow the nucleic acid encoding an antibody sequence of interest, for example a CDR having variant amino acids, to be easily removed from the vector system and placed into another vector system.
  • appropriate restriction sites can be engineered in a vector system to facilitate the removal of the nucleic acid sequence encoding an antibody or antibody variable domain having variant amino acids.
  • the restriction sequences are usually chosen to be unique in the vectors to facilitate efficient excision and ligation into new vectors.
  • Antibodies or antibody variable domains can then be expressed from vectors without extraneous fusion sequences, such as viral coat proteins or other sequence tags.
  • DNA encoding a termination or stop codon may be inserted, such termination codons including UAG (amber), UAA (ocher) and UGA (opel).
  • UAG amber
  • UAA ocher
  • UGA opel
  • the termination or stop codon expressed in a wild type host cell results in the synthesis of the gene 1 protein product without the gene 2 protein attached.
  • growth in a suppressor host cell results in the synthesis of detectable quantities of fused protein.
  • Such suppressor host cells are well known and described, such as E. coli suppressor strain (Bullock et al., BioTechniques 5:376-379 (1987)). Any acceptable method may be used to place such a termination codon into the mRNA encoding the fusion polypeptide.
  • the suppressible codon may be inserted between the first gene encoding an antibody variable or constant domain, and a second gene encoding at least a portion of a phage coat protein.
  • the suppressible termination codon may be inserted adjacent to the fusion site by replacing the last amino acid triplet in the antibody variable domain or the first amino acid in the phage coat protein.
  • the suppressible termination codon may be located at or after the C-terminal end of a dimerization domain.
  • the antibody variable domain When the plasmid is grown in a non-suppressor host cell, the antibody variable domain is synthesized substantially without fusion to the phage coat protein due to termination at the inserted suppressible triplet UAG, UAA, or UGA. In the non-suppressor cell the antibody variable domain is synthesized and secreted from the host cell due to the absence of the fused phage coat protein which otherwise anchored it to the host membrane.
  • the CDR being diversified may have a stop codon engineered in the template sequence (referred to herein as a “stop template”).
  • stop template a stop codon engineered in the template sequence
  • This feature provides for detection and selection of successfully diversified sequences based on successful repair of the stop codon(s) in the template sequence due to incorporation of the oligonucleotide(s) comprising the sequence(s) for the variant amino acids of interest. This feature is further illustrated in the Examples below.
  • the light and/or heavy chain antibody variable or constant domains can also be fused to an additional peptide sequence, the additional peptide sequence providing for the interaction of one or more fusion polypeptides on the surface of the viral particle or cell.
  • additional peptide sequences are herein referred to as “dimerization domains”.
  • Dimerization domains may comprise at least one or more of a dimerization sequence, or at least one sequence comprising a cysteine residue or both.
  • Suitable dimerization sequences include those of proteins having amphipathic alpha helices in which hydrophobic residues are regularly spaced and allow the formation of a dimer by interaction of the hydrophobic residues of each protein; such proteins and portions of proteins include, for example, leucine zipper regions.
  • Dimerization domains can also comprise one or more cysteine residues (e.g. as provided by inclusion of an antibody hinge sequence within the dimerization domain).
  • the cysteine residues can provide for dimerization by formation of one or more disulfide bonds.
  • the dimerization domain comprises at least one cysteine residue.
  • the dimerization domains are preferably located between the antibody variable or constant domain and the viral coat protein component.
  • the vector encodes a single antibody-phage polypeptide in a single chain form containing, for example, both the heavy and light chain variable regions fused to a coat protein.
  • the vector is considered to be “monocistronic”, expressing one transcript under the control of a certain promoter.
  • a vector may utilize a promoter (such as the alkaline phosphatase (AP) or Tac promoter) to drive expression of a monocistronic sequence encoding VL and VH domains, with a linker peptide between the VL and VH domains.
  • This cistronic sequence may be connected at the 5′ end to a signal sequence (such as an E.
  • a vector may further comprise a sequence encoding a dimerization domain (such as a leucine zipper) at its 3′ end, between the second variable domain sequence (for e.g., V H ) and the viral coat protein sequence. Fusion polypeptides comprising the dimerization domain are capable of dimerizing to form a complex of two scFv polypeptides (referred to herein as “(ScFv)2-pIII)”).
  • a dimerization domain such as a leucine zipper
  • variable regions of the heavy and light chains can be expressed as separate polypeptides, the vector thus being “bicistronic”, allowing the expression of separate transcripts.
  • a suitable promoter such as the Ptac or PhoA promoter, is used to drive expression of a bicistronic message.
  • a first cistron encoding for example, a light chain variable and constant domain, may be connected at the 5′ end to a signal sequence, such as E. coli malE or heat-stable enterotoxin II (STII) signal sequence, and at the 3′ end to a nucleic acid sequence encoding a tag sequence, such as gD tag.
  • a signal sequence such as E. coli malE or heat-stable enterotoxin II (STII) signal sequence
  • a second cistron encoding, for example, a heavy chain variable domain and constant domain CH1 is connected at its 5′ end to a signal sequence, such as E. coli malE or heat-stable enterotoxin II (STII) signal sequence, and at the 3′ end to all or a portion of a viral coat protein.
  • a signal sequence such as E. coli malE or heat-stable enterotoxin II (STII) signal sequence
  • a suitable promoter such as Ptac or PhoA (AP) promoter, drives expression of a first cistron encoding a light chain variable and constant domain operably linked at 5′ end to a signal sequence such as the E. coli malE or heat stable enteroxtoxin II (STII) signal sequence, and at the 3′ end to a nucleic acid sequence encoding a tag sequence such as gD tag.
  • the second cistron encodes, for example, a heavy chain variable and constant domain operatively linked at 5′ end to a signal sequence such as E. coli malE or heat stable enterotoxin II (STI) signal sequence, and at 3′ end has a dimerization domain comprising IgG hinge sequence and a leucine zipper sequence followed by at least a portion of viral coat protein.
  • Fusion polypeptides of a CDR-containing polypeptide can be displayed on the surface of a cell, virus, or phagemid particle in a variety of formats.
  • These formats include single chain Fv fragment (scFv), F(ab) fragment and multivalent forms of these fragments.
  • multivalent forms include a dimer of ScFv, Fab, or F(ab′), herein referred to as (ScFv) 2 , F(ab) 2 and F(ab′) 2 , respectively.
  • the multivalent forms of display are advantageous in some contexts in part because they have more than one antigen binding site which generally results in the identification of lower affinity clones and also allows for more efficient sorting of rare clones during the selection process.
  • nucleic acid sequences encoding an antibody variable light chain domain and an antibody variable heavy chain variable domain When a vector is constructed for display in a scFv format, it includes nucleic acid sequences encoding an antibody variable light chain domain and an antibody variable heavy chain variable domain. Typically, the nucleic acid sequence encoding an antibody variable heavy chain domain is fused to a viral coat protein component. One or both of the antibody variable domains can have variant amino acids in at least one CDR region.
  • the nucleic acid sequence encoding the antibody variable light chain is connected to the antibody variable heavy chain domain by a nucleic acid sequence encoding a peptide linker.
  • the peptide linker typically contains about 5 to 15 amino acids.
  • other sequences encoding, for example, tags useful for purification or detection can be fused at the 3′ end of either the nucleic acid sequence encoding the antibody variable light chain or antibody variable heavy chain domain or both.
  • a vector When a vector is constructed for F(ab) display, it includes nucleic acid sequences encoding antibody variable domains and antibody constant domains.
  • a nucleic acid encoding a variable light chain domain is fused to a nucleic acid sequence encoding a light chain constant domain.
  • a nucleic acid sequence encoding an antibody heavy chain variable domain is fused to a nucleic acid sequence encoding a heavy chain constant CH1 domain.
  • the nucleic acid sequence encoding the heavy chain variable and constant domains are fused to a nucleic acid sequence encoding all or part of a viral coat protein.
  • One or both of the antibody variable light or heavy chain domains can have variant amino acids in at least one CDR.
  • the heavy chain variable and constant domains are expressed as a fusion with at least a portion of a viral coat protein, and the light chain variable and constant domains are expressed separately from the heavy chain viral coat fusion protein.
  • the heavy and light chains associate with one another, which may be by covalent or non-covalent bonds.
  • other sequences encoding, for example, polypeptide tags useful for purification or detection can be fused at the 3′ end of either the nucleic acid sequence encoding the antibody light chain constant domain or antibody heavy chain constant domain or both.
  • a bivalent moiety for example, a F(ab) 2 dimer or F(ab′) 2 dimer, is used for displaying antibody fragments with the variant amino acid substitutions on the surface of a particle.
  • F(ab′) 2 dimers generally have the same affinity as F(ab) dimers in a solution phase antigen binding assay but the off rate for F(ab′) 2 are reduced because of a higher avidity. Therefore, the bivalent format (for example, F(ab′) 2 ) is a particularly useful format since it can allow for the identification of lower affinity clones and also allows more efficient sorting of rare clones during the selection process.
  • Vectors constructed as described in accordance with the invention are introduced into a host cell for amplification and/or expression.
  • Vectors can be introduced into host cells using standard transformation methods including electroporation, calcium phosphate precipitation and the like. If the vector is an infectious particle such as a virus, the vector itself provides for entry into the host cell. Transfection of host cells containing a replicable expression vector which encodes the gene fusion and production of phage particles according to standard procedures provides phage particles in which the fusion protein is displayed on the surface of the phage particle.
  • Replicable expression vectors are introduced into host cells using a variety of methods.
  • vectors can be introduced into cells using electroporation as described in WO/00106717.
  • Initial purification is preferably by resuspending the cell pellet in a buffer solution (e.g. 1.0 mM HEPES pH 7.4) followed by recentriguation and removal of supernatant.
  • the resulting cell pellet is resuspended in dilute glycerol (e.g. 5-20% v/v) and again recentrifuged to form a cell pellet and the supernatant removed.
  • the final cell concentration is obtained by resuspending the cell pellet in water or dilute glycerol to the desired concentration.
  • a particularly preferred recipient cell is the electroporation competent E. coli strain of the present invention, which is E. coli strain SS320 (Sidhu et al., Methods Enzymol . (2000), 328:333-363).
  • Strain SS320 was prepared by mating MC1061 cells with XL1-BLUE cells under conditions sufficient to transfer the fertility episome (F′ plasmid) or XL1-BLUE into the MC1061 cells.
  • Strain SS320 has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. USA, on Jun. 18, 1998 and assigned Deposit Accession No. 98795. Any F′ episome which enables phage replication in the strain may be used in the invention.
  • ATCC American Type Culture Collection
  • Suitable episomes are available from strains deposited with ATCC or are commercially available (CJ236, CSH18, DHF′, JM101, JM103, JM105, JM107, JM109, JM110), KS1000, XL1-BLUE, 71-18 and others).
  • phage display for identifying target antigen binders, with its various permutations and variations in methodology, are well established in the art.
  • One approach involves constructing a family of variant replicable vectors containing a transcription regulatory element operably linked to a gene fusion encoding a fusion polypeptide, transforming suitable host cells, culturing the transformed cells to form phage particles which display the fusion polypeptide on the surface of the phage particle, followed by a process that entails selection or sorting by contacting the recombinant phage particles with a target antigen so that at least a portion of the population of particles bind to the target with the objective to increase and enrich the subsets of the particles which bind from particles relative to particles that do not bind in the process of selection.
  • the selected pool can be amplified by infecting host cells, such as fresh XL1-Blue cells, for another round of sorting on the same target with different or same stringency.
  • the resulting pool of variants are then screened against the target antigens to identify novel high affinity binding proteins.
  • novel high affinity binding proteins can be useful as therapeutic agents as antagonists or agonists, and/or as as diagonostic and research reagents.
  • Fusion polypeptides such as antibody variable domains comprising the variant amino acids can be expressed on the surface of a phage, phagemid particle or a cell and then selected and/or screened for the ability of members of the group of fusion polypeptides to bind a target antigen which is typically an antigen of interest.
  • the processes of selection for binders to target can also be include sorting on a generic protein having affinity for antibody variable domains such as protein L or a tag specific antibody which binds to antibody or antibody fragments displayed on phage, which can be used to enrich for library members that display correctly folded antibody fragments (fusion polypeptides).
  • Target proteins such as receptors
  • Target antigens can include a number of molecules of therapeutic interest.
  • sorting for affinity can be used.
  • One example is a solid-support method or plate sorting or immobilized target sorting.
  • Another example is a solution-binding method.
  • the target protein may be attached to a suitable solid or semi solid matrix which are known in the art such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyalkyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like. Attachment of the target protein to the matrix may be accomplished by methods described in Methods in Enzymology, 44 (1976), or by other means known in the art.
  • the immobilized target After attachment of the target antigen to the matrix, the immobilized target is contacted with the library expressing the fusion polypeptides under conditions suitable for binding of at least a subset of the phage particle population with the immobilized target antigen. Normally, the conditions, including pH, ionic strength, temperature and the like will mimic physiological conditions. Bound particles (“binders”) to the immobilized target are separated from those particles that do not bind to the target by washing. Wash conditions can be adjusted to result in removal of all but the high affinity binders. Binders may be dissociated from the immobilized target by a variety of methods. These methods include competitive dissociation using the wild-type ligand (e.g.
  • binders typically involves elution from an affinity matrix with a suitable elution material such as acid like 0.1M HCl or ligand. Elution with increasing concentrations of ligand could elute displayed binding molecules of increasing affinity.
  • a suitable elution material such as acid like 0.1M HCl or ligand. Elution with increasing concentrations of ligand could elute displayed binding molecules of increasing affinity.
  • the binders can be isolated and then re-amplified in suitable host cells by infecting the cells with the viral particles that are binders (and helper phage if necessary, e.g. when viral particle is a phagemid particle) and the host cells are cultured under conditions suitable for amplification of the particles that display the desired fusion polypeptide.
  • the phage particles are then collected and the selection process is repeated one or more times until binders of the target antigen are enriched in a way. any number of rounds of selection or sorting can be utilized.
  • One of the selection or sorting procedures can involve isolating binders that bind to a generic affinity protein such as protein L or an antibody to a polypeptide tag present in a displayed polypeptide such as antibody to the gD protein or polyhistidine tag.
  • a generic affinity protein such as protein L
  • an antibody to a polypeptide tag present in a displayed polypeptide such as antibody to the gD protein or polyhistidine tag.
  • the invention allows solution phase sorting with much improved efficiency over conventional solution sorting methods.
  • the solution binding method may be used for finding original binders from a random library or finding improved binders from a library that was designated to improve affinity of a particular binding clone or group of clones.
  • the method comprises contacting a plurality of polypeptides, such as those displayed on phage or phagemid particles (library), with a target antigen labelled or fused with a tag molecule.
  • the tag could be biotin or other moieties for which specific binders are available.
  • the stringency of the solution phase can be varied by using decreasing concentrations of labelled target antigen in the first solution binding phase.
  • the first solution binding phase can be followed by a second solution phase having high concentration of unlabelled target antigen after the initial binding with the labelled target in the first solution phase.
  • 100 to 1000 fold of unlabelled target over labelled target is used in the second phase (if included).
  • the length of time of incubation of the first solution phase can vary from a few minutes to one to two hours or longer to reach equilibrium. Using a shorter time for binding in this first phase may bias or select for binders that have fast on-rate.
  • the length of time and temperature of incubation in second phase can be varied to increase the stringency.
  • This provides for a selection bias for binders that have slow rate of coming off the target (off-rate).
  • the phage or phagemid particles that are bound to labelled targets are separated from phage that do not bind.
  • the particle-target mixture from solution phase of binding is isolated by contacting it with the labelled target moiety and allowing for its binding to, a molecule that binds the labelled target moiety for a short period of time (eg. 2-5 minutes).
  • the initial concentration of the labelled target antigen can range from about 0.1 nM to about 1000 nM.
  • the bound particles are eluted and can be propagated for next round of sorting. Multiple rounds of sorting are preferred using a lower concentration of labelled target antigen with each round of sorting.
  • an initial sort or selection using about 100 to 250 nM labelled target antigen should be sufficient to capture a wide range of affinities, although this factor can be determined empirically and/or to suit the desire of the practitioner.
  • about 25 to 100 nM of labelled target antigen may be used.
  • about 0.1 to 25 nM of labled target antigen may be used.
  • the conventional solution sorting involves use of beads like strepavidin-coated beads, which is very cumbersome to use and often results in very low efficiency of phage binders recovery.
  • the conventional solution sorting with beads takes much longer than 2-5 minutes and is less feasible to adapt to high throughput automation than the invention described above.
  • combinations of solid support and solution sorting methods can be advantageously used to isolate binders having desired characteristics.
  • screening of individual clones from the selected pool generally is performed to identify specific binders with the desired properties/characteristics.
  • the process of screening is carried out by automated systems to allow for high-throughput screening of library candidates.
  • the first screening method comprises a phage ELISA assay with immobilized target antigen, which provides for identification of a specific binding clone from a non-binding clone. Specificity can be determined by simultaneous assay of the clone on target coated well and BSA or other non-target protein coated wells. This assay is automatable for high throughput screening.
  • One embodiment provides a method of selecting for an antibody variable domain that binds to a specific target antigen from a library of antibody variable domain by generating a library of replicable expression vectors comprising a plurality of polypeptides; contacting the library with a target antigen and at least one nontarget antigen under conditions suitable for binding; separating the polypeptide binders in the library from the nonbinders; identifying the binders that bind to the target antigen and do not bind to the nontarget antigen; eluting the binders from the target antigen; and amplifying the replicable expression vectors comprising the polypeptide binder that bind to a specific antigen.
  • the second screening assay is an affinity screening assay that provides for screening for clones that have high affinity from clones that have low affinity in a high throughput manner.
  • each clone is assayed with and without first incubating with target antigen of certain concentration for a period of time (for e.g 30-60 minutes) before application to target coated wells briefly (e.g. 5-15 minutes).
  • bound phage is measured by usual phage ELISA method, eg. using anti-M 13 HRP conjugates.
  • the ratio of binding signal of the two wells, one well having been preincubated with target and the other well not preincubated with target antigen is an indication of affinity.
  • the selection of the concentraion of target for first incubation depends on the affinity range of interest. For example, if binders with affinity higher than 10 nM are desired, 100 nM of target in the first incubation is often used. Once binders are found from a particular round of sorting (selection), these clones can be screened with affinity screening assay to identify binders with higher affinity.
  • polypeptide binders are first selected for binding to immobilized target antigen.
  • Polypeptide binders that bind to the immobilized target antigen can then be amplified and screened for binding to the target antigen and for lack of binding to nontarget antigens.
  • Polypeptide binders that bind specifically to the target antigen are amplified.
  • polypeptide binders can then selected for higher affinity by contact with a concentration of a labelled target antigen to form a complex, wherein the concentration ranges of labelled target antigen from about 0.1 nM to about 1000 nM, the complexes are isolated by contact with an agent that binds to the label on the target antigen.
  • the polypeptide binders are then eluted from the labled target antigen and optionally, the rounds of selection are repeated, each time a lower concentration of labelled target antigen is used.
  • the high affinity polypeptide binders isolated using this selection method can then be screened for high affinity using a variety of methods known in the art, some of which are described herein.
  • the nucleic acid can be extracted. Extracted DNA can then be used directly to transform E. coli host cells or alternatively, the encoding sequences can be amplified, for example using PCR with suitable primers, and sequenced by typical sequencing method. Variable domain DNA of the binders can be restriction enzyme digested and then inserted into a vector for protein expression.
