Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • 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
• • 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
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • 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
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
  • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
  • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
  • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
  • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• • 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
  • C07K2317/30—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
  • C07K2317/56—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
  • C07K2317/90—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
• • 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
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the present invention relates to multispecific antibodies, and methods of making and using such antibodies.
• Antibodies are specific immunoglobulin polypeptides produced by the vertebrate immune system in response to challenge by foreign proteins, glycoproteins, cells, or other antigenic foreign substances. An important part of this process is the generation of antibodies that bind specifically to a particular foreign substance.
• the binding specificity of such polypeptides to a particular antigen is highly refined, and the multitude of specificities capable of being generated by the individual vertebrate is remarkable in its complexity and variability. Thousands of antigens are capable of eliciting responses, each almost exclusively directed to the particular antigen which elicited it.
• Antibodies and antibody fragments can be used to target particular tissues, for example, a tumor, and thereby minimize the potential side effects of non-specific targeting. As such, there is a current and continuing need to identify and characterize therapeutic antibodies, especially antibodies, fragments, and derivatives thereof, useful in the treatment of cancer and other proliferative disorders. Summary of the Invention
• the present invention provides an isolated antibody comprising a hypervariable region (HVR) Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1 ), where the antibody specifically binds human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF).
• HVR hypervariable region
• the antibody further comprises an HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2) and/or an HVR-L3 comprising the sequence HYSSPP (SEQ ID NO: 3).
• the antibody further comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3 comprising the sequence WGGDGFY AMD (SEQ ID NO:6).
• the antibody further comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii) HVR-H3 comprising the sequence WVGVGFY AMD (SEQ ID NO:9).
• an antibody comprising the sequence XiI X 3 X 4 X 5 X 6 X 7 XsXgY (SEQ ID NO: 83) has an asparagine at X 1 , an alanine at X 3 , a lysine at X 4 , a threonine at X 5 , a serine at X 7 , and/or a glycine at X 8 , or any combination thereof.
• any of the HVR-Ll residues shown in Figure 57 to have an F value of greater than 1, 5, or 10 are residues that are preferably maintained as the same residue found in the same position of the HVR-Ll of bH 1-44 or bHl- 81 (SEQ ID NO: 1).
• any of the HVR-Ll residues shown in Table 14 to have ⁇ G values greater than 1 are residues that are preferably maintained as the same residue found in the same position of the HVR-Ll of bH 1-44 or bHl-81 (SEQ ID NO: 1).
• the antibody comprises an HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 84). In one embodiment, the antibody further comprises an HVR-L2 comprising the sequence WGSFLY (SEQ ID NO: 2) and/or an HVR- L3 comprising the sequence HYSSPP (SEQ ID NO: 3).
• the antibody further comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and (iii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the invention features an isolated antibody comprising an HVR-H2 sequence comprising the sequence RX2X3X4X5X6X7X8X9R (SEQ ID NO: 85), wherein X 5 is any amino acid except threonine and X6 is any amino acid except asparagine and where the antibody specifically binds HER2 and VEGF.
• an antibody comprising the sequence RX 2 X 3 X 4 X 5 X 6 X 7 X 8 XgR (SEQ ID NO: 84) has a tyrosine at X 8 .
• the antibodies further comprise, one or two HVR sequences selected from (i) HVR-Hl comprising the sequence NIKDTY (SEQ ID NO:4) and (ii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6).
• the antibodies further comprise, one or two HVR sequences selected from (i) HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7) and (ii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the antibody comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO: 1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NIKDTY (SEQ ID NO:4); an HVR-H2 sequence comprising RIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence comprising WGGDGFYAMD (SEQ ID NO:6) or comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO: 1 ); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NISGTY (SEQ ID NO:7); an HVR-H2 sequence comprising RIYPSEGYTR (SEQ ID NO:8);
• the isolated antibody comprises HVR-Ll, HVR- L2, HVR-L3, HVR-Hl, HVR-H2, and HVR-H3, wherein each, in order, comprises the sequence NIAKTISGY (SEQ ID NO: 1); WGSFLY (SEQ ID NO:2); HYSSPP (SEQ ID NO:3); NIKDTY (SEQ ID NO:4); RIYPTNGYTR (SEQ ID NO:5); and WGGDGFY AMD (SEQ ID NO:6) and specifically binds HER2 and VEGF.
• SEQ ID NO: 1 the sequence NIAKTISGY
• WGSFLY SEQ ID NO:2
• HYSSPP SEQ ID NO:3
• NIKDTY SEQ ID NO:4
• RIYPTNGYTR SEQ ID NO:5
• WGGDGFY AMD SEQ ID NO:6
• the invention features an isolated antibody that binds human and murine VEGF with a Kd of 150 nM or stronger and HER2 with a Kd of 7 nM or stronger and wherein the antibody inhibits VEGF-induced cell proliferation and proliferation of a HER2 expressing cell relative to a control.
• the antibody binds human and murine VEGF with a Kd of 36 nM or stronger and HER2 with a Kd of 1 nM or stronger.
• the invention provides an isolated antibody fragment that binds human VEGF with a Kd of 58 nM or stronger and HER2 with a Kd of 6 nM or stronger, and/or inhibits VEGF-induced cell proliferation and proliferation of a HER2 expressing cell relative to a control.
• the antibody fragment binds human and murine VEGF with a Kd of 33 nM or stronger and HER2 with a Kd of 0.7 nM or stronger.
• the fragment is a Fab fragment or a single chain variable fragment (scFv).
• the antibody may be a monoclonal antibody. In another embodiment of all the above aspects, the antibody may be an IgG antibody. In additional embodiments of all the above aspects, at least a portion of the framework sequence of the antibody may be a human consensus framework sequence.
• the invention features a fragment of an antibody any of the antibodies described herein.
• an antibody fragment is a fragment comprising a HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1) that specifically binds HER2 and VEGF.
• the antibody fragment further comprises one or two HVR sequences selected from (i) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO:2); and (ii) HVR-L3 comprising the sequence HYSSPP (SEQ ID NO:3).
• the antibody fragment further comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2 comprising the sequence RJYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6).
• the antibody fragment further comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and (iii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the antibody fragment comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO: 1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NIKDTY (SEQ ID NO:4); an HVR-H2 sequence comprising RrYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence comprising WGGDGFYAMD (SEQ ID NO:6) or comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO: 1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NISGTY (SEQ ID NO:7); an HVR-H2 sequence comprising RIYPSEGYTR (SEQ ID NO: 8);
• the polynucleotide may further encode an antibody comprising one, two, or three of an HVR-Hl sequence comprising NIKDTY (SEQ ID NO:4); an HVR-H2 sequence comprising RIYPTNG YTR (SEQ ID NO: 5); and an HVR-H3 sequence comprising WGGDGFYAMD (SEQ ID NO:6); or an antibody comprising one, two, or three of an HVR-Hl comprising NISGTY (SEQ ID NO:7); an HVR-H2 comprising RIYPSEGYTR (SEQ ID NO:8); and/or an HVR-H3 sequence comprising WVGVGFYAMD (SEQ ID NO:9).
• the polynucleotide encodes an HVR-Hl sequence comprising the sequence of NISGTY (SEQ ID NO: 7), an HVR-H2 comprising the sequence of RIYPSEGYTR (SEQ ID NO: 8), or an HVR-H3 comprising the sequence of WVGVGFYAMD (SEQ ID NO: 9), or any combination thereof.
• the invention features an isolated polynucleotide encoding an HVR- Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1) and, optionally, the polynucleotide further encodes one, two, or three HVR sequences selected from (i) an HVR- Hl comprising the sequence NIKDTY (SEQ ID NO:4); (ii) an HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO: 5); and (iii) an HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6).
• the invention features an isolated polynucleotide encoding an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1 ); and (i) an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO:2) or (ii) an HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID NO:3), or both, and, optionally, the polynucleotide further encodes one, two, or three HVR sequences selected from (i) an HVR-Hl comprising the sequence NIKDTY (SEQ ID NO:4); (ii) an HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) an HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6).
• the invention features an isolated polynucleotide encoding an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1); an HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); an HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and an HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the invention features an isolated polynucleotide encoding an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1); an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID NO:3); an HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); an HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO:8); and an HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the invention features an isolated polynucleotide encoding an HVR- Hl sequence comprising the sequence NISGTY (SEQ ID NO:7), an isolated polynucleotide encoding an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO:8), and an isolated polynucleotide encoding an HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the invention features an isolated polynucleotide encoding an polypeptide comprising an HVR-Hl sequence comprising the sequence NISGTY (SEQ ID NO:7); an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and an HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the isolated polynucleotide encodes an HVR-Ll sequence comprising the sequence X 1 I X 3 X 4 X 5 X 6 X 7 X 8 XsY (SEQ ID NO: 83), wherein Xi is any amino acid except aspartic acid, X 3 is any amino acid except proline, X 4 is any amino acid except arginine, and X 5 is any amino acid except serine.
• the polynucleotide encodes an HVR-Ll sequence comprising the sequence X
• the polynucleotide encodes an antibody comprising the sequence X 1 I X 3 X 4 X 5 X 6 X 7 X 8 X 9 Y (SEQ ID NO: 83) that has an asparagine at Xi , an alanine at X 3, a lysine at X 4, a threonine at X 5, a serine at X 7, and/or a glycine at Xg , or any combination thereof.
• any of the HVR-Ll residues shown in Figure 57 to have an F value of greater than 1, 5, or 10 are residues that are preferably maintained as the same residue found in the same position of the HVR-Ll of bH 1-44 or bHl -81 (SEQ ID NO: 1).
• any of the HVR-Ll residues shown in Table 14 to have ⁇ G values greater than 1 are residues that are preferably maintained as the same residue found in the same position of the HVR-Ll of bH 1 -44 or bHl-81 (SEQ ID NO: 1).
• the polynucleotide encodes an HVR- H2 sequence comprising the sequence RX 2 X 3 X 4 X 5 X 6 X 7 XgXgR (SEQ ID NO: 85), wherein X 5 is any amino acid except threonine and X 6 is any amino acid except asparagine.
• the invention provides a polynucleotide encoding an HVR-Hl sequence comprising the sequence NISGTY (SEQ ID NO: 7); an HVR-H2 sequence comprising the sequence RX 2 X 3 X 4 X 5 X O X 7 X S XQR (SEQ ID NO: 85), wherein wherein X 5 is any amino acid except threonine and X 6 is any amino acid except asparagine; and an HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO: 9).
• the polynucleotide encodes an HVR-H2 sequence comprising the sequence RX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 R (SEQ ID NO: 84) that has a serine at X 5 , a glutamic acid at X 6 , and/or a tyrosine at X 8 , or any combination thereof.
• any of the HVR-H2 residues shown in Figure 57 to have an F value of greater than 1, 5, or 10 are residues that are preferably maintained as the same residue found in the same position of the HVR-H2 of bHl-44 or bHl-81 (SEQ ID NOS: 8 and 5, respectively).
• any of the HVR-H2 residues shown in Table 14 to have ⁇ G values greater than 1 are residues that are preferably maintained as the same residue found in the same position of the HVR-H2 of bHl-44 or bHl-81 (SEQ ID NOS: 8 and 5, respectively).
• the invention features an isolated polypeptide comprising an HVR- Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1) or an isolated polypeptide comprising an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1); an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO:2); and/or an HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID NO:3).