  • binders comprising polypeptides having CDR(s) with restricted sequence diversity generated according to methods of the invention can be used to isolate binders against a variety of targets, including those listed in FIGS. 3, 4 , 5 , 8 .
  • These binders may comprise one or more variant CDRs comprising diverse sequences generated using restricted codons.
  • a variant CDR is CDRH3 comprising sequence diversity generated by amino acid substitution with restricted codon sets and/or amino acid insertions resulting from varying CDRH3 lengths.
  • Illustrative oligonucleotides useful for generating fusion polypeptides of the invention include those listed in FIGS. 2, 9 , 14 .
  • One or more variant CDRs may be combined.
  • only CDRH3 is diversified.
  • two or more heavy chain CDRs, including CDRH3, are variant.
  • one or more heavy chain CDRs, excluding CDRH3, are variant.
  • at least one heavy chain and at least one light chain CDR are variant.
  • at least one, two, three, four, five or all of CDRs H1, H2, H3, L1, L2 and L3 are variant.
  • binders generally lower affinity binders
  • Binders from a heavy chain library can then be randomized with CDR diversity in a light chain CDRs by, for e.g.
  • binders for example, low affinity binders
  • library of an L1/L2/L3 diversity to replace its original fixed L1/L2/L3, wherein the new libraries are then further sorted against a target of interest to obtain another set of binders (for example, high affinity binders).
  • Novel antibody sequences can be identified that display higher binding affinity to any of a variety of target antigens.
  • libraries comprising polypeptides of the invention are subjected to a plurality of sorting rounds, wherein each sorting round comprises contacting the binders obtained from the previous round with a target antigen distinct from the target antigen(s) of the previous round(s).
  • the target antigens are homologous in sequence, for example members of a family of related but distinct polypeptides, such as, but not limited to, cytokines (for example, alpha interferon subtypes).
  • Libraries of variant CDR polypeptides can be generated by mutating the solvent accessible and/or highly diverse positions in at least one CDR of an antibody variable domain. Some or all of the CDRs can be mutated using the methods of the invention. In some embodiments, it may be preferable to generate diverse antibody libraries by mutating positions in CDRH1, CDRH2 and CDRH3 to form a single library or by mutating positions in CDRL3 and CDRH3 to form a single library or by mutating positions in CDRL3 and CDRH1, CDRH2 and CDRH3 to form a single library.
  • a library of antibody variable domains can be generated, for example, having mutations in the solvent accessible and/or highly diverse positions of CDRH1, CDRH2 and CDRH3.
  • Another library can be generated having mutations in CDRL1, CDRL2 and CDRL3.
  • These libraries can also be used in conjunction with each other to generate binders of desired affinities. For example, after one or more rounds of selection of heavy chain libraries for binding to a target antigen, a light chain library can be replaced into the population of heavy chain binders for further rounds of selection to increase the affinity of the binders.
  • a library is created by substitution of original amino acids with a limited set of variant amino acids in the CDRH3 region of the variable region of the heavy chain sequence.
  • this library can contain a plurality of antibody sequences, wherein the sequence diversity is primarily in the CDRH3 region of the heavy chain sequence.
  • the library is created in the context of the humanized antibody 4D5 sequence, or the sequence of the framework amino acids of the humanized antibody 4D5 sequence.
  • the library is created by substitution of at least residues 95-100a of the heavy chain with amino acids encoded by the TMT, KMT or WMT codon set, wherein the TMT, KMT or WMT codon set is used to encode a limited set of variant amino acids for every one of these positions.
  • suitable oligonucleotide sequences include, but are not limited to, those listed in FIG. 2 and FIG. 9 and can be determined by one skilled in the art according to the criteria described herein.
  • CDRH3 designs are utilized to isolate high affinity binders and to isolate binders for a variety of epitopes.
  • multiple libraries can be constructed separately with different lengths of H3 and then combined to select for binders to target antigens.
  • the range of lengths of CDRH3 generated in this library can be 3-20; 5-20, 7-20, 5-18 or 7-18 amino acids, although lengths different from this can also be generated.
  • Diversity can also be generated in CDRH1 and CDRH2, as indicated above.
  • diversity in H1 and H2 is generated utilizing the oligonucleotides illustrated in FIGS. 2 and 9 .
  • Other oligonucleotides with varying sequences can also be used. Oligonucleotides can be used singly or pooled in any of a variety of combinations depending on practical needs and desires of the practitioner.
  • randomized positions in heavy chain CDRs include those listed in FIG. 1 .
  • Multiple libraries can be pooled and sorted using solid support selection and solution sorting methods as described herein. Multiple sorting strategies may be employed. For example, one variation involves sorting on target bound to a solid, followed by sorting for a tag that may be present on the fusion polypeptide (eg. anti-gD tag) and followed by another sort on target bound to solid. Alternatively, the libraries can be sorted first on target bound to a solid surface, the eluted binders are then sorted using solution phase binding with decreasing concentrations of target antigen. Utilizing combinations of different sorting methods provides for minimization of selection of only highly expressed sequences and provides for selection of a number of different high affinity clones.
  • Light chain diversity may be, but is not necessarily, generated in this embodiment as follows: in CDRL1, positions to be diversified include amino acid positions 28, 29, 30, 31, 32; in CDRL2, positions to be diversified include amino acid positions 50, 51, 53, 54, 55; in CDRL3, positions to be diversified include amino acid positions 91, 92, 93, 94, 95, 97. In one embodiment, the randomized positions are those listed in FIG. 13 .
  • High affinity binders isolated from the libraries of these embodiments are readily produced in bacterial and eukaryotic cell culture in high yield.
  • the vectors can be designed to readily remove sequences such as gD tags, viral coat protein component sequence, and/or to add in constant region sequences to provide for production of full length antibodies or antigen binding fragments in high yield.
  • Any combination of codon sets and CDRs can be diversified according to methods of the invention. Examples of suitable codons in various combinations of CDRs are illustrated in FIGS. 2, 6 , 9 , 13 .
  • an antibody polypeptide of the invention For recombinant production of an antibody polypeptide of the invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the antibody is 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 the antibody).
  • Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.
  • Polynucleotide sequences encoding polypeptide components of the antibody of the invention can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention.
  • Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector.
  • Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides.
  • the vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species.
  • pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells.
  • pBR322 its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.
  • promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Pat. No. 5,648,237.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • bacteriophage such as ⁇ GEM.TM.-11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • the expression vector of the invention may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components.
  • a promoter is an untranslated regulatory sequence located upstream (5′) to a cistron that modulates its expression.
  • Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g. the presence or absence of a nutrient or a change in temperature.
  • the selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention.
  • Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes.
  • heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.
  • Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the ⁇ -galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter.
  • trp tryptophan
  • other promoters that are functional in bacteria such as other known bacterial or phage promoters
  • Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.
  • each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane.
  • the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector.
  • the signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP.
  • STII heat-stable enterotoxin II
  • LamB, PhoE, PelB, OmpA and MBP are STII signal sequences or variants thereof.
  • the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron.
  • immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm.
  • Certain host strains e.g., the E. coli trxB ⁇ strains
  • the present invention provides an expression system in which the quantitative ratio of expressed polypeptide components can be modulated in order to maximize the yield of secreted and properly assembled antibodies of the invention. Such modulation is accomplished at least in part by simultaneously modulating translational strengths for the polypeptide components.
  • TIR translational initiation region
  • a series of amino acid or nucleic acid sequence variants can be created with a range of translational strengths, thereby providing a convenient means by which to adjust this factor for the desired expression level of the specific chain.
  • TIR variants can be generated by conventional mutagenesis techniques that result in codon changes which can alter the amino acid sequence, although silent changes in the nucleotide sequence are preferred.
  • Alterations in the TIR can include, for example, alterations in the number or spacing of Shine-Dalgarno sequences, along with alterations in the signal sequence.
  • One method for generating mutant signal sequences is the generation of a “codon bank” at the beginning of a coding sequence that does not change the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be accomplished by changing the third nucleotide position of each codon; additionally, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that can add complexity in making the bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.
  • a set of vectors is generated with a range of TIR strengths for each cistron therein.
  • This limited set provides a comparison of expression levels of each chain as well as the yield of the desired antibody products under various TIR strength combinations.
  • TIR strengths can be determined by quantifying the expression level of a reporter gene as described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Based on the translational strength comparison, the desired individual TIRs are selected to be combined in the expression vector constructs of the invention.
  • Prokaryotic host cells suitable for expressing antibodies of the invention include Archaebacteria and Eubacteria , such as Gram-negative or Gram-positive organisms.
  • useful bacteria include Escherichia (e.g., E. coli ), Bacilli (e.g., B. subtilis ), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa ), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla , or Paracoccus .
  • gram-negative cells are used.
  • E. coli cells are used as hosts for the invention. Examples of E.
  • coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology , vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W3110 ⁇ fhuA ( ⁇ tonA) ptr3 lac Iq lacL8 ⁇ ompT ⁇ (nmpc-fepE) degP4I kan R (U.S. Pat. No. 5,639,635).
  • Other strains and derivatives thereof such as E. coli 294 (ATCC 31,446), E. coli B, E. coli ⁇ 1776 (ATCC 31,537) and E.
  • coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium.
  • E. coli, Serratia , or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN4]0 are used to supply the replicon.
  • the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.
  • Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant.
  • transformation is done using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers.
  • Another method for transformation employs polyethylene glycol/DMSO.
  • Yet another technique used is electroporation.
  • Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in the art and suitable for culture of the selected host cells.
  • suitable media include luria broth (LB) plus necessary nutrient supplements.
  • the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.
  • any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.
  • the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.
  • the prokaryotic host cells are cultured at suitable temperatures.
  • the preferred temperature ranges from about 20° C. to about 39° C., more preferably from about 25° C. to about 37° C., even more preferably at about 30° C.
  • the pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism.
  • the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.
  • an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter.
  • PhoA promoters are used for controlling transcription of the polypeptides.
  • the transformed host cells are cultured in a phosphate-limiting medium for induction.
  • the phosphate-limiting medium is the C.R.A.P medium (see, for e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147).
  • a variety of other inducers may be used, according to the vector construct employed, as is known in the art.
  • the expressed polypeptides of the present invention are secreted into and recovered from the periplasm of the host cells.
  • Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.
  • PAGE polyacrylamide gel electrophoresis
  • antibody production is conducted in large quantity by a fermentation process.
  • Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins.
  • Large-scale fermentations have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source).
  • Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.
  • induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD 550 of about 180-220, at which stage the cells are in the early stationary phase.
  • a desired density e.g., an OD 550 of about 180-220
  • inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.
  • various fermentation conditions can be modified.
  • additional vectors overexpressing chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells.
  • the chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al.
  • certain host strains deficient for proteolytic enzymes can be used for the present invention.
  • host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof.
  • E. coli protease-deficient strains are available and described in, for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).
  • E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the invention.
  • the antibody protein produced herein is further purified to obtain preparations that are substantially homogeneous for further assays and uses.
  • Standard protein purification methods known in the art can be employed. The following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.
  • Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody products of the invention.
  • Protein A is a 41 kD cell wall protein from Staphylococcus aureas which binds with a high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth. 62:1-13.
  • the solid phase to which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column has been coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adherence of contaminants.
  • the preparation derived from the cell culture as described above is applied onto the Protein A immobilized solid phase to allow specific binding of the antibody of interest to Protein A.
  • the solid phase is then washed to remove contaminants non-specifically bound to the solid phase.
  • the antibody of interest is recovered from the solid phase by elution.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • a vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal are available.
  • the DNA for such precursor region is ligated in reading frame to DNA encoding the antibody.
  • an origin of replication component is not needed for mammalian expression vectors.
  • the SV40 origin may typically be used only because it contains the early promoter.
  • Selection genes may contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media.
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
  • Mtx methotrexate
  • An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).
  • host cells transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3′-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.
  • APH aminoglycoside 3′-phosphotransferase
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the antibody polypeptide nucleic acid.
  • Promoter sequences are known for eukaryotes.
  • Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated.
  • Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide.
  • N may be any nucleotide.
  • AATAAA sequence At the 3′ end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3′ end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • Antibody polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from hetero
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment.
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) on expression of human ⁇ -interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.
  • Enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5′ or 3′ to the antibody polypeptide-encoding sequence, but is preferably located at a site 5′ from the promoter.
  • Expression vectors used in eukaryotic host cells will typically also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci.
  • COS-7 monkey kidney CV1 line transformed by SV40
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)
  • baby hamster kidney cells
  • mice sertoli cells TM4 , Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce an antibody of this invention may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody can be produced intracellularly, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human ⁇ 1, ⁇ 2, or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).
  • Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al., EMBO J. 5:15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a C H 3 domain
  • the Bakerbond ABXTMresin J. T. Baker, Phillipsburg, N.J.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • the antibodies of the present invention can be characterized for their physical/chemical properties and biological functions by various assays known in the art.
  • the purified immunoglobulins can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.
  • assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.
  • the immunoglobulins produced herein are analyzed for their biological activity.
  • the immunoglobulins of the present invention are tested for their antigen binding activity.
  • the antigen binding assays that are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays.
  • the present invention contemplates an altered antibody that possesses some but not all effector functions, which make it a desired candidate for many applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • the Fc activities of the produced immunoglobulin are measured to ensure that only the desired properties are maintained.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc ⁇ R binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • An example of an in vitro assay to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 or 5,821,337.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity.
  • a CDC assay for e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art, for e.g. those desribed in the Examples section.
  • the present invention encompasses humanized antibodies.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901.
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
  • the invention provides antibody fragment comprising modifications in the interface of Fc polypeptides comprising the Fc region, wherein the modifications facilitate and/or promote heterodimerization.
  • modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance is positionable in the cavity so as to promote complexing of the first and second Fc polypeptides.
  • amino acid sequence modification(s) of the antibodies described herein are contemplated.
  • Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made.
  • a useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085.
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed immunoglobulins are screened for the desired activity.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated.
  • Conservative substitutions are shown in Table 2 under the heading of “preferred substitutions”. If such substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions” in the table below, or as further described below in reference to amino acid classes, may be introduced and the products screened.
  • Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):
  • Naturally occurring residues may be divided into groups based on common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino acid substitutions at each site.
  • the antibodies thus generated are displayed from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed.
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions including that of a hinge cysteine.
  • a human Fc region sequence e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region
  • an amino acid modification e.g. a substitution
  • an antibody used in methods of the invention may comprise one or more alterations as compared to the wild type counterpart antibody, for e.g. in the Fc region.
  • These antibodies would nonetheless retain substantially the same characteristics required for therapeutic utility as compared to their wild type counterpart.
  • certain alterations can be made in the Fc region that would result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), for e.g., as described in WO99/51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351 concerning other examples of Fc region variants.
  • the invention also pertains to immunoconjugates, or antibody-drug conjugates (ADC), comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an en
  • Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer Inst. 92(19): 1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res.
  • bacterial toxins such as diphtheria toxin
  • plant toxins such as ricin
  • small molecule toxins such as geldanamycin
  • maytansinoids EP 1391213; Liu et al., (1996)
  • the toxins may effect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
  • ZEVALIN® is an antibody-radioisotope conjugate composed of a murine IgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and 111 In or 90 Y radioisotope bound by a thiourea linker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
  • ZEVALIN has activity against B-cell non-Hodgkin's Lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients.
  • MYLOTARGTM (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos.
  • Cantuzumab mertansine an antibody drug conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the maytansinoid drug moiety, DM1
  • CanAg such as colon, pancreatic, gastric, and others.
  • MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody drug conjugate composed of the anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DM1, is under development for the potential treatment of prostate tumors.
  • PSMA anti-prostate specific membrane antigen
  • auristatin peptides auristatin E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, were conjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784) and are under therapeutic development.
  • AE auristatin E
  • MMAE monomethylauristatin
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate),
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • Conjugates of an antibody and one or more small molecule toxins such as a calicheamicin, maytansinoids, a trichothecene, and CC 1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.
  • an antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules.
  • Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Pat. Nos.
  • maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens.
  • Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, the disclosures of which are hereby expressly incorporated by reference.
  • the conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay.
  • Chari et al., Cancer Research 52:127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene.
  • the cytotoxicity of the TA.1-maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3 ⁇ 10 5 HER-2 surface antigens per cell.
  • the drug conjugate achieved a degree of cytotoxicity similar to the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule.
  • the A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.
  • Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule.
  • An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody.
  • Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.
  • Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.
  • linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al., Cancer Research 52:127-131 (1992).
  • the linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.
  • Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5
  • Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 [1978]) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.
  • SPDP N-succinimidyl-3-(2-pyridyldithio) propionate
  • SPP N-succinimidyl-4-(2-pyridylthio)pentanoate
  • the linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link.
  • an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group.
  • the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.
  • Another immunoconjugate of interest comprises an antibody conjugated to one or more calicheamicin molecules.
  • the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations.
  • For the preparation of conjugates of the calicheamicin family see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
  • Structural analogues of calicheamicin which may be used include, but are not limited to, ⁇ 1 1 , ⁇ 2 1 , ⁇ 3 1 , N-acetyl- ⁇ 1 1 , PSAG and ⁇ 1 1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid).
  • Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate.
  • QFA is an antifolate.
  • Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.
  • the present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
  • a compound with nucleolytic activity e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase.
  • the antibody may comprise a highly radioactive atom.
  • radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • the conjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • the radio- or other labels may be incorporated in the conjugate in known ways.
  • the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen.
  • Labels such as tc 99m or I 123 , Re 186 , Re 188 and In 111 can be attached via a cysteine residue in the peptide.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal,CRC Press 1989) describes other methods in detail.
  • Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-d
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the linker may be a “cleavable linker” facilitating release of the cytotoxic drug in the cell.
  • an acid-labile linker for example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
  • the compounds of the invention expressly contemplate, but are not limited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook and Catalog.
  • an antibody (Ab) is conjugated to one or more drug moieties (D), e.g. about 1 to about 20 drug moieties per antibody, through a linker (L).
  • the ADC of Formula I may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form Ab-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody.
  • Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
  • Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol).
  • a reducing agent such as DTT (dithiothreitol).
  • Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.
  • Antibody drug conjugates of the invention may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic subsituents on the linker reagent or drug.
  • the sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties.
  • the resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages.
  • reaction of the carbohydrate portion of a glycosylated antibody with either glactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques ).
  • proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852).
  • Such aldehyde can be reacted with a drug moiety or linker nucleophile.
  • nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
  • a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis.
  • the length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
  • the antibody may be conjugated to a “receptor” (such streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide).
  • a receptor such streptavidin
  • a ligand e.g., avidin
  • cytotoxic agent e.g., a radionucleotide
  • the antibodies of the present invention can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody are water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), poly
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymers are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • Therapeutic formulations comprising an antibody of the invention are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions, lyophilized or other dried formulations.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, hist
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulin of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and y ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-( ⁇ )-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated immunoglobulins When encapsulated immunoglobulins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • an antibody of the present invention may be used in, for example, in vitro, ex vivo and in vivo therapeutic methods.
  • Antibodies of the invention can be used as an antagonist to partially or fully block the specific antigen activity in vitro, ex vivo and/or in vivo.
  • at least some of the antibodies of the invention can neutralize antigen activity from other species.
  • the antibodies of the invention can be used to inhibit a specific antigen activity, e.g., in a cell culture containing the antigen, in human subjects or in other mammalian subjects having the antigen with which an antibody of the invention cross-reacts (e.g.
  • the antibody of the invention can be used for inhibiting antigen activities by contacting the antibody with the antigen such that antigen activity is inhibited.
  • the antigen is a human protein molecule.
  • an antibody of the invention can be used in a method for inhibiting an antigen in a subject suffering from a disorder in which the antigen activity is detrimental, comprising administering to the subject an antibody of the invention such that the antigen activity in the subject is inhibited.
  • the antigen is a human protein molecule and the subject is a human subject.
  • the subject can be a mammal expressing the antigen with which an antibody of the invention binds.
  • the subject can be a mammal into which the antigen has been introduced (e.g., by administration of the antigen or by expression of an antigen transgene).
  • An antibody of the invention can be administered to a human subject for therapeutic purposes.
  • an antibody of the invention can be administered to a non-human mammal expressing an antigen with which the immunoglobulin cross-reacts (e.g., a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time courses of administration).
  • Blocking antibodies of the invention that are therapeutically useful include, for example but are not limited to, anti-HER2, anti-VEGF, anti-IgE, anti-CD11, anti-interferon, anti-interferon receptor, anti-hepatocyte growth factor (HGF), anti-c-met, and anti-tissue factor antibodies.
  • the antibodies of the invention can be used to treat, inhibit, delay progression of, prevent/delay recurrence of, ameliorate, or prevent diseases, disorders or conditions associated with abnormal expression and/or activity of one or more antigen molecules, including but not limited to malignant and benign tumors; non-leukemias and lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.
  • a blocking antibody of the invention is specific to a ligand antigen, and inhibits the antigen activity by blocking or interfering with the ligand-receptor interaction involving the ligand antigen, thereby inhibiting the corresponding signal pathway and other molecular or cellular events.
  • the invention also features receptor-specific antibodies which do not necessarily prevent ligand binding but interfere with receptor activation, thereby inhibiting any responses that would normally be initiated by the ligand binding.
  • the invention also encompasses antibodies that either preferably or exclusively bind to ligand-receptor complexes.
  • An antibody of the invention can also act as an agonist of a particular antigen receptor, thereby potentiating, enhancing or activating either all or partial activities of the ligand-mediated receptor activation.
  • an immunoconjugate comprising an antibody conjugated with a cytotoxic agent is administered to the patient.
  • the immunoconjugate and/or antigen to which it is bound is/are internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the target cell to which it binds.
  • the cytotoxic agent targets or interferes with nucleic acid in the target cell. Examples of such cytotoxic agents include any of the chemotherapeutic agents noted herein (such as a maytansinoid or a calicheamicin), a radioactive isotope, or a ribonuclease or a DNA endonuclease.
  • Antibodies of the invention can be used either alone or in combination with other compositions in a therapy.
  • an antibody of the invention may be co-administered with another antibody, chemotherapeutic agent(s) (including cocktails of chemotherapeutic agents), other cytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or growth inhibitory agent(s).
  • chemotherapeutic agent(s) including cocktails of chemotherapeutic agents
  • other cytotoxic agent(s) include anti-angiogenic agent(s), cytokines, and/or growth inhibitory agent(s).
  • an antibody of the invention inhibits tumor growth, it may be particularly desirable to combine it with one or more other therapeutic agent(s) which also inhibits tumor growth.
  • an antibody of the invention may be combined with an anti-VEGF antibody (e.g., AVASTIN) and/or anti-ErbB antibodies (e.g.
  • HERCEPTIN® anti-HER2 antibody in a treatment scheme, e.g. in treating any of the diseases described herein, including colorectal cancer, metastatic breast cancer and kidney cancer.
  • the patient may receive combined radiation therapy (e.g. external beam irradiation or therapy with a radioactive labeled agent, such as an antibody).
  • combined therapies noted above include combined administration (where the two or more agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, and/or following, administration of the adjunct therapy or therapies.
  • the antibody of the invention (and adjunct therapeutic agent) is/are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. Dosing can be by any suitable route, for e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • the antibody composition of the invention will be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question.
  • the effective amount of such other agents depends on the amount of antibodies of the invention present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
  • an antibody of the invention when used alone or in combination with other agents such as chemotherapeutic agents, will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.
  • One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, e.g. about six doses of the antibody).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • An exemplary dosing regimen comprises administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the antibody.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or when combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an antibody of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the first and second antibody compositions can be used to treat a particular condition, for e.g. cancer.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • Ringer's solution such as phosphate
  • Phage-displayed Fab libraries were constructed using a phagemid vector that resulted in the display of bivalent Fab moieties dimerized by a leucine zipper domain inserted between the Fab heavy chain and the C-terminal domain of the gene-3 minor coat protein (P3C).
  • This vector comprises the sequence shown in FIG. 18 (SEQ ID NO:4).
  • the vector (schematically illustrated in FIG. 19 ) comprises the humanized antibody 4D5 variable domains under the control of the IPTG-inducible Ptac promoter.
  • the humanized antibody 4D5 is an antibody which has mostly human consensus sequence framework regions in the heavy and light chains, and CDR regions from a mouse monoclonal antibody specific for Her-2.
  • the method of making the anti-Her-2 antibody and the identity of the variable domain sequences are provided in U.S. Pat. Nos. 5,821,337 and 6,054,297.
  • Library YS-A was constructed with randomized residues in all three heavy chain CDRs
  • Library YS-B was constructed with randomized residues in all three heavy chain CDRs and light chain CDR3. The specific residues that were randomized are shown in the FIG. 1 .
  • the length of CDRH3 was varied by using oligonucleotides that replaced the 7 wild-type codons between positions 101 to 107 with varying numbers of TMT codons (7 to 20 for Library YS-A and 7 to 15 for Library YS-B).
  • the CDRL3 of Library YS-B was randomized so that 50% of the library members contained a deletion at position number 91 while the other 50% contained the wildtype Gln residue at this position.
  • the mutagenesis reactions were electroporated into E. coli SS320 (Sidhu et al., supra), and the transformed cells were grown overnight in the presence of M13-KO7 helper phage (New England Biolabs, Beverly, Mass.) to produce phage particles that encapsulated the phagemid DNA and displayed Fab fragments on their surfaces.
  • M13-KO7 helper phage New England Biolabs, Beverly, Mass.
  • Phage from library YS-A or YS-B were cycled through rounds of binding selection to enrich for clones binding to targets of interest. Eight target proteins were analyzed separately with each library: human VEGF, murine VEGF, neutravidin, an apoptosis protein (AP), maltose binding protein, erbin-GST fusion, and Insulin. The binding selections were conducted using previously described methods (Sidhu et al., supra).
  • NUNC 96-well Maxisorp immunoplates were coated overnight at 4° C. with capture target (5 ⁇ g/mL) and blocked for 2 h with Superblock TBS (tris-buffered saline) (Pierce). After overnight growth at 37° C., phage were concentrated by precipitation with PEG/NaCl and resuspended in Superblock TBS, 0.05% Tween 20 (Sigma), as described previously (Sidhu et al., supra). Phage solutions ( ⁇ 10 12 phage/mL) were added to the coated immunoplates. Following a 2 h incubation to allow for phage binding, the plates were washed 10 times with PBS, 0.05% Tween 20.
  • Bound phage were eluted with 0.1 M HCl for 10 min and the eluant was neutralized with 1.0 M Tris base. Eluted phage were amplified in E. coli XL1-blue and used for further rounds of selection.
  • the libraries were subjected to 5 rounds of selection against each target protein, and at each round, titers were obtained for phage binding to either the target protein or blank wells coated with Superblock TBS.
  • the titer of phage bound to target-coated wells divided by the titer of phage bound to the blank wells was defined as an enrichment ratio used to quantify specific binding of phage pools to the target protein; larger enrichment ratios indicate higher specific binding.
  • the enrichment ratios observed after 3, 4, or 5 rounds of selection are shown in FIG. 3 .
  • BIAcore data was obtained according to Chen et al., J Mol Biol . (1999), 293(4):865-81. Briefly, binding affinities of hVEGF binders for hVEGF and mVEGF were calculated from association and dissociation rate constants measured using a BIAcoreTM-2000 surface plasmon resonance system (BIAcore, Inc., Piscataway, N.J.).
  • a biosensor chip was activated for covalent coupling of VEGF using N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's (BIAcore, Inc., Piscataway, N.J.) instructions.
  • EDC N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • IC50 values for selected anti-AP clones were determined by phage ELISA, as described previously (Sidhu et al., supra). The values are shown in FIG. 11 .
  • Phage displayed libraries were constructed, as described in Example 1, with a previously described phagemid designed to display bivalent Fab moieties dimerized by a leucine zipper domain inserted between the Fab heavy chain and the C-terminal domain of the gene-3 minor coat protein (P3C) (as described in Example 1).
  • CDR positions in the heavy chain were randomized, positions as shown in FIG. 1 .
  • Eleven separate mutagenesis reactions were performed with each mutagenesis reaction designed to randomize the CDR positions with a tetranomial codon that encoded for only four amino acids. In each mutagenesis reaction, the CDR positions were simultaneously replaced with only one type of tetranomial codon.
  • the eleven tetranomial codons used for the eleven mutagenesis reactions and the amino acids they encode are shown in FIG. 6 .
  • three mutagenic oligonucleotides were used, with each designed to introduce diversity into one of the three heavy chain CDRs.
  • the eleven mutagenesis reactions were pooled and electroporated into E. coli SS320 (Sidhu et al., supra), and the transformed cells were grown overnight in the presence of M13-KO7 helper phage (New England Biolabs, Beverly, Mass.) to produce phage particles that encapsulated the phagemid DNA and displayed Fab fragments on their surfaces.
  • the library contained 2.6 ⁇ 10 10 unique members, and it was named library F0505.
  • Phage from library F0505 (Example 3) were cycled through rounds of binding selection to enrich for clones binding four different targets: IGF, h-VEGF, anti-hGH, hGH binding protein.
  • the binding selections were conducted using previously described methods (Sidhu et al., supra).
  • NUNC 96-well Maxisorp immunoplates were coated overnight at 4° C. with capture target (5 ⁇ g/mL) and blocked for 2 h with BSA (Sigma). After overnight growth at 37° C., phage were concentrated by precipitation with PEG/NaCl and resuspended in PBS, 0.5% BSA, 0.05% Tween 20 (Sigma), as described previously (Sidhu et al., supra). Phage solutions ( ⁇ 10 12 phage/mL) were added to the coated immunoplates. Following a 2 h incubation to allow for phage binding, the plates were washed 10 times with PBS, 0.05% Tween20.
  • Bound phages were eluted with 0.1 M HCl for 10 min and the eluant was neutralized with 1.0 M Tris base. Eluted phage were amplified in E. coli XL1-blue and used for further rounds of selection.
  • the libraries were subjected to 4 rounds of selection against each target protein. After rounds 2 and 3, individual clones from each round and each target selection were grown in a 96-well format in 500 ⁇ L of 2YT broth supplemented with carbenicillin and M13-VCS, and the culture supernatants were used directly in phage ELISAs (Sidhu et al., supra) to detect phage-displayed Fabs that bound to plates coated with target protein but not to plates coated with BSA.
  • a clone is considered to be a specific binder if the ELISA signal on plates coated with target protein was at least 10 times greater than the signal on BSA coated plates.
  • the number of specific binders for each round and each target is tabulated in FIG. 7 .
  • the specific clones were subjected to DNA sequence analysis.
  • the library of origin for each of the unique sequence were determined and summarized in FIG. 8 .
  • YADS-A and YADS-B Two phage displayed libraries (YADS-A and YADS-B) were constructed, as described in Example 1, with a previously described phagemid designed to display bivalent Fab moieties dimerized by a leucine zipper domain inserted between the Fab heavy chain and the C-terminal domain of the gene-3 minor coat protein (P3C) (as described in Example 1). CDR positions in the heavy chain were randomized, positions as shown in FIG. 1 .
  • the oligonucleotide sequences are shown in FIG. 9 .
  • oligonucleotides were used to introduce diversity into CDR-H3: YADS-H3-3, YADS-H3-4, YADS-H3-5, YADS-H3-6, YADS-H3-7, YADS-H3-8, YADS-H3-9, YADS-H3-10, YADS-H3-11, YADS-H3-12, YADS-H3-13, YADS-H3-14, or YADS-H3-15.
  • the 13 reactions were pooled.
  • Phage from library YADS-A and YADS-B were cycled seperately through rounds of binding selection to enrich for clones binding to h-VEGF.
  • the binding selections were conducted using previously described methods (Sidhu et al., supra).
  • NUNC 96-well Maxisorp immunoplates were coated overnight at 4° C. with capture target (5 ⁇ g/mL) and blocked for 2 h with BSA (Sigma). After overnight growth at 37° C., phage were concentrated by precipitation with PEG/NaCl and resuspended in PBS, 0.5% BSA, 0.05% Tween 20 (Sigma), as described previously (Sidhu et al., supra). Phage solutions ( ⁇ 10 12 phage/mL) were added to the coated immunoplates. Following a 2 h incubation to allow for phage binding, the plates were washed 10 times with PBS, 0.05% Tween20.
  • Bound phages were eluted with 0.1 M HCl for 10 min and the eluant was neutralized with 1.0 M Tris base. Eluted phage were amplified in E. coli XL1-blue and used for further rounds of selection.
  • the libraries were subjected to 4 rounds of selection against each target protein. Individual clones from each round were grown in a 96-well format in 500 ⁇ L of 2YT broth supplemented with carbenicillin and M13-VCS, and the culture supernatants were used directly in phage ELISAs (Sidhu et al., supra) to detect phage-displayed Fabs that bound to plates coated with target protein but not to plates coated with BSA. A clone was considered to be a specific binder if the ELISA signal on target coated plates was at least 20 times greater than that on BSA coated plates. The results are tabulated in FIG. 10 . Multiple unique sequences of specific binders were obtained (data not shown).
  • VEGF-binding clones selected from libraries YADS-A and YADS-B revealed 24 unique clones in which the randomized heavy chain CDR positions contained only tyrosine, alanine, asparte, or serine.
  • the Kunkel method of site-directed mutagenesis was used to construct 16 “stop template” versions of phagemids used in this Example. Codons in the light chain CDRs (positions 29, 32, 51, 54, 55, 93, 94 and 97) were replaced with TAA stop codons. Sixteen separate mutagenesis reactions (one with each template) were performed with three oligonucleotides designed to simultaneously repair the stop codons and introduce degenerate codons encoding for tyrosine, alanine, aspartate, and serine.
  • the mutagenic oligonucleotides YADS-L1, YADS-L2, and YADS-L3 were used to introduce diversity into CDR-L 1, CDR-L2, and CDR-L3, respectively.
  • the oligonucleotide sequences are shown in FIG. 13 and the light chain CDR sites that were randomized are shown in FIG. 12 .
  • the 16 mutagenesis reactions were pooled and electroporated into E. coli SS320 (Sidhu et al., supra).
  • the transformed cells were grown overnight in the presence of M13-KO7 helper phage (New England Biolabs, Beverly, Mass.) to produce phage particles that encapsulated the phagemid DNA and displayed Fab fragments on their surfaces.
  • the library contained 6.5 ⁇ 10 9 unique members, and it was named library YADS-II.
  • Phage from library YADS-II (Example 7) were cycled through rounds of binding selection to enrich for clones binding h-VEGF.
  • the binding selections were conducted as follows.
  • phage were concentrated by precipitation with PEG/NaCl and resuspended in Superblock 1% TBS (Pierce), 0.05% Tween 20 (Sigma), as described above. Phage solutions (200 ⁇ L at a concentration close to 10 12 phage/mL) were incubated with biotinylated h-VEGF at a concentration of 25 nM. After 2 hours of incubation at room temperature with gentle shaking, 800 uL of Superblock plus 0.05% Tween 20 was added.
  • binders Based on the amount of inhibition of binding by 100 nM of hVEGF, three binders were further analyzed. The measurement of binding on other proteins ( FIG. 16 ) was determined for these three binders. These binders were expressed as Fab proteins in E. coli , and their binding affinities to hVEGF and mVEGF measured by Biacore as described in Example 2. Data is summarized in FIG. 17 .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • Dermatology (AREA)
  • Virology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Rheumatology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Transplantation (AREA)
US10/901,011 2003-08-01 2004-07-28 Binding polypeptides with restricted diversity sequences Abandoned US20050106667A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/901,011 US20050106667A1 (en) 2003-08-01 2004-07-28 Binding polypeptides with restricted diversity sequences
US12/015,451 US20090023602A1 (en) 2003-08-01 2008-01-16 Binding polypeptides with restricted diversity sequences