• the polypeptide comprises the HVR-Ll sequence X 1 I X 3 X 4 X 5 X 6 X 7 XgXgY (SEQ ID NO: 83), wherein X 1 is any amino acid except aspartic acid, X 3 is any amino acid except proline, X 4 is any amino acid except arginine, and X 5 is any amino acid except serine.
• the polypeptide further comprises an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO: 2) and/or an HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID NO: 3).
• the polypeptide comprising the HVR-H2 sequence comprising the sequence RX 2 X 3 X 4 X 5 X 6 X 7 XsXgR (SEQ ID NO: 84) has a serine at X 5 , a glutamic acid at X 6 , and/or a tyrosine at X 8, or any combination thereof.
• any of the HVR-H2 residues shown in Figure 57 to have an F value of greater than 1 , 5, or 10 are residues that are preferably maintained as the same residue found in the same position of the HVR-H2 of bHl-44 or bHl-81 (SEQ ID NOS: 8 and 5, respectively).
• any of the HVR-H2 residues shown in Table 14 to have ⁇ G values greater than 1 are residues that are preferably maintained as the same residue found in the same position of the HVR-H2 of bH 1-44 or bHl-81 (SEQ ID NOS: 8 and 5, respectively).
• the invention also provides a polypeptide comprising one, two, or three of an HVR- Hl sequence comprising the sequence NISGTY (SEQ ID NO: 7), a HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO: 8), and/or an HVR-H3 sequence comprising the sequence WVGVGFY AMD (SEQ ID NO: 9), or any combination thereof.
• the isolated polypeptide may further comprise one, two, or three of an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO:2); and/or an HVR-L3 sequence comprising the sequence HYSSPP (SEQ ID NO:3), or any combination thereof.
• the isolated polypeptide may further comprise an HVR- Hl comprising the sequence NIKDTY (SEQ ID NO:4); an HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO:5); and/or an HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6), or any combination thereof.
• the invention features features an isolated polypeptide comprising an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO:1) and (i) an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO:2) or (ii) an HVR- L3 sequence comprising the sequence HYSSPP (SEQ ID NO:3), or both; and one, two, of three HVR sequences selected from (i) an HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); (ii) an HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and/or (iii) an HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9), or any combination thereof.
• the invention features features an isolated polypeptide comprising an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO:1) and (i) an HVR-L2 sequence comprising the sequence WGSFLY (SEQ ID NO:2) or (ii) an HVR- L3 sequence comprising the sequence HYSSPP (SEQ ID NO:3), or both; and one, two, of three HVR sequences selected from (i) an HVR-Hl comprising the sequence NIKDTY (SEQ ID NO:4); (ii) an HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO:5); and/or (iii) an HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6), or any combination thereof.
• the invention features an isolated polypeptide comprising an HVR-Hl sequence comprising the sequence NISGTY (SEQ ID NO:7); an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO:8); and an HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the invention provides a vector comprising any of the above described polynucleotides of the invention.
• the invention features a host cell comprising any of the vectors of the invention, hi one embodiment, the host cell is prokaryotic. In another embodiment, the host cell is eukaryotic, for example, a mammalian cell.
• the invention features a method of producing any of the antibodies or antibody fragments described above.
• This method comprises culturing a host cell that comprises a vector comprising a polynucleotide encoding the antibody and recovering the antibody.
• the polynucleotide encodes an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO:1) and, optionally, the polynucleotide further encodes an HVR-Hl comprising the sequence NIKDTY (SEQ ID NO:4); an HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO: 6).
• the polynucleotide encodes an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO:1); an HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); an HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and an HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the polynucleotide encodes an HVR-Hl sequence comprising the sequence NISGTY (SEQ ID NO:7), an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO:8), or an HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the polynucleotide encodes a polypeptide comprising an HVR-Hl sequence comprising the sequence NISGTY (SEQ ID NO:7); an HVR-H2 sequence comprising the sequence RIYPSEGYTR (SEQ ID NO:8); and an HVR-H3 sequence comprising the sequence WVGVGFYAMD (SEQ ID NO: 9).
• the host cell is prokaryotic and in another embodiment, the host cell is eukaryotic, such as a mammalian cell.
• the invention features a method of treating a tumor in a subject.
• This method comprises administering to the subject an antibody or antibody fragment described herein, where the administering is for a time and in an amount sufficient to treat or prevent the tumor in the subject.
• the tumor is a colorectal tumor, a breast cancer, a lung cancer, a renal cell carcinoma, a glioma, a glioblastoma, or an ovarian cancer.
• the antibody comprises an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO:1) and specifically binds HER2 and VEGF.
• the antibody further comprises one or two HVR sequences selected from (i) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO:2); and (ii) HVR-L3 comprising the sequence HYSSPP (SEQ ID NO:3).
• the antibody comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NTKDTY (SEQ ID NO:4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6).
• the antibody comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the antibody comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NIKDTY (SEQ ID NO:4); an HVR-H2 sequence comprising RIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence comprising WGGDGFYAMD (SEQ ID NO:6) and specifically binds HER2 and VEGF.
• the antibody comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NISGTY (SEQ ID NO:7); an HVR-H2 sequence comprising RIYPSEGYTR (SEQ ID NO:8); and an HVR-H3 sequence comprising WVGVGFYAMD (SEQ ID NO:9) and specifically binds HER2 and VEGF.
• the method further comprises administering to the subject an additional anti-cancer therapy.
• the additional anti-cancer therapy comprises another antibody, a chemotherapeutic agent, a cytotoxic agent, an anti-angiogenic agent, an immunosuppressive agent, a prodrug, a cytokine, a cytokine antagonist, cytotoxic radiotherapy, a corticosteroid, an anti-emetic, a cancer vaccine, an analgesic, or a growth- inhibitory agent.