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US49187703P 2003-08-01 2003-08-01
US51649503P 2003-11-01 2003-11-01
US57091204P 2004-05-12 2004-05-12
US57123904P 2004-05-13 2004-05-13
US57631504P 2004-06-01 2004-06-01
US58075704P 2004-06-18 2004-06-18
US10/901,011 US20050106667A1 (en) 2003-08-01 2004-07-28 Binding polypeptides with restricted diversity sequences

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/015,451 Continuation US20090023602A1 (en) 2003-08-01 2008-01-16 Binding polypeptides with restricted diversity sequences

Publications (1)

Publication Number Publication Date
US20050106667A1 true US20050106667A1 (en) 2005-05-19

Family

ID=34120173

Family Applications (9)

Application Number Title Priority Date Filing Date
US10/901,011 Abandoned US20050106667A1 (en) 2003-08-01 2004-07-28 Binding polypeptides with restricted diversity sequences
US11/342,249 Expired - Lifetime US7811785B2 (en) 2003-08-01 2006-01-27 Anti-VEGF antibodies
US11/583,994 Expired - Lifetime US7691977B2 (en) 2003-08-01 2006-10-19 Anti-VEGF antibodies
US12/700,609 Expired - Lifetime US8092797B2 (en) 2003-08-01 2010-02-04 Anti-VEGF antibodies
US12/844,616 Expired - Lifetime US8101177B2 (en) 2003-08-01 2010-07-27 Anti-VEGF antibodies
US13/335,044 Expired - Fee Related US8492527B2 (en) 2003-08-01 2011-12-22 Anti-VEGF antibodies
US13/926,801 Expired - Lifetime US9018357B2 (en) 2003-08-01 2013-06-25 Anti-VEGF antibodies
US14/636,782 Expired - Lifetime US9353177B2 (en) 2003-08-01 2015-03-03 Anti-VEGF antibodies
US15/137,577 Abandoned US20170073405A1 (en) 2003-08-01 2016-04-25 Anti-vegf antibodies

Family Applications After (8)

Application Number Title Priority Date Filing Date
US11/342,249 Expired - Lifetime US7811785B2 (en) 2003-08-01 2006-01-27 Anti-VEGF antibodies
US11/583,994 Expired - Lifetime US7691977B2 (en) 2003-08-01 2006-10-19 Anti-VEGF antibodies
US12/700,609 Expired - Lifetime US8092797B2 (en) 2003-08-01 2010-02-04 Anti-VEGF antibodies
US12/844,616 Expired - Lifetime US8101177B2 (en) 2003-08-01 2010-07-27 Anti-VEGF antibodies
US13/335,044 Expired - Fee Related US8492527B2 (en) 2003-08-01 2011-12-22 Anti-VEGF antibodies
US13/926,801 Expired - Lifetime US9018357B2 (en) 2003-08-01 2013-06-25 Anti-VEGF antibodies
US14/636,782 Expired - Lifetime US9353177B2 (en) 2003-08-01 2015-03-03 Anti-VEGF antibodies
US15/137,577 Abandoned US20170073405A1 (en) 2003-08-01 2016-04-25 Anti-vegf antibodies

Country Status (17)

Country Link
US (9) US20050106667A1 (enrdf_load_stackoverflow)
EP (2) EP1648939A2 (enrdf_load_stackoverflow)
JP (2) JP4845732B2 (enrdf_load_stackoverflow)
KR (6) KR101359142B1 (enrdf_load_stackoverflow)
CN (1) CN104761643A (enrdf_load_stackoverflow)
AU (1) AU2004262006C1 (enrdf_load_stackoverflow)
BR (1) BRPI0412637A (enrdf_load_stackoverflow)
CA (2) CA2932216A1 (enrdf_load_stackoverflow)
EA (1) EA012835B1 (enrdf_load_stackoverflow)
HK (1) HK1211600A1 (enrdf_load_stackoverflow)
IL (2) IL173317A (enrdf_load_stackoverflow)
MX (1) MXPA06001319A (enrdf_load_stackoverflow)
NO (1) NO20061014L (enrdf_load_stackoverflow)
NZ (2) NZ545135A (enrdf_load_stackoverflow)
OA (1) OA13225A (enrdf_load_stackoverflow)
SG (2) SG193170A1 (enrdf_load_stackoverflow)
WO (1) WO2005012359A2 (enrdf_load_stackoverflow)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050281894A1 (en) * 2002-11-08 2005-12-22 Kin-Ping Wong Extract of Trapa natans and methods of using the same
US20090011060A1 (en) * 2007-07-06 2009-01-08 Peter Koepke Campsiandra angustifolia extract and methods of extracting and using such extract
US20090075378A1 (en) * 2007-02-20 2009-03-19 Anaptysbio, Inc. Somatic hypermutation systems
US20100267050A1 (en) * 2005-12-15 2010-10-21 Genentech, Inc. Methods and compositions for targeting polyubiquitin
US7879369B2 (en) 2007-09-18 2011-02-01 Selvamedica, Llc Combretum laurifolium Mart. extract and methods of extracting and using such extract
US20140154743A1 (en) * 2011-02-03 2014-06-05 Raphael D. Levy Methods and materials for enhancing functional protein expression in bacteria
US8992919B2 (en) 2010-04-15 2015-03-31 Genentech, Inc. Anti-polyubiquitin antibodies and methods of use
US9081015B2 (en) 2008-01-18 2015-07-14 Genentech, Inc. Methods and compositions for targeting polyubiquitin
EP2861064A4 (en) * 2012-06-11 2015-12-02 Syngenta Participations Ag MANUFACTURE OF SOLIDS AND ASSOCIATED MOTHER TUBES
US9321844B2 (en) 2011-08-05 2016-04-26 Genentech, Inc. Anti-polyubiquitin antibodies
US11008387B2 (en) 2016-01-06 2021-05-18 Order-Made Medical Research Inc. Antibody inhibiting binding of VEGF to NRP1
US11007259B2 (en) 2016-01-06 2021-05-18 Order-Made Medical Research Inc. High-affinity anti-VEGF antibody