• the additional anti-cancer therapy is administered prior to or subsequent to the administration of an antibody. In a further embodiment, the additional anti-cancer therapy is administered concurrently with an antibody.
• the invention features a method of treating an autoimmune disease in a subject.
• This method comprises administering to the subject an antibody or antibody fragment described herein, where the administering is for a time and in an amount sufficient to treat or prevent the autoimmune disease in the subject.
• the antibody comprises an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO:1) and specifically binds HER2 and VEGF.
• the antibody comprises one or two HVR sequences selected from (i) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO:2); and (ii) HVR-L3 comprising the sequence HYSSPP (SEQ ID NO:3).
• the antibody comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6).
• the antibody comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:7)
• the antibody comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NIKDTY (SEQ ID NO:4); an HVR-H2 sequence comprising RIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence comprising WGGDGFYAMD (SEQ ID NO:6) and specifically binds HER2 and VEGF or the antibody comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NISGTY (SEQ ID NO:7); an HVR-H2 sequence
• the invention features a method of treating a non-malignant disease involving abnormal activation of HER2 in a subject.
• This method comprises administering to the subject an antibody or antibody fragment described herein, where the administering is for a time and in an amount sufficient to treat or prevent the non-malignant disease in the subject.
• the antibody comprises an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO:1) and specifically binds HER2 and VEGF.
• the antibody comprises one or two HVR sequences selected from (i) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO:2); and (ii) HVR- L3 comprising the sequence HYSSPP (SEQ ID NO:3).
• the antibody further comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6).
• the antibody comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO:8); and (iii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the antibody comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NIKDTY (SEQ ID NO:4); an HVR-H2 sequence comprising RIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence comprising WGGDGFYAMD (SEQ ID NO:6) and specifically binds HER2 and VEGF or the antibody comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NISGTY (SEQ ID NO:7); an HVR-H2 sequence comprising RI
• the antibodies and antibody fragments described herein in the treatment of a tumor, an autoimmune disease, or a non- malignant disease involving abnormal activation of HER2 in a subject, as well as use in the manufacture of a medicament for the treatment of a tumor, an autoimmune disease, or a non- malignant disease involving abnormal activation of HER2 in a subject.
• the antibody comprises an HVR-Ll sequence comprising the sequence NIAKTISGY (SEQ ID NO: 1) and specifically binds HER2 and VEGF.
• the antibody further comprises one or two HVR sequences selected from (i) HVR-L2 comprising the sequence WGSFLY (SEQ ID NO:2); and (ii) HVR-L3 comprising the sequence HYSSPP (SEQ ID NO:3).
• the antibody comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NIKDTY (SEQ ID NO:4); (ii) HVR-H2 comprising the sequence RIYPTNGYTR (SEQ ID NO:5); and (iii) HVR-H3 comprising the sequence WGGDGFYAMD (SEQ ID NO:6).
• the antibody comprises, one, two, or three HVR sequences selected from (i) HVR-Hl comprising the sequence NISGTY (SEQ ID NO:7); (ii) HVR-H2 comprising the sequence RIYPSEGYTR (SEQ ID NO: 8); and (iii) HVR-H3 comprising the sequence WVGVGFYAMD (SEQ ID NO:9).
• the antibody comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NIKDTY (SEQ ID NO:4); an HVR-H2 sequence comprising RIYPTNGYTR (SEQ ID NO:5); and an HVR-H3 sequence comprising WGGDGFYAMD (SEQ ID NO: 6) and specifically binds HER2 and VEGF or the antibody comprises an HVR-Ll sequence comprising NIAKTISGY (SEQ ID NO:1); an HVR-L2 sequence comprising WGSFLY (SEQ ID NO:2); an HVR-L3 sequence comprising HYSSPP (SEQ ID NO:3); an HVR-Hl sequence comprising NISGTY (SEQ ID NO:7); an HVR-H2 sequence comprising
• the subject is a human.
• kits, compositions, and articles of manufacture comprising the antibodies and antibody fragments described herein.
• Figure 1 shows the designed diversity in various LC libraries.
• Figure 2 shows a summary of four light chain libraries used to alter anti-VEGF antibodies or anti-Her2 antibodies to bind to an additional target.
• the italicized NNK and XYZ refer to codon sets.
• Ys, Ds, Ts and Ss refer to soft randomizations by having tyrosine, aspartic acid, threonine and serine, respectively, occuring 50% of the time and any one of the 20 amino acids occurring the other 50% of the time.
• D/Ds and T/Ts refer to a soft randomization having D or T, respectively, occurring 75% of the time and any one of the 20 amino acids occurring the other 25% of the time.
• Figure 3 shows sequences of HC, LC CDR residues of light chain templates.
• Figure 4 shows the natural and designed diversity of light chain CDRs. At each position, the Herceptin® antibody sequence is shown in parenthesis. An "*" denotes an insertion not present in the Herceptin® antibody.
• Figure 6 is a graph showing binding specificity of the antibodies derived from the LC library. The results for antibodies bHl, bH3, 3-1, bDl, bD2, 4-1, and 4-5 are shown. Bound IgG antibodies were detected spectrophotometrically (optical density at 450 nm, y-axis).
• the proteins included in the assay were (left to right for each antibody) human vascular endothelial growth factor A (hVEGF-A), hVEGF-C, hVEGF-D, hHER2 extracellular domain (ECD), epidermal growth factor receptor extracellular domain (hEGFR), human death receptor 5 (hDR5), bovine serum albumin (BSA), casein, fetal bovine serum (FBS), WIL2 cell lysate, and NR6 cell lysate.
• hVEGF-A human vascular endothelial growth factor A
• ECD epidermal growth factor receptor extracellular domain
• hDR5 human death receptor 5
• BSA bovine serum albumin
• FBS fetal bovine serum
• WIL2 cell lysate fetal bovine serum
• NR6 cell lysate NR6 cell lysate.