Families Citing this family (337)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7183387B1 (en) 1999-01-15 2007-02-27 Genentech, Inc. Polypeptide variants with altered effector function
US20050123925A1 (en) 2002-11-15 2005-06-09 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
HUP0600342A3 (en) * 2001-10-25 2011-03-28 Genentech Inc Glycoprotein compositions
US7696320B2 (en) 2004-08-24 2010-04-13 Domantis Limited Ligands that have binding specificity for VEGF and/or EGFR and methods of use therefor
US7758859B2 (en) 2003-08-01 2010-07-20 Genentech, Inc. Anti-VEGF antibodies
US20050106667A1 (en) 2003-08-01 2005-05-19 Genentech, Inc Binding polypeptides with restricted diversity sequences
CN102641503A (zh) 2004-07-20 2012-08-22 健泰科生物技术公司 血管生成素样4蛋白抑制剂，组合，以及其用途
US8604185B2 (en) 2004-07-20 2013-12-10 Genentech, Inc. Inhibitors of angiopoietin-like 4 protein, combinations, and their use
SG158089A1 (en) * 2004-12-17 2010-01-29 Genentech Inc Antiangiogenesis therapy of autoimmune disease in patients who have failed prior therapy
FR2893622B1 (fr) 2005-11-24 2007-12-21 Commissariat Energie Atomique Composition a base de caprolactame,procede de fabrication d'un element d'etancheite,et reservoir
US20070237764A1 (en) * 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
AU2012204022C1 (en) * 2005-12-02 2014-02-20 Genentech, Inc. Binding polypeptides and uses thereof
AU2006338198B2 (en) * 2005-12-02 2012-04-26 Genentech, Inc. Binding polypeptides and uses thereof
CN101370832B (zh) * 2005-12-02 2014-07-02 健泰科生物技术公司 结合多肽及其用途
TW200804425A (en) * 2005-12-06 2008-01-16 Domantis Ltd Ligands that have binding specificity for EGFR and/or VEGF and methods of use therefor
DK1973950T3 (en) 2006-01-05 2014-12-15 Genentech Inc ANTI-EphB4 ANTIBODIES AND METHODS FOR USING SAME
AU2007215334B2 (en) 2006-02-13 2013-11-21 Daiichi Sankyo Company, Limited Polynucleotides and polypeptide sequences involved in the process of bone remodeling
US8168181B2 (en) 2006-02-13 2012-05-01 Alethia Biotherapeutics, Inc. Methods of impairing osteoclast differentiation using antibodies that bind siglec-15
AR059851A1 (es) * 2006-03-16 2008-04-30 Genentech Inc Anticuerpos de la egfl7 y metodos de uso
ES2544957T3 (es) 2006-03-21 2015-09-07 Genentech, Inc. Terapia combinada que implica antagonistas alfa5beta1
KR100981202B1 (ko) * 2006-05-31 2010-09-10 한화케미칼 주식회사 Ｖｃａｍ－１ 특이적 단일클론항체
RU2415869C2 (ru) * 2006-06-06 2011-04-10 Дженентек, Инк. Антитела против dll4 и способы их применения
US7803377B2 (en) 2006-06-06 2010-09-28 Genentech, Inc. Anti-DLL4 antibodies and methods using same
SG174090A1 (en) * 2006-08-21 2011-09-29 Hoffmann La Roche Tumor therapy with an anti-vegf antibody
DK2059533T3 (da) * 2006-08-30 2013-02-25 Genentech Inc Multispecifikke antistoffer
HUE028379T2 (en) 2006-09-29 2016-12-28 Oncomed Pharm Inc Preparations and procedures for the diagnosis and treatment of cancer
US20090098529A1 (en) 2006-10-16 2009-04-16 Nanhai Chen Methods for attenuating virus strains for diagnostic and therapeutic uses
CU23636A1 (es) 2006-11-01 2011-03-21 Ct Ingenieria Genetica Biotech Anticuerpos recombinantes contra el factor de crecimiento del endotelio vascular (vegf)
US20100086544A1 (en) 2006-12-11 2010-04-08 Mass Robert D Compositions and methods for treating a neoplasm
MX2009006779A (es) 2006-12-19 2009-08-27 Genentech Inc Antagonistas especificos para terapia con adyuvantes y neo-adyuvantes y el tratamiento de tumores en etapa temprana.
CN101646458A (zh) * 2007-02-01 2010-02-10 健泰科生物技术公司 使用血管发生抑制剂的联合疗法
WO2008100805A2 (en) 2007-02-09 2008-08-21 Genentech, Inc. Anti-robo4 antibodies and uses therefor
US8642067B2 (en) 2007-04-02 2014-02-04 Allergen, Inc. Methods and compositions for intraocular administration to treat ocular conditions
US7977313B2 (en) * 2007-04-27 2011-07-12 Affinergy, Inc. Methods and compositions for promoting localization of pharmaceutically active agents to bone
GB0709333D0 (en) 2007-05-15 2007-06-20 Smart Targeting Ltd Binding protein
MX2009012282A (es) * 2007-05-17 2009-11-25 Genentech Inc Estructuras cristalinas de fragmentos de neuropilina y complejos de neuropilina-anticuerpos.
PE20090321A1 (es) 2007-06-04 2009-04-20 Genentech Inc Anticuerpos anti-notch1 nrr, metodo de preparacion y composicion farmaceutica
DK2219672T3 (en) * 2007-11-09 2016-05-17 Peregrine Pharmaceuticals Inc The anti-VEGF antibody compositions and methods
AU2013205269B2 (en) * 2007-11-09 2016-05-19 Affitech Research As Anti-VEGF antibody compositions and methods
BRPI0817063A2 (pt) 2007-11-09 2015-03-24 Genentch Inc "métodos de inibição, para o tratamento de uma condição patológica, para a inibição de linfagiogênese tumoral, de inibição ou prevenção,de desorganização, para inibição de crescimento de tumor, de tratamento, de acentuação da eficácia de um agente anti-angiogênese e antagonista de alk-1
AR069501A1 (es) * 2007-11-30 2010-01-27 Genentech Inc Anticuerpos anti- vegf (factor de crecimiento endotelial vascular)
AU2015200714B2 (en) * 2007-11-30 2017-01-12 Genentech, Inc. Anti-VEGF antibodies
AU2013204108B2 (en) * 2007-11-30 2014-11-20 Genentech, Inc. Anti-VEGF antibodies
US8227577B2 (en) * 2007-12-21 2012-07-24 Hoffman-La Roche Inc. Bivalent, bispecific antibodies
US9266967B2 (en) 2007-12-21 2016-02-23 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
US20090162359A1 (en) 2007-12-21 2009-06-25 Christian Klein Bivalent, bispecific antibodies
PL3269366T3 (pl) 2008-03-18 2020-07-27 Genentech, Inc. Kombinacje koniugatu przeciwciało anty-HER2-lek i lapatinibu, i sposoby zastosowania
CA2716882A1 (en) * 2008-03-27 2009-10-01 Zymogenetics, Inc. Compositions and methods for inhibiting pdgfr-beta and vegf-a
WO2009134776A2 (en) * 2008-04-29 2009-11-05 Abbott Laboratories Dual variable domain immunoglobulins and uses thereof
CN102112495A (zh) 2008-06-03 2011-06-29 雅培制药有限公司 双重可变结构域免疫球蛋白及其用途
BRPI0913366A8 (pt) 2008-06-03 2017-07-11 Abbott Lab Imunoglobulinas de domínio variável duplo e seus usos
BRPI0914251B1 (pt) 2008-06-25 2022-07-19 Novartis Ag Imunoligante recombinante, seu fragmento de ligação ao antígeno, uso dos mesmos, e composição
NZ590667A (en) * 2008-07-02 2013-01-25 Emergent Product Dev Seattle Tgf-b antagonist multi-target binding proteins
CA2729949A1 (en) 2008-07-08 2010-01-14 Abbott Laboratories Prostaglandin e2 dual variable domain immunoglobulins and uses thereof
US20100029491A1 (en) * 2008-07-11 2010-02-04 Maike Schmidt Methods and compositions for diagnostic use for tumor treatment
AU2009285540A1 (en) * 2008-08-29 2010-03-04 Genentech, Inc. Diagnostics and treatments for VEGF-independent tumors
US8268314B2 (en) * 2008-10-08 2012-09-18 Hoffmann-La Roche Inc. Bispecific anti-VEGF/anti-ANG-2 antibodies
PE20110707A1 (es) 2008-10-14 2011-10-11 Genentech Inc Variantes de inmunoglobulinas
WO2010056893A1 (en) * 2008-11-13 2010-05-20 Imclone Llc Humanization and affinity-optimization of antibodies
PL2361085T5 (pl) 2008-11-22 2018-12-31 F.Hoffmann-La Roche Ag Zastosowanie przeciwciała anty-vegf w połączeniu z chemioterapią w leczeniu raka sutka
SG172355A1 (en) 2008-12-23 2011-07-28 Genentech Inc Methods and compositions for diagnostic use in cancer patients
DK2411411T3 (en) 2009-03-25 2016-11-07 Hoffmann La Roche New anti-alpha5beta1 antibodies and uses thereof
AU2010230563A1 (en) * 2009-04-02 2011-09-22 Roche Glycart Ag Multispecific antibodies comprising full length antibodies and single chain Fab fragments
AU2010234031B2 (en) 2009-04-07 2015-10-01 Roche Glycart Ag Trivalent, bispecific antibodies
US9181339B2 (en) 2009-04-20 2015-11-10 Oxford Bio Therapeutics Ltd. Antibodies specific to cadherin-17
KR20120096401A (ko) * 2009-04-20 2012-08-30 제넨테크, 인크. 아주반트 암 요법
US9676845B2 (en) 2009-06-16 2017-06-13 Hoffmann-La Roche, Inc. Bispecific antigen binding proteins
US20110076271A1 (en) 2009-07-13 2011-03-31 Genentech, Inc. Diagnostic methods and compositions for treatment of cancer
AR077595A1 (es) 2009-07-27 2011-09-07 Genentech Inc Tratamientos de combinacion
RU2567803C2 (ru) 2009-07-31 2015-11-10 Дженентек, Инк. ИНГИБИРОВАНИЕ МЕТАСТАЗИРОВАНИЯ ОПУХОЛИ ПРИ ПОМОЩИ АНТАГОНИСТОВ Bv8 ИЛИ G-CSF
EP2464220A4 (en) 2009-08-13 2014-05-07 Crystal Bioscience Inc Transgenic animal for the production of antibodies with minimal complementarity-determining regions
KR20120059553A (ko) 2009-08-14 2012-06-08 제넨테크, 인크. Ｖｅｇｆ 길항제에 대한 환자 반응을 모니터링하기 위한 생물학적 마커
SG178419A1 (en) 2009-08-15 2012-04-27 Genentech Inc Anti-angiogenesis therapy for the treatment of previously treated breast cancer
UY32870A (es) 2009-09-01 2011-02-28 Abbott Lab Inmunoglobulinas con dominio variable dual y usos de las mismas
WO2011032013A1 (en) 2009-09-11 2011-03-17 Genentech, Inc. Method to identify a patient with an increased likelihood of responding to an anti-cancer agent
CA2781519A1 (en) 2009-09-16 2011-03-24 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
BR112012005670A2 (pt) 2009-09-17 2017-01-10 Hoffmann La Roche ''método para a identificação de um paciente com câncer que pode se beneficiar com a terapia antiagiogênica, método para a predição da responsividade de um paciente com câncer à terapia antiangiogênica, uso de um anticorpo anto- vegf para melhorar o efeito do tratamento de um paciente sofrendo de câncer, método ou uso, kit útil para realizar o método, uso de uma proteína ou usom kit útil para realizar o método, uso de uma proteína ou oligonucleotídeo ou matriz de polinecleotídeo para determinar o nível de expressão de bfgf em um método e kit ou uso
JP2013506435A (ja) * 2009-10-05 2013-02-28 オプソニック セラピューティクス インコーポレイテッド 抗体特異性をリダイレクトするための高親和性アダプター分子
TW201119676A (en) 2009-10-15 2011-06-16 Abbott Lab Dual variable domain immunoglobulins and uses thereof
ES2575152T3 (es) 2009-10-16 2016-06-24 Oncomed Pharmaceuticals, Inc. Combinación terapéutica y uso de anticuerpos antagonistas de DLL4 y agentes antihipertensores
NZ599110A (en) * 2009-10-21 2013-09-27 Genentech Inc Genetic polymorphisms in age-related macular degeneration
WO2011056497A1 (en) 2009-10-26 2011-05-12 Genentech, Inc. Activin receptor type iib compositions and methods of use
WO2011056502A1 (en) 2009-10-26 2011-05-12 Genentech, Inc. Bone morphogenetic protein receptor type ii compositions and methods of use
UY32979A (es) 2009-10-28 2011-02-28 Abbott Lab Inmunoglobulinas con dominio variable dual y usos de las mismas
EP3778917A3 (en) * 2009-12-04 2021-06-09 F. Hoffmann-La Roche AG Multispecific antibodies, antibody analogs, compositions, and methods
JP5818805B2 (ja) 2009-12-11 2015-11-18 ジェネンテック， インコーポレイテッド 抗ｖｅｇｆ−ｃ抗体及びその使用方法
US8765432B2 (en) 2009-12-18 2014-07-01 Oligasis, Llc Targeted drug phosphorylcholine polymer conjugates
TW201636048A (zh) 2009-12-21 2016-10-16 建南德克公司 抗體調配物
SG181905A1 (en) 2009-12-23 2012-07-30 Genentech Inc Anti-bv8 antibodies and uses thereof
WO2011088111A1 (en) * 2010-01-12 2011-07-21 Genentech, Inc. ANTI-PlGF ANTIBODIES AND METHODS USING SAME
KR20190143480A (ko) 2010-02-23 2019-12-30 제넨테크, 인크. 난소암의 치료를 위한 항혈관신생 요법
CN102167740B (zh) * 2010-02-25 2014-06-04 上海百迈博制药有限公司 一种全人源抗vegf单克隆抗体、其制备方法及用途
AR080794A1 (es) * 2010-03-26 2012-05-09 Hoffmann La Roche Anticuerpos bivalentes biespecificos anti- vegf/ anti-ang-2
AR080793A1 (es) 2010-03-26 2012-05-09 Roche Glycart Ag Anticuerpos biespecificos
WO2011153243A2 (en) 2010-06-02 2011-12-08 Genentech, Inc. Anti-angiogenesis therapy for treating gastric cancer
WO2011153224A2 (en) 2010-06-02 2011-12-08 Genentech, Inc. Diagnostic methods and compositions for treatment of cancer
WO2011159704A1 (en) 2010-06-14 2011-12-22 Vaccinex, Inc. Anti-vegf antibodies and uses thereof
EP2848940A1 (en) 2010-07-19 2015-03-18 F. Hoffmann-La Roche AG Method to identify a patient with an increased likelihood of responding to an anti-cancer therapy
WO2012010548A1 (en) 2010-07-19 2012-01-26 F. Hoffmann-La Roche Ag Method to identify a patient with an increased likelihood of responding to an anti-cancer therapy
ES2979168T3 (es) 2010-07-23 2024-09-24 Univ Boston Inhibidores anti-DEspR como productos terapéuticos para inhibición de angiogénesis patológica e invasividad de células tumorales y para imagenología molecular y administración dirigida
NZ607480A (en) 2010-08-03 2014-10-31 Abbott Lab Dual variable domain immunoglobulins and uses thereof
AR082693A1 (es) 2010-08-17 2012-12-26 Roche Glycart Ag Terapia de combinacion de un anticuerpo anti-cd20 afucosilado con un anticuerpo anti-vegf
RU2013110875A (ru) 2010-08-24 2014-09-27 Ф.Хоффманн-Ля Рош Аг БИСПЕЦИФИЧЕСКИЕ АНТИТЕЛА, СОДЕРЖАЩИЕ СТАБИЛИЗИРОВАННЫЙ ДИСУЛЬФИДОМ ФРАГМЕНТ Fv
MX2013002270A (es) 2010-08-26 2013-05-14 Abbvie Inc Inmonoglubinas de dominio variable doble y usos de las mismas.
JP2013537966A (ja) 2010-08-31 2013-10-07 ジェネンテック， インコーポレイテッド バイオマーカー及び治療の方法
US8551479B2 (en) 2010-09-10 2013-10-08 Oncomed Pharmaceuticals, Inc. Methods for treating melanoma
KR20130138802A (ko) * 2010-10-20 2013-12-19 옥스포드 바이오테라퓨틱스 리미티드 항체
AR083819A1 (es) 2010-11-10 2013-03-27 Genentech Inc UN ANTICUERPO QUE SE UNE A BACE1 (ENZIMA 1 DE DISOCIACION DE PROTEINA PRECURSORA DEL AMILOIDE DE SITIO b), METODOS Y COMPOSICIONES PARA INMUNOTERAPIA PARA ENFERMEDAD NEURAL
NZ610538A (en) 2010-11-15 2015-02-27 Five Prime Therapeutics Inc Fgfr1 extracellular domain combination therapies
CA2818621A1 (en) * 2010-11-24 2012-05-31 Glaxo Group Limited Multispecific antigen binding proteins targeting hgf
EP2655413B1 (en) 2010-12-23 2019-01-16 F.Hoffmann-La Roche Ag Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery
CU23895B1 (es) 2010-12-28 2013-05-31 Biorec Sa Anticuerpos recombinantes contra el factor de crecimiento del endotelio vascular (vegf) obtenidos mediante mutagénesis de regiones variables
WO2012092539A2 (en) 2010-12-31 2012-07-05 Takeda Pharmaceutical Company Limited Antibodies to dll4 and uses thereof
HUE053720T2 (hu) 2011-01-14 2021-07-28 Univ California ROR-1 fehérje elleni terápiás antitestek és eljárások alkalmazásukra
MX341921B (es) 2011-02-28 2016-09-07 Hoffmann La Roche Proteinas de union a antigeno.
CN103403025B (zh) 2011-02-28 2016-10-12 弗·哈夫曼-拉罗切有限公司 单价抗原结合蛋白
JP5926791B2 (ja) 2011-03-29 2016-05-25 ロシュ グリクアート アーゲー 抗体Ｆｃ変種
RS56759B2 (sr) 2011-04-01 2024-10-31 Genentech Inc Kombinacija akt inhibitor jedinjenja i abiraterona za upotrebu pri terapeutskim tretiranjima
EP2699590B8 (en) 2011-04-20 2019-04-17 Acceleron Pharma Inc. Endoglin polypeptides and uses thereof
WO2012151317A1 (en) 2011-05-03 2012-11-08 Genentech, Inc. Vascular disruption agents and uses thereof
US20130004484A1 (en) 2011-06-30 2013-01-03 Genentech, Inc. Anti-c-met antibody formulations
US20130022551A1 (en) 2011-07-22 2013-01-24 Trustees Of Boston University DEspR ANTAGONISTS AND AGONISTS AS THERAPEUTICS
US10466160B2 (en) 2011-08-01 2019-11-05 Celsee Diagnostics, Inc. System and method for retrieving and analyzing particles
CN103998394B (zh) 2011-08-01 2016-08-17 德诺弗科学公司 细胞捕获系统和使用方法
RU2014109985A (ru) 2011-08-17 2015-09-27 Дженентек, Инк. Ингибирование ангиогенеза в рефрактерных опухолях
US20130078252A1 (en) 2011-09-19 2013-03-28 Genentech, Inc. Combination treatments comprising c-met antagonists and b-raf antagonists
SI2758073T1 (sl) 2011-09-23 2019-02-28 Oncomed Pharmaceuticals, Inc. Sredstva, ki vežejo VEGF/DLL4, in njihove uporabe
KR20140095096A (ko) 2011-11-21 2014-07-31 제넨테크, 인크. 항-c-met 항체의 정제
WO2013082511A1 (en) 2011-12-02 2013-06-06 Genentech, Inc. Methods for overcoming tumor resistance to vegf antagonists
SG11201402737SA (en) 2011-12-05 2014-06-27 Hoffmann La Roche Blood plasma biomarkers for bevacizumab combination therapies for treatment of breast cancer
US20130195851A1 (en) 2011-12-23 2013-08-01 Genentech, Inc. Articles of manufacture and methods for co-administration of antibodies
TW201333035A (zh) 2011-12-30 2013-08-16 Abbvie Inc 針對ｉｌ－１３及／或ｉｌ－１７之雙特異性結合蛋白
CN104203268A (zh) 2012-01-13 2014-12-10 霍夫曼-拉罗奇有限公司 用于识别适于用vegf拮抗剂治疗的患者的生物标记物
MX2014009565A (es) 2012-02-10 2014-11-10 Genentech Inc Anticuerpos monocatenarios y otros heteromultimeros.
BR112014018964A2 (pt) 2012-03-13 2020-05-12 F. Hoffmann-La Roche Ag Anticorpo anti-vegf e uso de um anticorpo anti-vegf
MX2014011582A (es) 2012-03-30 2014-11-21 Genentech Inc Metodos y composiciones de diagnostico para el tratamiento de cancer.
WO2013152252A1 (en) 2012-04-06 2013-10-10 OSI Pharmaceuticals, LLC Combination anti-cancer therapy
TW201823460A (zh) * 2012-05-29 2018-07-01 美商再生元醫藥公司 生產細胞株增強子
MX363872B (es) 2012-05-31 2019-04-05 Genentech Inc Anticuerpos anti-pd-l1 y antagonistas de vegf y su uso en el tratamiento del cáncer.
KR101909801B1 (ko) 2012-06-08 2018-10-18 에프. 호프만-라 로슈 아게 암의 치료를 위한 포스포이노시타이드 3 키나제 억제제 화합물 및 화학치료제의 돌연변이체 선택성 및 조합물
EP2864788A1 (en) 2012-06-26 2015-04-29 F. Hoffmann-La Roche AG Blood plasma biomarkers for bevacizumab combination therapies for treatment of breast cancer
CA2871882A1 (en) 2012-06-27 2014-01-03 F. Hoffmann-La Roche Ag Method for making antibody fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof
WO2014001324A1 (en) 2012-06-27 2014-01-03 Hoffmann-La Roche Ag 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
ES2896493T3 (es) 2012-07-13 2022-02-24 Roche Glycart Ag Anticuerpos biespecíficos anti-VEGF/anti-ANG-2 y su uso en el tratamiento de enfermedades vasculares oculares
JP6268173B2 (ja) 2012-07-19 2018-01-24 第一三共株式会社 抗Ｓｉｇｌｅｃ−１５抗体
MX360189B (es) 2012-08-07 2018-10-24 Genentech Inc Terapia de combinacion para el tratamiento de glioblastoma.
CA2880271C (en) 2012-08-13 2021-10-19 Genentech, Inc. Anti-jagged anitbodies and methods of use
US12024568B2 (en) 2012-09-13 2024-07-02 Cornell University Treatment of brain cancers using central nervous system mediated gene transfer of monoclonal antibodies
CA2889638A1 (en) 2012-10-31 2014-05-08 Oncomed Pharmaceuticals, Inc. Methods and monitoring of treatment with a dll4 antagonist
TW202014439A (zh) 2012-11-01 2020-04-16 美商艾伯維有限公司 抗-vegf/dll4雙重可變區域免疫球蛋白及其用途
US20140154255A1 (en) * 2012-11-30 2014-06-05 Abbvie Biotherapeutics Inc. Anti-vegf antibodies and their uses
WO2014106076A2 (en) * 2012-12-28 2014-07-03 Quest Diagnostics Investments Incorporated Universal sanger sequencing from next-gen sequencing amplicons
JP6408492B2 (ja) 2013-02-18 2018-10-17 ヴェジェニクス プロプライエタリー リミテッドＶｅｇｅｎｉｃｓ Ｐｔｙ Ｌｉｍｉｔｅｄ リガンド結合分子およびその使用
KR20150118159A (ko) 2013-02-22 2015-10-21 에프. 호프만-라 로슈 아게 암의 치료 방법 및 약물 내성의 예방 방법
RU2015137610A (ru) 2013-03-06 2017-04-10 Дженентек, Инк. Способы лечения и профилактики лекарственной резистентности злокачественных опухолей
CA2906057A1 (en) 2013-03-13 2014-10-02 Genentech, Inc. Antibody formulations
KR20150127216A (ko) 2013-03-14 2015-11-16 제넨테크, 인크. 암의 치료 및 암 약물 내성의 예방 방법
HK1214533A1 (zh) 2013-03-15 2016-07-29 基因泰克公司 治疗癌症和预防癌症耐药性的方法
EP2970459A2 (en) 2013-03-15 2016-01-20 AbbVie Inc. Dual specific binding proteins directed against il-1beta and il-17
CN113491690B (zh) 2013-04-09 2023-03-14 北京强新生物科技有限公司 2-乙酰基萘并[2,3-b]呋喃-4,9-二酮用于治疗癌症的用途
RU2015154985A (ru) 2013-05-23 2017-06-29 Файв Прайм Терапьютикс, Инк. Способы лечения рака
KR101541478B1 (ko) 2013-05-31 2015-08-05 동아쏘시오홀딩스 주식회사 항-vegf 항체 및 이를 포함하는 암 또는 신생혈관형성관련 질환의 예방, 진단 또는 치료용 약학 조성물
US10391490B2 (en) 2013-05-31 2019-08-27 Celsee Diagnostics, Inc. System and method for isolating and analyzing cells
BR112016002845A2 (pt) 2013-08-12 2017-09-12 Genentech Inc composições e métodos para tratar condições associadas ao complemento
US10456470B2 (en) 2013-08-30 2019-10-29 Genentech, Inc. Diagnostic methods and compositions for treatment of glioblastoma