• Figure 7 shows sorting conditions and enrichment of Library C and D.
• FIG 8 shows VEGF binders.
• Residues 28, 30, 30a, 31, 92, 93, and 93a were fully diverse.
• Residues 32, 50, 53, 91 and 94 were restricted.
• Residues 29, 33, and 51 were limited ( ⁇ 3).
• Figure 9 shows human VEGF binders, combined plate and solution selection.
• Figures 1OA and 1OB show clones that bind both VEGF and HER2.
• Figure 11 shows clones that only bind VEGF and lost the binding activity with HER2.
• Figure 12 shows clones binding to VEGF.
• Figures 13 A and 13B show clones that block VEGF binding to VEGFRl -D2 or Dl .
• Figures 14A and 14B show VEGF binders and the affinities of VEGF binders from library L1/L2/L3-C,D.
• Figure 15 shows clones that can bind both hVEGF and HER2.
• Figure 16 shows the LC library binders used in scFv'2 formation and displayed on phage.
• Figure 17 shows the expression of various clones in Fab or hlgG form.
• Figure 20 shows competitive ELISAs of clones in hlgG form in the presence of Her2 and VEGF or DR5.
• Figure 21 shows a Biacore Analysis of binding to VEGF or HER2.
• Figure 22 shows binding to HER2-ECD or hVEGF with an IgG or Fab having a light chain obtained from a different binding clone.
• Figures 23A and 23B show an anti-VEGF antibody blocking VEGF interaction with VEGFRl D 1-3 and KDR Dl -7.
• Figure 24 shows antibodies blocking B20-4.1 and VEGF binding.
• Figure 25 shows antibodies blocking Avastin® antibody and VEGF binding.
• Figure 26 shows crystal structures of the bispecific bHl Fab bound to HER2 or VEGF.
• Figure 27 is a graph showing that anti-VEGF antibodies block hVEGF binding to VEGF receptor 2 (VEGFR2).
• Figure 28 shows crystal structures of the bispecific bHl Fab bound to HER2 or VEGF.
• Figure 29 is a series of pie charts showing the individual CDR contributions to the structural paratope for bHl .
• the paratope size for VEGF is 73 ⁇ A 2 and for HER2 is 690A 2 .
• the heavy chain CDRs are indicated in gray and the light chain CDRs in white.
• Figure 30 shows the superposition of the CDR loops of VEGF/HER2-bound bHl or HER2- bound Herceptin® antibody in the same orientation as Figure 28.
• Figure 31 shows crystal structures of the bispecific bHl Fab bound to HER2 or VEGF. CDR- Ll of the two bHl complexes are shown in the same orientation.
• Figure 32 shows the energetically important binding sites of bHl for VEGF and HER2 binding.
• Figure 33 shows codons of bHl that were shotgun scanned.
• Figure 34 shows a library construction
• Figure 35 shows an antibody clone with shotgun scan mutations screened by binding to VEGF.
• Figure 36 shows an antibody clone with shotgun scan mutations screened by binding to HER2.
• Figures 37A-37D show alanine scanning results.
• Figures 37A and 37B show the results of an alanine scan of bHl for ( Figure 37A) VEGF binding or ( Figure 37B) HER2 binding and the results of a homolog scan of bHl for ( Figure 37C) VEGF binding or ( Figure 37D) HER2 binding.
• Figure 38 shows alanine scanning results of bHl or the Herceptin® antibody mutants.
• Figures 39A1-39A3 and 39B1 -39B3 show shotgun alanine- and homolog scanning of bHl Fab for binding to VEGF and HER2.
• Figures 40A-40B show the energetically important binding sites of bHl for VEGF and HER2 binding.
• Figure 41 shows bHl VEGF-affinity matured clone sequences and binding affinity for VEGF or HER2.
• Figure 42 shows the inhibition of VEGF induced HUVEC cell proliferation with anti-VEGF antibodies.
• Figure 43 shows binding of bispecific antibodies to HER2 expressed on NR6 cells.
• Figure 44 shows the results of competitive binding experiments for bHl to VEGF or HER2.
• Figure 45 shows that bHl and affinity improved variants bHl -44 and bHl -81 IgG inhibit HER2 and VEGF-mediated cell proliferation in vitro.
• Figures 48 A and 48B show that VEGF and HER2 compete for binding to bHl -44 bispecific IgG in solution.
• Figures 49A and 49B show that the bispecific antibodies bHl and bHl -44 bind to HER2 expressing mouse fibroblast cells (NR6; Figure 49B), but not to HER2 negative NR6 cells ( Figure 49A).
• Figure 51 shows tumor inhibition of bHl -44 in Colo205 and BT474M1 xenografts in immuno-compromised mice.
• Figures 52A, 52B, and 53 depict exemplary acceptor human consensus framework sequences for use in practicing the instant invention with sequence identifiers as follows:
• VH consensus frameworks (FIG. 52A and 52B) human VH subgroup I consensus framework regions FRl, FR2, FR3, and FR4 minus Kabat CDRs (IA: SEQ ID NOS: 42-45, respectively) human VH subgroup I consensus framework regions FRl, FR2, FR3, and FR4 minus extended hypervariable regions (IB: SEQ ID NOS: 46, 47, 44, and 45, respectively; IC: SEQ ID NOS: 46-48 and 45, respectively; ID: SEQ ID NOS: 42, 47, 49, and 45, respectively) human VH subgroup II consensus framework regions FRl, FR2, FR3, and FR4 minus Kabat CDRs (IIA: SEQ ID NOS: 50-52 and 45, respectively) human VH subgroup II consensus framework regions FRl , FR2, FR3, and FR4 minus extended hypervariable regions (IIB: SEQ ID NOS: 53, 54, 52, and 45, respectively;
• CDRs (IHA: SEQ ID NOS: 57-59 and 45, respectively) human VH subgroup III consensus framework regions FRl, FR2, FR3, and FR4 minus extended hypervariable regions (IHB: SEQ ID NOS: 60, 61, 59, and 45, respectively; IIIC:
• VL consensus frameworks (FIG. 53) human VL kappa subgroup I consensus framework regions FRl , FR2, FR3, and FR4 (kvl : SEQ ID NOS: 70-73, respectively) human VL kappa subgroup II consensus framework regions FRl, FR2, FR3, and FR4 (kv2: SEQ ID NOS: 74-76 and 73, respectively) human VL kappa subgroup III consensus framework regions FRl, FR2, and FR3 (kv3: SEQ ID NOS: 77-79 and 73, respectively) human VL kappa subgroup IV consensus framework regions FRl, FR2, and FR3 (kv4: SEQ ID NOS: 80-82 and 73, respectively)
• Figure 54 shows the residues that make structural contacts or an energetic interaction with HER2, VEGF, or both.
• the residues that make structural contacts (>25% buried) or an energetic interaction ( ⁇ G > 10% total binding energy) with HER2 (light grey), VEGF (grey), or both (shared, black) are mapped on the surface of HER2-bound bHl.
• Figure 55 shows the bHl/VEGF and bHl/HER2 binding interfaces.
• a close-up of the bHl/VEGF (A) and the bHl/HER2 (B) binding interface illustrates the structural differences between VEGF and HER2 in the regions of antibody binding.
• Surface representations of VEGF (C) and HER2-ECD (D) are shown in the same orientation relative to bHl Fab. The residues in contact with bHl Fab (closer than 4.5 A) are highlighted. There is no apparent similarity between the two epitopes for bHl in terms of chemical composition or topology.
• FIG 56 shows that bHl and bHl-44 antibodies block human VEGF binding to VEGFRl.
• Biotinylated human VEGFi 65 was incubated with increasing concentrations of IgG (x-axis), then captured on immobilized human VEGFRl -Fc, and detected with horseradish peroxidase- conjugated streptavidin with added substrate (normalized % OD 450 , y-axis).
• Figure 57 shows alanine scanning results of bHl and bHl-44 mutants.
• Alanine scanning mutagenesis identified the functionally important residues for VEGF and/or HER2 binding.
• F values represent the relative contribution of each scanned residue to antigen binding.
• F values were determined for bHl-44 binding to VEGF and HER2 (black bars), and compared to the F values of bHl (white bars).
• the amino acids in parenthesis denote bHl-44 residues that differ from bHl . This graph was adapted from Figure 56.
• Figure 58 shows the binding of bHl-44 I29A Y32A bHl-44 and R50A R58A bHl-44 antibodies to VEGF and HER2.
• the ELISA binding assays show the ability of bHl-44 IgG and the two double mutants to bind to biotinylated VEGFi 09 (left) or HER2-ECD (right), and compete with the immobilized anti-VEGF antibody or Herceptin, respectively.
• the I29A/Y32A LC mutant has lost binding of VEGF, while maintaining similar affinity for HER2 as bHl-44.
• the R50A/R58A HC mutant has lost affinity for HER2, but retains VEGF binding.
• Figures 59A-D Solutions OfVEGF 109 of HER2-ECD at concentrations ranging from 10-20 ⁇ M were titrated by 15 injections of bHl or bHl-44 Fab at concentrations from 100 to 200 ⁇ M.
• Figures 59E-F Solutions OfVEGFi 09 or HER2-ECD at concentrations of 10 to 20 ⁇ M were titrated by 20 injections of bHl-44 LC-I29A+Y32A Fab or bHl-44 HC-R50A+R58A Fab at concentrations of 150 and 250 ⁇ M. Titrations number 1 and 13 in ( Figure 59E) were excluded from the analysis due to instrument noise.
• FIG 60 shows the thermodynamic profiles of the bHl variants and the Herceptin® antibody.
• Each dual specific variant (bHl, bHl -81, and bHl-44) has thermodynamic profiles characterized by favorable enthalpy and entropy for both VEGF and HER2 binding.
• the thermodynamic profiles of the bHl-44/HER2 interaction are distinct from Herceptin/HER2.
• Figure 62 shows the estimated heat capacity changes associated with bHl-44 Fab binding with VEGF or HER2.
• the ⁇ Cp for Herceptin®/HER2 was previously determined by Kelley et al. (Biochemistry, 1992).
• the variant bHl- 44-Y32A displayed significantly weakened binding to VEGF compared to the wild type bHl- 44.
• Figure 65 shows the expression of the Herceptin® mutant Fabs (R50A, R58A, and R50A/R58A).
• the present invention provides methods of making multispecific antibodies and antibody fragments, as well as antibodies identified using these methods and their use.
• the methods of the invention involve diversifying the light chain variable domain or the heavy chain variable domain of an antibody to generate variants that can be stably expressed in a library. Diversified antibodies that are capable of specifically binding two epitopes are then selected from this library and further characterized.
• Exemplary antibodies identified using the methods of the invention include antibodies that bind both HER2 (human epidermal growth factor receptor 2) and VEGF (vascular endothelial growth factor).
• the data described herein show that mutations in the light chain complementarity determining regions (CDRs) of a HER2 antibody confer dual binding capabilities for unrelated protein antigens as well as HER2.
• CDRs light chain complementarity determining regions
• One bi-specific high affinity HER2/VEGF antibody is extensively characterized.
• the crystal structures of this bi-specific Fab in complex with HER2 and VEGF are shown and the energetic contribution of the Fab residues by mutagenesis is evaluated.
• the binding sites for the two antigens overlap extensively; most of the CDR residues that contact HER2 also engage VEGF. Energetically, however, the residues of the heavy chain dominate the HER2 specificity while the light chain dominates VEGF specificity.