US10617755B2 (en) 2013-08-30 2020-04-14 Genentech, Inc. Combination therapy for the treatment of glioblastoma
ES2819217T3 (es) 2013-09-08 2021-04-15 Kodiak Sciences Inc Conjugados de polímeros iónicos dipolares y factor VIII
JP6422956B2 (ja) 2013-10-11 2018-11-14 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 多重特異性ドメイン交換共通可変軽鎖抗体
AU2014339898B2 (en) 2013-10-25 2020-07-23 Acceleron Pharma, Inc. Endoglin peptides to treat fibrotic diseases
EP3074037B1 (en) 2013-11-27 2020-10-07 Inis Biotech LLC Methods for modulating angiogenesis of cancers refractory to anti-vegf treatment
JP2017505756A (ja) 2013-12-13 2017-02-23 ザ ジェネラル ホスピタル コーポレイション 可溶性高分子量（ｈｍｗ）タウ種およびその用途
MX2015014724A (es) * 2013-12-31 2016-06-02 Dev Center Biotechnology Anticuerpos anti-factor de crecimiento endotelial vascular (vegf) y uso de los mismos.
JOP20200094A1 (ar) 2014-01-24 2017-06-16 Dana Farber Cancer Inst Inc جزيئات جسم مضاد لـ pd-1 واستخداماتها
JOP20200096A1 (ar) 2014-01-31 2017-06-16 Children’S Medical Center Corp جزيئات جسم مضاد لـ tim-3 واستخداماتها
TWI558399B (zh) 2014-02-26 2016-11-21 美國禮來大藥廠 癌症之組合療法
TW201622744A (zh) 2014-03-04 2016-07-01 美國禮來大藥廠 癌症之組合療法
EA201691765A1 (ru) 2014-03-14 2016-12-30 Новартис Аг Молекулы антител против lag-3 и их применения
CN107002119A (zh) 2014-03-24 2017-08-01 豪夫迈·罗氏有限公司 使用c‑met拮抗剂的癌症治疗及前者与hgf表达的关联
CA2943834A1 (en) 2014-03-31 2015-10-08 Genentech, Inc. Combination therapy comprising anti-angiogenesis agents and ox40 binding agonists
TW201623337A (zh) 2014-05-01 2016-07-01 建南德克公司 抗－因子ｄ抗體變異體及其用途
US9388239B2 (en) 2014-05-01 2016-07-12 Consejo Nacional De Investigation Cientifica Anti-human VEGF antibodies with unusually strong binding affinity to human VEGF-A and cross reactivity to human VEGF-B
JP2017517552A (ja) 2014-06-13 2017-06-29 ジェネンテック， インコーポレイテッド 抗癌剤耐性の治療及び防止方法
US9840553B2 (en) 2014-06-28 2017-12-12 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
EP3169801A1 (en) 2014-07-14 2017-05-24 F. Hoffmann-La Roche AG Diagnostic methods and compositions for treatment of glioblastoma
US20170281624A1 (en) 2014-09-13 2017-10-05 Novartis Ag Combination therapies of alk inhibitors
EP3193932B1 (en) 2014-09-15 2023-04-26 F. Hoffmann-La Roche AG Antibody formulations
EP3200775B1 (en) 2014-10-03 2019-11-20 Novartis AG Combination therapies
MA41044A (fr) 2014-10-08 2017-08-15 Novartis Ag Compositions et procédés d'utilisation pour une réponse immunitaire accrue et traitement contre le cancer
JP6877339B2 (ja) 2014-10-14 2021-05-26 ノバルティス アーゲー Ｐｄ−ｌ１に対する抗体分子およびその使用
CN107208076A (zh) 2014-10-17 2017-09-26 科达制药 丁酰胆碱酯酶两性离子聚合物缀合物
US20160176962A1 (en) 2014-10-31 2016-06-23 Oncomed Pharmaceuticals, Inc. Combination Therapy For Treatment Of Disease
SG11201703767XA (en) 2014-11-10 2017-06-29 Genentech Inc Anti-interleukin-33 antibodies and uses thereof
EP3221361B1 (en) 2014-11-19 2021-04-21 Genentech, Inc. Anti-transferrin receptor / anti-bace1 multispecific antibodies and methods of use
EP3221364B1 (en) 2014-11-19 2020-12-16 Genentech, Inc. Antibodies against bace1 and use thereof for neural disease immunotherapy
PL3227332T3 (pl) 2014-12-03 2020-06-15 F. Hoffmann-La Roche Ag Wielospecyficzne przeciwciała
WO2016094881A2 (en) 2014-12-11 2016-06-16 Abbvie Inc. Lrp-8 binding proteins
US20170340733A1 (en) 2014-12-19 2017-11-30 Novartis Ag Combination therapies
AU2015369624A1 (en) 2014-12-23 2017-06-08 Genentech, Inc. Compositions and methods for treating and diagnosing chemotherapy-resistant cancers
CA3022547A1 (en) 2015-05-12 2016-11-17 Syntimmune, Inc. Humanized affinity matured anti-fcrn antibodies
HK1245632A1 (zh) 2015-06-03 2018-08-31 波士顿生物医药有限公司 包含癌症干性抑制剂的组合物和用於治疗癌症的免疫治疗剂
TW201711702A (zh) 2015-06-04 2017-04-01 應克隆公司 使用針對纖維母細胞生長因子受體３（ｆｇｆｒ３）之化合物的療法
JP2018518483A (ja) 2015-06-08 2018-07-12 ジェネンテック， インコーポレイテッド 抗ｏｘ４０抗体及びｐｄ−１軸結合アンタゴニストを使用して癌を治療する方法
TW201710286A (zh) 2015-06-15 2017-03-16 艾伯維有限公司 抗ｖｅｇｆ、ｐｄｇｆ及／或其受體之結合蛋白
TW201716439A (zh) 2015-07-20 2017-05-16 美國禮來大藥廠 Ｈｅｒ３抗體
PL3317301T3 (pl) 2015-07-29 2021-11-15 Novartis Ag Terapie skojarzone zawierające cząsteczki przeciwciał przeciw lag-3
EP3878465A1 (en) 2015-07-29 2021-09-15 Novartis AG Combination therapies comprising antibody molecules to tim-3
WO2017019896A1 (en) 2015-07-29 2017-02-02 Novartis Ag Combination therapies comprising antibody molecules to pd-1
ES2968074T3 (es) 2015-09-23 2024-05-07 Mereo Biopharma 5 Inc Anticuerpo bi-específico anti-VEGF/DLL4 para su uso en el tratamiento del cáncer de ovario resistente al platino
US10072075B2 (en) 2015-09-23 2018-09-11 Genentech, Inc. Optimized variants of anti-VEGF antibodies and methods of treatment thereof by reducing or inhibiting angiogenesis
HUE054093T2 (hu) 2015-10-30 2021-08-30 Hoffmann La Roche Anti-HtrA1 antitestek és azok alkalmazási eljárásai
BR112018008769A2 (pt) 2015-10-30 2018-12-04 Genentech Inc formulações farmacêuticas, formulações líquidas aquosa reconstituída e liofilizada, seringa, método de fabricação de uma formulação farmacêutica, método para tratamento de uma doença e uso de uma formulação reconstruída
US10654932B2 (en) 2015-10-30 2020-05-19 Genentech, Inc. Anti-factor D antibody variant conjugates and uses thereof
JP2018534930A (ja) 2015-10-30 2018-11-29 ジェネンテック， インコーポレイテッド 抗ｄ因子抗体及びコンジュゲート
CA3004117A1 (en) 2015-11-03 2017-05-11 Janssen Biotech, Inc. Antibodies specifically binding pd-1 and their uses
WO2017106656A1 (en) 2015-12-17 2017-06-22 Novartis Ag Antibody molecules to pd-1 and uses thereof
KR102799807B1 (ko) 2015-12-30 2025-04-24 코디악 사이언시스 인코포레이티드 항체 및 이의 접합체
WO2017165681A1 (en) 2016-03-24 2017-09-28 Gensun Biopharma Inc. Trispecific inhibitors for cancer treatment
ES2850428T3 (es) 2016-04-15 2021-08-30 Hoffmann La Roche Procedimientos de monitorización y tratamiento del cáncer
JP2019513767A (ja) 2016-04-15 2019-05-30 イーライ リリー アンド カンパニー マントル細胞リンパ腫の治療における使用のためのラムシルマブ及びアベマシクリブの組み合わせ療法
US20190336599A1 (en) 2016-04-15 2019-11-07 Eli Lilly And Company Combination of ramucirumab and merestinib for use in treatment of colorectal cancer
MX2018012493A (es) 2016-04-15 2019-06-06 Genentech Inc Métodos para controlar y tratar el cáncer.
EP3448429B1 (en) 2016-04-25 2021-01-13 Syntimmune Inc. Humanized affinity matured anti-fcrn antibodies
WO2017210119A1 (en) 2016-06-03 2017-12-07 Imclone Llc Combination of ramucirumab and pembrolizumab for the treatment of certain cancers
WO2018007615A1 (en) 2016-07-07 2018-01-11 The European Molecular Biology Laboratory Viral polypeptide fragments that bind cellular pol ii c-terminal domain (ctd) and their uses
JP7050702B2 (ja) 2016-07-08 2022-04-08 ジェネンテック， インコーポレイテッド Ｎｒｆ２及びその遺伝子の下流標的遺伝子の発現状態及び変異状態によるがんの診断及び治療方法
JP2019524706A (ja) 2016-07-08 2019-09-05 ジェネンテック， インコーポレイテッド Ｍｕｃ１６陽性癌治療の応答性を評価するためのヒト精巣上体タンパク質４（ｈｅ４）の使用
JP2019523404A (ja) 2016-07-15 2019-08-22 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト 全ｖｅｇｆ−ａのレベルを検出するための方法及び手段
WO2018030193A1 (ja) * 2016-08-08 2018-02-15 コニカミノルタ株式会社 実験動物の腫瘍組織の評価に関する方法
EP3509623A4 (en) * 2016-09-07 2020-06-03 Saksin Lifesciences PVT Ltd SYNTHETIC ANTIBODIES TO VEGF AND USES THEREOF
BR112019006074A2 (pt) 2016-09-29 2019-06-18 Beijing Hanmi Pharmaceutical Co Ltd heterodímero, método de produção do heterodímero, métodos de produção de um heterodímero, ácido nucleico, vetor ou sistema de vetores, célula, composição farmacêutica e método de tratamento ou prevenção de uma doença ou distúrbio em um sujeito em necessidade do mesmo
JP2019534888A (ja) 2016-10-28 2019-12-05 イムクローン・リミテッド・ライアビリティ・カンパニーＩｍｃｌｏｎｅ ＬＬＣ 癌の治療のための抗ｖｅｇｆｒ−２抗体と抗ｐｄ−ｌ１抗体との組み合わせ
EP3541838A1 (en) 2016-11-16 2019-09-25 Eli Lilly and Company Therapy for metatastic colorectal cancer using anti-vegfr-2 and anti-vegf-d antibodies
WO2018093669A1 (en) 2016-11-16 2018-05-24 Eli Lilly And Company Combination therapy for cancer with exon 14 skipping mutation(s) or exon 14 skipping phenotype
TN2019000161A1 (en) 2016-11-18 2020-10-05 Beijing Hanmi Pharmaceutical Co Ltd Anti-pd-1/anti-her2 natural antibody structure-like bispecific antibody of heterodimeric form and preparation thereof
CA3045306A1 (en) 2016-11-29 2018-06-07 Boston Biomedical, Inc. Naphthofuran derivatives, preparation, and methods of use thereof
US10537637B2 (en) 2017-01-05 2020-01-21 Gensun Biopharma Inc. Checkpoint regulator antagonists
AU2018228873A1 (en) 2017-03-01 2019-08-29 Genentech, Inc. Diagnostic and therapeutic methods for cancer
AU2018240375C1 (en) 2017-03-22 2024-02-01 Ascendis Pharma A/S Hydrogel cross-linked hyaluronic acid prodrug compositions and methods
BR112019019706A2 (pt) 2017-03-22 2020-04-28 Genentech Inc conjugado de anticorpo, anticorpo, composição farmacêutica, métodos para reduzir ou inibir angiogênese e para tratar um distúrbio ocular
JP2020520923A (ja) 2017-05-17 2020-07-16 ボストン バイオメディカル， インコーポレイテッド がんを処置するための方法
CU24606B1 (es) 2017-06-22 2022-06-06 Novartis Ag Moléculas de anticuerpo que se unen a cd73
EP3642240A1 (en) 2017-06-22 2020-04-29 Novartis AG Antibody molecules to cd73 and uses thereof
CN112980667B (zh) 2017-08-29 2024-09-17 伯乐实验室有限公司 用于分离和分析细胞的系统和方法
WO2019057946A1 (en) 2017-09-25 2019-03-28 F. Hoffmann-La Roche Ag MULTI-CYCLIC AROMATIC COMPOUNDS AS D-FACTOR INHIBITORS
CN107703290A (zh) * 2017-09-30 2018-02-16 安徽伊普诺康生物技术股份有限公司 一种血管内皮因子检测试剂盒的制备方法
CN107727847A (zh) * 2017-09-30 2018-02-23 安徽伊普诺康生物技术股份有限公司 一种血管内皮因子检测试剂盒及其使用方法
JPWO2019098385A1 (ja) 2017-11-20 2020-12-24 コニカミノルタ株式会社 薬剤評価方法
US12398209B2 (en) 2018-01-22 2025-08-26 Janssen Biotech, Inc. Methods of treating cancers with antagonistic anti-PD-1 antibodies
WO2019153200A1 (zh) 2018-02-08 2019-08-15 北京韩美药品有限公司 抗pd-1/抗her2天然抗体结构样异源二聚体形式双特异抗体及其制备
WO2019154349A1 (zh) 2018-02-11 2019-08-15 北京韩美药品有限公司 抗pd-1/抗vegf天然抗体结构样异源二聚体形式双特异抗体及其制备
MX2020009152A (es) 2018-03-02 2020-11-09 Kodiak Sciences Inc Anticuerpos de il-6 y constructos de fusion y conjugados de los mismos.
AU2019237774B2 (en) * 2018-03-19 2023-03-23 Pharmabcine Inc Anti-VEGFR-2 antibody
WO2019195770A1 (en) 2018-04-06 2019-10-10 Atyr Pharma, Inc. Compositions and methods comprising anti-nrp2 antibodies
WO2019201195A1 (zh) 2018-04-16 2019-10-24 上海岸阔医药科技有限公司 预防或治疗肿瘤疗法副作用的方法
WO2019225777A1 (ko) 2018-05-23 2019-11-28 에이비엘바이오 주식회사 항-ror1 항체 및 그 용도
WO2019225787A1 (ko) 2018-05-24 2019-11-28 에이비엘바이오 주식회사 항-b7-h3 항체 및 그 용도
JOP20200303A1 (ar) 2018-05-24 2020-11-23 Janssen Biotech Inc عوامل ربط psma واستخداماتها
AR126019A1 (es) 2018-05-30 2023-09-06 Novartis Ag Anticuerpos frente a entpd2, terapias de combinación y métodos de uso de los anticuerpos y las terapias de combinación
US20210214459A1 (en) 2018-05-31 2021-07-15 Novartis Ag Antibody molecules to cd73 and uses thereof
US11001635B2 (en) 2018-06-29 2021-05-11 Gensun Biopharma Inc. Antitumor antagonists
TW202021618A (zh) 2018-08-17 2020-06-16 美商２３與我有限公司 抗ｉｌ１ｒａｐ抗體及其使用方法
CN113196061A (zh) 2018-10-18 2021-07-30 豪夫迈·罗氏有限公司 肉瘤样肾癌的诊断和治疗方法
AR117327A1 (es) 2018-12-20 2021-07-28 23Andme Inc Anticuerpos anti-cd96 y métodos de uso de estos
EP3898673A1 (en) 2018-12-21 2021-10-27 23Andme, Inc. Anti-il-36 antibodies and methods of use thereof
US10633693B1 (en) 2019-04-16 2020-04-28 Celsee Diagnostics, Inc. System and method for leakage control in a particle capture system
AU2020259404A1 (en) 2019-04-19 2021-09-23 Janssen Biotech, Inc. Methods of treating prostate cancer with an anti- PSMA/CD3 antibody
MX2021013222A (es) 2019-05-03 2022-01-06 Genentech Inc Metodos para tratar el cancer con un anticuerpo anti-pd-l1.
US11273439B2 (en) 2019-05-07 2022-03-15 Bio-Rad Laboratories, Inc. System and method for target material retrieval from microwells
SG11202112151UA (en) 2019-05-07 2021-12-30 Bio Rad Laboratories System and method for automated single cell processing
WO2020237092A2 (en) * 2019-05-21 2020-11-26 The Regents Of The University Of California Mmp-9 antibodies and methods of use thereof
US11332546B2 (en) 2019-05-21 2022-05-17 The Regents Of The University Of California Protease inhibitory antibodies and methods of use thereof
JP2022539739A (ja) * 2019-06-26 2022-09-13 バイオ－ラッド ラボラトリーズ インコーポレイテッド マイクロウェルからの標的物質の回収システムおよび方法
WO2020260595A1 (en) 2019-06-26 2020-12-30 Oncurious Nv Combination treatment of medulloblastoma using a placental growth factor inhibitor and a chemotherapeutic agent
JP2022540571A (ja) 2019-06-28 2022-09-16 ゲンスン バイオファーマ、インコーポレーテッド 変異したＴＧＦβ１－ＲＩＩ細胞外ドメインおよび免疫グロブリン足場で構成される抗腫瘍アンタゴニスト
WO2021013065A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 人源化抗VEGF Fab抗体片段及其用途
CA3147921C (en) * 2019-07-19 2023-02-21 Sinocelltech Ltd. Humanized anti-vegf monoclonal antibody
WO2021013781A1 (en) 2019-07-22 2021-01-28 F. Hoffmann-La Roche Ag Substance p as blood biomarker for the non-invasive diagnosis of endometriosis
BR112022001140A2 (pt) 2019-07-22 2022-03-15 Hoffmann La Roche Métodos para avaliar se um paciente tem endometriose ou está em risco de desenvolver endometriose, selecionar um paciente para terapia e monitorar um paciente que sofre de endometriose ou que está sendo tratado para endometriose
CN114144675A (zh) 2019-07-22 2022-03-04 豪夫迈·罗氏有限公司 S100a9作为血液生物标志物用于子宫内膜异位症的非侵入性诊断
WO2021013784A1 (en) 2019-07-22 2021-01-28 F. Hoffmann-La Roche Ag S100a8 as blood biomarker for the non-invasive diagnosis of endometriosis
WO2021013786A1 (en) 2019-07-22 2021-01-28 F. Hoffmann-La Roche Ag S100a6 as blood biomarker for the non-invasive diagnosis of endometriosis
WO2021053559A1 (en) 2019-09-18 2021-03-25 Novartis Ag Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies
AU2020358854A1 (en) * 2019-10-03 2022-05-26 Atyr Pharma, Inc. Compositions and methods comprising anti-NRP2 antibodies
CN114786731A (zh) 2019-10-10 2022-07-22 科达制药股份有限公司 治疗眼部病症的方法
JP2023502360A (ja) 2019-11-15 2023-01-24 エフ．ホフマン－ラ ロシュ アーゲー 患者試料における質量分析測定のためのβ－ラクタム抗生物質の誘導体化
EP4061852A4 (en) * 2019-11-21 2024-03-13 Unity Biotechnology ANTIBODIES TARGETED TO TIE-2 AND METHODS OF USE
CN115605507A (zh) 2020-03-13 2023-01-13 基因泰克公司（Us） 抗白介素-33抗体及其用途
EP4127139A1 (en) 2020-03-27 2023-02-08 Mendus B.V. Ex vivo use of modified cells of leukemic origin for enhancing the efficacy of adoptive cell therapy
WO2021202959A1 (en) 2020-04-03 2021-10-07 Genentech, Inc. Therapeutic and diagnostic methods for cancer
JP2023522278A (ja) 2020-04-23 2023-05-29 エフ． ホフマン－ラ ロシュ アーゲー 抗体－免疫アッセイでの使用のためのコロナヌクレオカプシド抗原
AU2021281417A1 (en) 2020-05-29 2022-12-08 23Andme, Inc. Anti-CD200R1 antibodies and methods of use thereof
CN116234824A (zh) 2020-06-22 2023-06-06 阿尔米雷尔有限公司 抗il-36抗体及其使用方法
CN116133679A (zh) 2020-06-30 2023-05-16 门德斯有限公司 白血病衍生细胞在卵巢癌疫苗中的用途
WO2022003156A1 (en) 2020-07-02 2022-01-06 Oncurious Nv Ccr8 non-blocking binders
EP3943946A1 (en) 2020-07-20 2022-01-26 F. Hoffmann-La Roche AG Gdf-15 for predicting the disease severity of a patient with covid-19
WO2022053715A1 (en) 2020-09-14 2022-03-17 Ichnos Sciences SA Antibodies that bind to il1rap and uses thereof
JP2023547505A (ja) 2020-11-02 2023-11-10 エフ． ホフマン－ラ ロシュ アーゲー ＳＡＲＳ－ＣｏＶ－２ヌクレオカプシド抗体
EP4243828B1 (en) 2020-11-13 2025-04-02 Genentech, Inc. Methods and compositions comprising a krasg12c inhibitor and a vegf inhibitor for treating solid tumors
EP4255929A2 (en) 2020-12-02 2023-10-11 Vib Vzw An ltbr agonist in combination therapy against cancer
WO2022117569A1 (en) 2020-12-02 2022-06-09 Oncurious Nv A ccr8 antagonist antibody in combination with a lymphotoxin beta receptor agonist antibody in therapy against cancer
EP4259146A1 (en) 2020-12-08 2023-10-18 Genentech, Inc. Methods and compositions comprising a krasg12c inhibitor and a egfr-inhibitor for treating solid tumors
EP4267617A1 (en) 2020-12-24 2023-11-01 Vib Vzw Human ccr8 binders
EP4267618A1 (en) 2020-12-24 2023-11-01 Vib Vzw Non-blocking human ccr8 binders
US20240052045A1 (en) 2020-12-24 2024-02-15 Vib Vzw Murine cross-reactive human ccr8 binders
US12397055B2 (en) 2021-01-22 2025-08-26 Mendus B.V. Methods of tumor vaccination
WO2022162518A2 (en) 2021-01-28 2022-08-04 Janssen Biotech, Inc. Psma binding proteins and uses thereof
AR124914A1 (es) * 2021-02-18 2023-05-17 Mitsubishi Tanabe Pharma Corp Nuevo anticuerpo anti-pad4
EP4304633A1 (en) 2021-03-12 2024-01-17 Mendus B.V. Methods of vaccination and use of cd47 blockade
CN112961243B (zh) * 2021-03-24 2022-04-29 山东兴瑞生物科技有限公司 一种vegf抗体、重组aav病毒及其应用
WO2022207628A1 (en) 2021-03-30 2022-10-06 F. Hoffmann-La Roche Ag Scf as blood biomarker for the non-invasive diagnosis of endometriosis
WO2022207685A1 (en) 2021-04-01 2022-10-06 F. Hoffmann-La Roche Ag Psp94 as blood biomarker for the non-invasive diagnosis of endometriosis
JP2024516230A (ja) 2021-04-30 2024-04-12 ジェネンテック， インコーポレイテッド がんのための治療及び診断方法並びに組成物
JP7727015B2 (ja) 2021-05-17 2025-08-20 エフ． ホフマン－ラ ロシュ アーゲー 腺筋症の非侵襲的診断のための血液バイオマーカーとしてのｓＦＲＰ４
WO2022256820A1 (en) 2021-06-03 2022-12-08 Gensun Biopharma Inc. Multispecific antagonists
BR112023026966A2 (pt) 2021-07-02 2024-03-12 Hoffmann La Roche Métodos para tratar um indivíduo com melanoma, para alcançar uma resposta clínica, para tratar um indivíduo com linfoma não hodgkin, para tratar uma população de indivíduos com linfoma não hodgkin e para tratar um indivíduo com câncer colorretal metastático
AU2022317820A1 (en) 2021-07-28 2023-12-14 F. Hoffmann-La Roche Ag Methods and compositions for treating cancer
WO2023009529A2 (en) 2021-07-28 2023-02-02 Trustees Of Boston University Novel polypeptides and uses thereof
US20250042976A1 (en) 2021-10-26 2025-02-06 Roche Diagnostics Operations, Inc. Monoclonal antibodies specific for sars-cov-2 rbd
WO2023080900A1 (en) 2021-11-05 2023-05-11 Genentech, Inc. Methods and compositions for classifying and treating kidney cancer
JPWO2023144973A1 (enrdf_load_stackoverflow) 2022-01-27 2023-08-03
CN118742325A (zh) 2022-01-28 2024-10-01 上海岸阔医药科技有限公司 预防或治疗与抗肿瘤剂相关的疾病或病症的方法
WO2023155905A1 (zh) 2022-02-21 2023-08-24 上海岸阔医药科技有限公司 化合物及其用途
CN116925234B (zh) * 2022-04-02 2024-05-31 合肥星眸生物科技有限公司 一种编码抗vegf-a和ang-2双特异性抗体的aav载体
US20250235534A1 (en) 2022-04-13 2025-07-24 Vib Vzw An LTBR Agonist In Combination Therapy Against Cancer
EP4519285A1 (en) 2022-05-03 2025-03-12 F. Hoffmann-La Roche AG Hiv gp41 variants for immunodiagnostic assays
CN119404103A (zh) 2022-06-23 2025-02-07 豪夫迈·罗氏有限公司 用于诊断子宫内膜异位症及用于对子宫内膜异位症的分期进行分类的方法
JP2025524027A (ja) 2022-07-22 2025-07-25 エフ． ホフマン－ラ ロシュ アーゲー 多嚢胞性卵巣症候群の診断のための（血液）バイオマーカーとしてのメテオリン様タンパク質（ｍｅｔｒｎｌ）
EP4558830A1 (en) 2022-07-22 2025-05-28 F. Hoffmann-La Roche AG Leukotriene a4 hydrolase (lta4h) as (blood) biomarker for the diagnosis of polycystic ovarian syndrome
JP2025523884A (ja) 2022-07-22 2025-07-25 エフ． ホフマン－ラ ロシュ アーゲー 多嚢胞性卵巣症候群の診断のための（血液）バイオマーカーとしての線維芽細胞増殖因子結合タンパク質１（ｆｇｆｂｐ１）
CN115850470B (zh) * 2022-12-12 2023-07-07 三门峡市眼科医院 Vegf抗体及其应用
WO2024263904A1 (en) 2023-06-23 2024-12-26 Genentech, Inc. Methods for treatment of liver cancer
WO2024263195A1 (en) 2023-06-23 2024-12-26 Genentech, Inc. Methods for treatment of liver cancer
WO2025026908A1 (en) 2023-07-28 2025-02-06 Roche Diagnostics Gmbh Serum epha1 as biomarker for endometriosis
WO2025049972A1 (en) 2023-09-01 2025-03-06 Cargo Therapeutics, Inc. Compositions and methods for immunotherapies
WO2025120867A1 (en) 2023-12-08 2025-06-12 Astellas Pharma Inc. Combination therapy involving bispecific binding agents binding to cldn18.2 and cd3 and anti-vegfr2 antibodies
WO2025120866A1 (en) 2023-12-08 2025-06-12 Astellas Pharma Inc. Combination therapy involving bispecific binding agents binding to cldn18.2 and cd3 and agents stabilizing or increasing expression of cldn18.2
WO2025121445A1 (en) 2023-12-08 2025-06-12 Astellas Pharma Inc. Combination therapy involving bispecific binding agents binding to cldn18.2 and cd3 and agents stabilizing or increasing expression of cldn18.2

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5427908A (en) * 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
US5432018A (en) * 1990-06-20 1995-07-11 Affymax Technologies N.V. Peptide library and screening systems
US5498530A (en) * 1991-10-16 1996-03-12 Affymax Technologies, N.V. Peptide library and screening method
US5534615A (en) * 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
US5650727A (en) * 1995-04-07 1997-07-22 Wizaed Devices, Inc. Circuit continuity testing device
US5733743A (en) * 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5750373A (en) * 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5821337A (en) * 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US5837242A (en) * 1992-12-04 1998-11-17 Medical Research Council Multivalent and multispecific binding proteins, their manufacture and use
US5969108A (en) * 1990-07-10 1999-10-19 Medical Research Council Methods for producing members of specific binding pairs
US6054297A (en) * 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
US6172197B1 (en) * 1991-07-10 2001-01-09 Medical Research Council Methods for producing members of specific binding pairs
US6190908B1 (en) * 1998-08-12 2001-02-20 The Scripps Research Institute Modulation of polypeptide display on modified filamentous phage
US20030091995A1 (en) * 1999-10-02 2003-05-15 Joe Buechler Human antibodies
US20050119455A1 (en) * 2002-06-03 2005-06-02 Genentech, Inc. Synthetic antibody phage libraries

Family Cites Families (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4318980A (en) 1978-04-10 1982-03-09 Miles Laboratories, Inc. Heterogenous specific binding assay employing a cycling reactant as label
US4376110A (en) 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
US4419446A (en) 1980-12-31 1983-12-06 The United States Of America As Represented By The Department Of Health And Human Services Recombinant DNA process utilizing a papilloma virus DNA as a vector
NZ201705A (en) 1981-08-31 1986-03-14 Genentech Inc Recombinant dna method for production of hepatitis b surface antigen in yeast
US4601978A (en) 1982-11-24 1986-07-22 The Regents Of The University Of California Mammalian metallothionein promoter system
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4675187A (en) 1983-05-16 1987-06-23 Bristol-Myers Company BBM-1675, a new antibiotic complex
DD266710A3 (de) 1983-06-06 1989-04-12 Ve Forschungszentrum Biotechnologie Verfahren zur biotechnischen Herstellung van alkalischer Phosphatase
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US4965199A (en) 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
US4879231A (en) 1984-10-30 1989-11-07 Phillips Petroleum Company Transformation of yeasts of the genus pichia
US4737456A (en) 1985-05-09 1988-04-12 Syntex (U.S.A.) Inc. Reducing interference in ligand-receptor binding assays
GB8516415D0 (en) 1985-06-28 1985-07-31 Celltech Ltd Culture of animal cells
FR2596047B1 (fr) 1986-03-21 1988-05-13 Charbonnages Ste Chimique Procede de production de styrene
GB8607679D0 (en) 1986-03-27 1986-04-30 Winter G P Recombinant dna product
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
GB8610600D0 (en) 1986-04-30 1986-06-04 Novo Industri As Transformation of trichoderma
IL85035A0 (en) 1987-01-08 1988-06-30 Int Genetic Eng Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same
JP3040121B2 (ja) 1988-01-12 2000-05-08 ジェネンテク，インコーポレイテッド 増殖因子レセプターの機能を阻害することにより腫瘍細胞を処置する方法
ATE135397T1 (de) 1988-09-23 1996-03-15 Cetus Oncology Corp Zellenzuchtmedium für erhöhtes zellenwachstum, zur erhöhung der langlebigkeit und expression der produkte
US5688666A (en) 1988-10-28 1997-11-18 Genentech, Inc. Growth hormone variants with altered binding properties
FI903489A0 (fi) 1988-11-11 1990-07-10 Medical Res Council Ligander med en enda sektion, receptorer innehaollande naemnda ligander, foerfaranden foer deras framstaellning samt anvaendning av liganderna och receptorerna.
FR2646437B1 (fr) 1989-04-28 1991-08-30 Transgene Sa Nouvelles sequences d'adn, leur application en tant que sequence codant pour un peptide signal pour la secretion de proteines matures par des levures recombinantes, cassettes d'expression, levures transformees et procede de preparation de proteines correspondant
EP0402226A1 (en) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Transformation vectors for yeast yarrowia
DE3920358A1 (de) 1989-06-22 1991-01-17 Behringwerke Ag Bispezifische und oligospezifische, mono- und oligovalente antikoerperkonstrukte, ihre herstellung und verwendung
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US5658727A (en) 1991-04-10 1997-08-19 The Scripps Research Institute Heterodimeric receptor libraries using phagemids
DE69233528T2 (de) 1991-11-25 2006-03-16 Enzon, Inc. Verfahren zur Herstellung von multivalenten antigenbindenden Proteinen
ZA936128B (en) 1992-08-21 1995-02-20 Genentech Inc Method for treating a LFA-1 mediated disorder
ES2152483T3 (es) 1992-11-13 2001-02-01 Idec Pharma Corp Aplicacion terapeutica en anticuerpos quimericos y radiomarcados contra el antigeno de diferenciacion restringida de los linfocitos b humanos para el tratamiento del linfoma de celulas b.
US5635388A (en) 1994-04-04 1997-06-03 Genentech, Inc. Agonist antibodies against the flk2/flt3 receptor and uses thereof
US5739277A (en) 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
ES2169864T3 (es) * 1996-05-31 2002-07-16 Health Research Inc Anticuerpos monoclonales anti-endoglina y su uso en terapia anti-angiogenesis.
ES2236634T3 (es) 1997-04-07 2005-07-16 Genentech, Inc. Anticuerpos anti-vegf.
US6884879B1 (en) * 1997-04-07 2005-04-26 Genentech, Inc. Anti-VEGF antibodies
WO2000034337A1 (en) * 1998-12-10 2000-06-15 Tsukuba Research Laboratory, Toagosei Co., Ltd. Humanized monoclonal antibodies against vascular endothelial cell growth factor
NZ539776A (en) 1999-01-15 2006-12-22 Genentech Inc Polypeptide variants with altered effector function
WO2000064946A2 (en) * 1999-04-28 2000-11-02 Board Of Regents, The University Of Texas System Compositions and methods for cancer treatment by selectively inhibiting vegf
DE60025526T2 (de) 1999-11-16 2006-08-24 Genentech, Inc., South San Francisco Elisa für vegf
CN1299833A (zh) * 1999-12-30 2001-06-20 华西医科大学口腔医学研究所 抗人血管内皮生长因子单链抗体及其制备方法
US7083784B2 (en) 2000-12-12 2006-08-01 Medimmune, Inc. Molecules with extended half-lives, compositions and uses thereof
CA2444624A1 (en) 2001-04-13 2002-10-24 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
EP2228389B1 (en) * 2001-04-13 2015-07-08 Human Genome Sciences, Inc. Antibodies against vascular endothelial growth factor 2
CN1187373C (zh) * 2002-03-20 2005-02-02 上海中信国健药业有限公司 人源化抗血管内皮生长因子单克隆抗体及其制法和药物组合物
AU2004205631A1 (en) * 2003-01-16 2004-08-05 Genentech, Inc. Synthetic antibody phage libraries
KR20180132969A (ko) * 2003-05-30 2018-12-12 제넨테크, 인크. 항-ｖｅｇｆ 항체를 사용한 치료
WO2005044853A2 (en) 2003-11-01 2005-05-19 Genentech, Inc. Anti-vegf antibodies
US7758859B2 (en) * 2003-08-01 2010-07-20 Genentech, Inc. Anti-VEGF antibodies
US20050106667A1 (en) 2003-08-01 2005-05-19 Genentech, Inc Binding polypeptides with restricted diversity sequences
US20070237764A1 (en) * 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
AR069501A1 (es) * 2007-11-30 2010-01-27 Genentech Inc Anticuerpos anti- vegf (factor de crecimiento endotelial vascular)
BRPI0918648A2 (pt) * 2008-09-03 2019-09-03 Genentech Inc anticorpos multiespecíficos
ES2826883T3 (es) 2009-10-14 2021-05-19 Janssen Biotech Inc Métodos de maduración por afinidad de anticuerpos
US9321844B2 (en) 2011-08-05 2016-04-26 Genentech, Inc. Anti-polyubiquitin antibodies