• the HER2/VEGF bi-specific antibody inhibits both HER2 and VEGF-mediated cell proliferation in vitro and in vivo.
• These results demonstrate that altering the sequence of the light chain variable domain of an antibody can generate antibodies with dual specificity and function.
• bHl-44 and bHl-81 have the potential to target two mechanisms of tumor progression: tumor cell proliferation mediated by HER2 and tumor angiogenesis mediated by VEGF.
• Co-targeting two antigens with a single antibody is an alternative to combination therapy.
• multispecific antibody is used in the broadest sense and specifically covers an antibody comprising a heavy chain variable domain (V H ) and a light chain variable domain (V L ), where the V H V L unit has polyepitopic specificity (i.e., is capable of binding to two different epitopes on one biological molecule or each epitope on a different biological molecule).
• Such multispecific antibodies include, but are not limited to, full length antibodies, antibodies having two or more V L and V H domains, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, antibody fragments that have been linked covalently or non-covalently.
• Polyepitopic specificity refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).
• “Monospecific” refers to the ability to bind only one epitope.
• the multispecific antibody is an IgGl form binds to each epitope with an affinity of 5 /M to 0.001 pM, 3 jiM to 0.00IpM, 1 /M to 0.00IpM, 0.5 /M to 0.001 pM or 0.1 /M to 0.001 pM.
• Each H and L chain also has regularly spaced intrachain disulfide bridges.
• Each H chain has, at the N-terminus, a variable domain (VJJ) followed by three constant domains (C JJ) for each of the ⁇ and ⁇ chains and four CJJ domains for ⁇ and ⁇ isotypes.
• Each L chain has, at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VJJ and the CL is aligned with the first constant domain of the heavy chain (C jj l).
• immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
• variable refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies.
• the V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
• variability is not evenly distributed across the 110-amino acid span of the variable domains.
• the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long.
• FRs framework regions
• hypervariable regions that are each 9-12 amino acids long.
• the variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
• the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
• the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
• hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
• the hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the V L , and around about residues 26-35 (Hl), 50-65 (H2) and 95-102 (H3) in the V H (in one embodiment, Hl is around about residues 31-35); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
• residues from a "hypervariable loop” e.g., residues 26-32 (Ll), 50-52 (L2), and 91-96 (L3) in the V L , and 26-32 (Hl), 53-55 (H2), and 96-101 (H3) in the V H ; Chothia and Lesk, J.
• FR Framework regions
• Each variable domain typically has four FRs identified as FRl , FR2, FR3 and FR4. If the CDRs are defined according to Kabat, the light chain FR residues are positioned at about residues 1-23 (LCFRl), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are positioned about at residues 1-30 (HCFRl), 36-49 (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 (LCFRl), 33-49 (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 (HCFRI), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain residues.
• the FR residues will be adjusted accordingly.
• CDRHl includes amino acids H26-H35
• the heavy chain FRl residues are at positions 1-25 and the FR2 residues are at positions 36-49.
• a "human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
• the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
• the subgroup of sequences is a subgroup as in Kabat.
• the subgroup is subgroup kappa I as in Kabat.
• the subgroup is subgroup III as in Kabat.
• monoclonal antibody refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are substantially similar and bind the same epitope(s), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts.
• Such monoclonal antibody typically includes an antibody comprising a variable region that binds a target, wherein the antibody was obtained by a process that includes the selection of the antibody from a plurality of antibodies.
• the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones.
• the selected antibody can be further altered, for example, to improve affinity for the target, to humanize the antibody, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered variable region sequence is also a monoclonal antibody of this invention, m addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
• the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including the hybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 , (Elsevier,
• An "intact” antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, C jj l, C H 2, and C H 3.
• the constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.
• the intact antibody has one or more effector functions.
• linear antibodies generally 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 I ) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
• Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
• the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V JJ), and the first constant domain of one heavy chain (C jj l).
• the Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
• the effector functions of antibodies are determined by sequences in the Fc region; this region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
• Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VJJ and VL antibody domains connected into a single polypeptide chain.
• the sFv polypeptide further comprises a polypeptide linker between the VJJ and VL domains which enables the sFv to form the desired structure for antigen binding.
• V L domains of the two antibodies are present on different polypeptide chains.
• Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
• Humanized forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
• donor antibody such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
• framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
• the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• 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 "non-random codon set”, as used herein, thus refers to a codon set that encodes select amino acids that fulfill partially, preferably completely, the criteria for amino acid selection as described herein.
• oligonucleotides with selected nucleotide "degeneracy" at certain positions is well known in that art, for example the TRIM approach (Knappek et al., J. MoI. Biol. 296:57-86, 1999); Garrard and Henner, Gene 128:103, 1993).
• Such sets of oligonucleotides having certain codon sets can be synthesized using commercial nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, CA), 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 do not necessarily, include restriction enzyme sites useful for, for example, cloning purposes.
• an antibody of this invention "which binds" an antigen of interest is one that binds the antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting a protein or a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins.
• the extent of binding of the antibody to a "non-target" protein will be less than about 10% of the binding of the antibody to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) or ELISA.
• the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction (e.g., for bHl-44 or bHl- 81 , a non-specific interaction is binding to bovine serum albumin, casein, fetal bovine serum, or neuravidin).
• Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target.
• binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
• the term “specific binding” or “specifically binds to” or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or greater.
• the term "specific binding" refers
• the Kd is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or stronger.
• Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.
• the "Kd” or “Kd value” according to this invention is measured by using surface plasmon resonance assays using a BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ⁇ 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions.
• CM5 carboxymethylated dextran biosensor chips
• EDC N-ethyl-N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride
• NHS N-hydroxysuccinimide
• Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 ⁇ g/ml ( ⁇ 0.2 ⁇ M) before injection at a flow rate of 5 ⁇ l/minute to achieve approximately 10 response units (RU) of coupled protein.