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5427908A (en) * 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
US5580717A (en) * 1990-05-01 1996-12-03 Affymax Technologies N.V. Recombinant library screening methods
US5723286A (en) * 1990-06-20 1998-03-03 Affymax Technologies N.V. Peptide library and screening systems
US5432018A (en) * 1990-06-20 1995-07-11 Affymax Technologies N.V. Peptide library and screening systems
US5969108A (en) * 1990-07-10 1999-10-19 Medical Research Council Methods for producing members of specific binding pairs
US5750373A (en) * 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5821337A (en) * 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US6054297A (en) * 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
US6172197B1 (en) * 1991-07-10 2001-01-09 Medical Research Council Methods for producing members of specific binding pairs
US5498530A (en) * 1991-10-16 1996-03-12 Affymax Technologies, N.V. Peptide library and screening method
US5733743A (en) * 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5837242A (en) * 1992-12-04 1998-11-17 Medical Research Council Multivalent and multispecific binding proteins, their manufacture and use
US5534615A (en) * 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
US5650727A (en) * 1995-04-07 1997-07-22 Wizaed Devices, Inc. Circuit continuity testing device
US6190908B1 (en) * 1998-08-12 2001-02-20 The Scripps Research Institute Modulation of polypeptide display on modified filamentous phage
US20030091995A1 (en) * 1999-10-02 2003-05-15 Joe Buechler Human antibodies
US20050119455A1 (en) * 2002-06-03 2005-06-02 Genentech, Inc. Synthetic antibody phage libraries

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050281894A1 (en) * 2002-11-08 2005-12-22 Kin-Ping Wong Extract of Trapa natans and methods of using the same
US7494671B2 (en) * 2002-11-08 2009-02-24 Kin-Ping Wong Extract of Trapa natans and methods of using the same
EP2325208A1 (en) 2005-12-15 2011-05-25 Genentech, Inc. Polyubiquitin antibodies
US9487578B2 (en) 2005-12-15 2016-11-08 Genentech, Inc. Methods and compositions for targeting polyubiquitin
EP3309170A1 (en) 2005-12-15 2018-04-18 Genentech, Inc. Polyubiquitin antibodies
US20100267050A1 (en) * 2005-12-15 2010-10-21 Genentech, Inc. Methods and compositions for targeting polyubiquitin
US8603475B2 (en) 2005-12-15 2013-12-10 Genentech, Inc. Methods and compositions for targeting polyubiquitin
US20090093024A1 (en) * 2007-02-20 2009-04-09 Anaptysbio, Inc. Methods of generating libraries and uses thereof
US20110183855A1 (en) * 2007-02-20 2011-07-28 Anaptysbio, Inc. Somatic hypermutation systems
US8603950B2 (en) 2007-02-20 2013-12-10 Anaptysbio, Inc. Methods of generating libraries and uses thereof
US8685897B2 (en) 2007-02-20 2014-04-01 Anaptysbio, Inc. Methods of generating libraries and uses thereof
US9260533B2 (en) 2007-02-20 2016-02-16 Anaptysbio, Inc. Methods of generating libraries and uses thereof
US9637556B2 (en) 2007-02-20 2017-05-02 Anaptysbio, Inc. Methods of generating libraries and uses thereof
US20090075378A1 (en) * 2007-02-20 2009-03-19 Anaptysbio, Inc. Somatic hypermutation systems
US20090011060A1 (en) * 2007-07-06 2009-01-08 Peter Koepke Campsiandra angustifolia extract and methods of extracting and using such extract
US7879369B2 (en) 2007-09-18 2011-02-01 Selvamedica, Llc Combretum laurifolium Mart. extract and methods of extracting and using such extract
US9365642B2 (en) 2008-01-18 2016-06-14 Genentech, Inc. Methods and compositions for targeting polyubiquitin
US10035849B2 (en) 2008-01-18 2018-07-31 Genentech, Inc. Methods and compositions for targeting polyubiquitin
US11753464B2 (en) 2008-01-18 2023-09-12 Genentech, Inc. Methods and compositions for targeting polyubiquitin
US9081015B2 (en) 2008-01-18 2015-07-14 Genentech, Inc. Methods and compositions for targeting polyubiquitin
US10808028B2 (en) 2008-01-18 2020-10-20 Genentech, Inc. Methods and compositions for targeting polyubiquitin
US10100105B2 (en) 2010-04-15 2018-10-16 Genentech, Inc. Anti-polyubiquitin antibodies and methods of use
US11066463B2 (en) 2010-04-15 2021-07-20 Genentech, Inc. Anti-polyubiquitin antibodies and methods of use
US8992919B2 (en) 2010-04-15 2015-03-31 Genentech, Inc. Anti-polyubiquitin antibodies and methods of use
US12037385B2 (en) 2010-04-15 2024-07-16 Genentech, Inc. Anti-polyubiquitin antibodies and methods of use
US9556262B2 (en) 2010-04-15 2017-01-31 Genentech, Inc. Anti-polyubiquitin antibodies and methods of use
US20140154743A1 (en) * 2011-02-03 2014-06-05 Raphael D. Levy Methods and materials for enhancing functional protein expression in bacteria
US9732143B2 (en) * 2011-02-03 2017-08-15 Xoma Technology Ltd. Methods and materials for enhancing functional protein expression in bacteria
US11597761B2 (en) 2011-08-05 2023-03-07 Genentech Inc. Methods of detecting polyubiquitin using anti-polyubiquitin antibodies
US10738106B2 (en) 2011-08-05 2020-08-11 Genentech, Inc. Nucleic acids encoding anti-polyubiquitin antibodies
US9321844B2 (en) 2011-08-05 2016-04-26 Genentech, Inc. Anti-polyubiquitin antibodies
US12286470B2 (en) 2011-08-05 2025-04-29 Genentech, Inc. Methods of determining the function of polyubiquitin using anti-polyubiquitin antibodies
EP2861064A4 (en) * 2012-06-11 2015-12-02 Syngenta Participations Ag MANUFACTURE OF SOLIDS AND ASSOCIATED MOTHER TUBES
US11007259B2 (en) 2016-01-06 2021-05-18 Order-Made Medical Research Inc. High-affinity anti-VEGF antibody
US11008387B2 (en) 2016-01-06 2021-05-18 Order-Made Medical Research Inc. Antibody inhibiting binding of VEGF to NRP1
US11872271B2 (en) 2016-01-06 2024-01-16 Order-Made Medical Research Inc. High-affinity anti-VEGF antibody KLHa505

Also Published As

Publication number Publication date
WO2005012359A2 (en) 2005-02-10
US20100189719A1 (en) 2010-07-29
US20110020346A1 (en) 2011-01-27
US20150232549A1 (en) 2015-08-20
US20120322982A1 (en) 2012-12-20
AU2004262006A1 (en) 2005-02-10
AU2004262006C1 (en) 2012-10-18
CA2533297C (en) 2016-06-07
JP2007526756A (ja) 2007-09-20
IL173317A (en) 2016-02-29
WO2005012359A3 (en) 2005-12-15
EA200600344A1 (ru) 2006-08-25
JP4845732B2 (ja) 2011-12-28
MXPA06001319A (es) 2006-05-04
KR20160052799A (ko) 2016-05-12
EP1648939A2 (en) 2006-04-26
KR20130098436A (ko) 2013-09-04
US8101177B2 (en) 2012-01-24
CN104761643A (zh) 2015-07-08
US8092797B2 (en) 2012-01-10
AU2004262006A2 (en) 2005-02-10
EA012835B1 (ru) 2009-12-30
CA2533297A1 (en) 2005-02-10
JP2011046732A (ja) 2011-03-10
US9353177B2 (en) 2016-05-31
OA13225A (en) 2006-12-13
EP2420512A3 (en) 2013-08-21
JP5162644B2 (ja) 2013-03-13
KR101644717B1 (ko) 2016-08-01
IL173317A0 (en) 2006-06-11
BRPI0412637A (pt) 2006-09-26
NO20061014L (no) 2006-04-24
US20130266581A1 (en) 2013-10-10
US20070141065A1 (en) 2007-06-21
US20060280747A1 (en) 2006-12-14
US8492527B2 (en) 2013-07-23
KR20110059806A (ko) 2011-06-03
CA2932216A1 (en) 2005-02-10
KR20060067950A (ko) 2006-06-20
AU2004262006B2 (en) 2011-12-22
NZ575674A (en) 2010-08-27
EP2420512A2 (en) 2012-02-22
HK1211600A1 (en) 2016-05-27
US7691977B2 (en) 2010-04-06
IL243914A0 (en) 2016-04-21
KR20140048973A (ko) 2014-04-24
US20170073405A1 (en) 2017-03-16
KR101359142B1 (ko) 2014-02-07
SG145725A1 (en) 2008-09-29
US7811785B2 (en) 2010-10-12
NZ545135A (en) 2009-10-30
KR101446849B1 (ko) 2014-10-15
KR20120074336A (ko) 2012-07-05
SG193170A1 (en) 2013-09-30
US9018357B2 (en) 2015-04-28

Similar Documents

Publication Publication Date Title
US8679490B2 (en) Binding polypeptides with diversified and consensus VH/VL hypervariable sequences
US20050106667A1 (en) Binding polypeptides with restricted diversity sequences
US8957187B2 (en) Binding polypeptides and uses thereof
US20090023602A1 (en) Binding polypeptides with restricted diversity sequences
US20070237764A1 (en) Binding polypeptides with restricted diversity sequences
EP1973950B1 (en) Anti-ephb4 antibodies and methods using the same
JP2009518011A5 (enrdf_load_stackoverflow)
US9845354B2 (en) Anti-EPHRINB2 antibodies and methods using same
AU2012204022B2 (en) Binding polypeptides and uses thereof
HK1121040A (en) Anti-ephb4 antibodies and methods using the same
HK1121040B (en) Anti-ephb4 antibodies and methods using the same
HK1161888A (en) Anti-ephb4 antibodies and methods using same
BRPI0620505A2 (pt) polipeptìdeos, anticorpo, polipeptìdeo de fusão, composição, polinucleotìdeo, vetor, célula hospedeira, método de produção de um polipeptìdeo, biblioteca de polipeptìdeos, métodos para gerar uma composição, método para a seleção de um domìnio variável ligante ao antìgeno, método para selecionar polipeptìdeos, método para isolar um ou mais polipeptìdeos, método para o tratamento de cáncer e método para tratar

Legal Events

Date Code Title Description
AS Assignment

Owner name: GENENTECH, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FELLOUSE, FREDERIC;SIDHU, SACHDEV;REEL/FRAME:015611/0224;SIGNING DATES FROM 20041115 TO 20041123

STCB Information on status: application discontinuation

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