• IM ethanolamine is injected to block unreacted groups.
• two-fold serial dilutions of Fab e.g., 0.78 nM to 500 nM
• PBST Tween 20
• Association rates (Ic 0n ) and dissociation rates (Ic 0Jf ) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneous fitting the association and dissociation sensorgram.
• the equilibrium dissociation constant (Kd) is calculated as the ratio Wk 0n . See, e.g., Chen, Y., et al., (1999) J. MoI. Biol. 293:865-881.
• Antigen is diluted with 10 niM sodium acetate, pH 4.8, into 5 ⁇ g/ml ( ⁇ 0.2 uM) before injection at a flow rate of 5 ⁇ l/minute to achieve approximately 10 response units (RU) of coupled protein.
• IM ethanolamine is injected to block unreacted groups.
• two-fold serial dilutions of Fab e.g., 0.78 nM to 500 nM
• PBST Tween 20
• Association rates (Ic 0n ) and dissociation rates (k off ) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneous fitting the association and dissociation sensorgram.
• the equilibrium dissociation constant (Kd) is calculated as the ratio Wk 0n . See, e.g., Chen, Y., et al., (1999) J. MoI. Biol. 293:865-881.
• Bio molecule refers to a nucleic acid, a protein, a carbohydrate, a lipid, and combinations thereof. In one embodiment, the biologic molecule exists in nature.
• Nucleic acid is "operably linked" when it is placed into 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 promoter 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, contiguous and in reading phase. 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 adaptors or linkers are used in accordance with conventional practice.
• Percent (%) amino acid sequence identity with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
• amino acid sequences described herein are contiguous amino acid sequences unless otherwise specified.
• Structurally unsimilar biological molecules refers to biological molecules that are not in the same class (protein, nucleic acid, lipid, carbohydrates, etc.) or, for example, when referring to proteins, having less than 60% amino acid identity, less than 50% amino acid identity, less than 40% amino acid identity, less than 30% amino acid identity, less than 20% amino acid identity or less than 10% amino acid identity compared to each other.
• Stringency conditions can be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 5O 0 C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/5 OmM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) overnight hybridization in a solution that employs 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),
• Modely stringent conditions can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength, and %SDS) less stringent that those described above.
• washing solution and hybridization conditions e.g., temperature, ionic strength, and %SDS
• moderately stringent conditions is overnight incubation at 37 0 C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-5O 0 C.
• a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-5O 0 C.
• the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as
• Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: CIq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
• ADCC activity of a molecule of interest is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991).
• an in vitro ADCC assay such as that described in US Patent No. 5,500,362 or 5,821,337 can be performed.
• Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
• PBMC peripheral blood mononuclear cells
• NK Natural Killer
• ADCC activity of the molecule of interest can be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. (Proc. Natl. Acad. Sci. USA) 95:652-656 (1998).
• Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
• the preferred FcR is a native sequence human FcR.
• a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
• Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
• Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
• Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
• FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).
• Human effector cells are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc ⁇ RIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; with PBMCs and NK cells being preferred.
• PBMC peripheral blood mononuclear cells
• NK natural killer cells
• monocytes cytotoxic T cells
• neutrophils neutrophils
• the effector cells can be isolated from a native source, e.g., from blood.
• “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (CIq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen.
• CIq first component of the complement system
• a CDC assay e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed.
• the term "therapeutically effective amount” refers to an amount of an antibody or antibody fragment to treat a disease or disorder in a subject.
• the therapeutically effective amount of the antibody or antibody fragment may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
• the antibody or antibody fragment may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
• efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.
• Reduce or inhibit is meant the ability to cause an overall decrease preferably of 20% or greater, more preferably of 50% or greater, and most preferably of 75%, 85%, 90%, 95%, or greater.
• Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, the size of the primary tumor, or the size or number of the blood vessels in angiogenic disorders.
• cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Included in this definition are benign and malignant cancers. 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, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (e.g., renal cell carcinoma), liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, and various types of head and neck cancer.
• gastrointestinal cancer pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer
• liver cancer, bladder cancer hepatoma, breast cancer, colon cancer
• colorectal cancer endometrial or
• stage cancer is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, 1, or II cancer.
• precancerous refers to a condition or a growth that typically precedes or develops into a cancer.
• psoriasis see definition below; endometriosis; scleroderma; restenosis; polyps such as colon polyps, nasal polyps or gastrointestinal polyps; fibroadenoma; respiratory disease (e.g., chronic bronchitis, asthma including acute asthma and allergic asthma, cystic fibrosis, bronchiectasis, allergic or other rhinitis or sinusitis, ⁇ l-anti-trypsin deficiency, coughs, pulmonary emphysema, pulmonary fibrosis or hyper-reactive airways, chronic obstructive pulmonary disease, and chronic obstructive lung disorder); cholecystitis; neurofibromatosis; polycystic kidney disease; inflammatory diseases; skin disorders including psoriasis and dermatitis; vascular disease; conditions involving abnormal proliferation of vascular epithelial cells; gastrointestinal ulcers; Menetrier's disease, secreting adenomas or
• microbial infections including microbial pathogens selected from adenovirus, hantaviruses, Borrelia burgdorferi, Yersinia spp. and Bordetella pertussis; thrombus caused by platelet aggregation; reproductive conditions such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or menometrorrhagia; synovitis; atheroma; acute and chronic nephropathies (including proliferative glomerulonephritis and diabetes-induced renal disease); eczema; hypertrophic scar formation; endotoxic shock and fungal infection; familial adenomatosis polyposis; neurodedenerative diseases (e.g.
• Alzheimer's disease AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration); myelodysplastic syndromes; aplastic anemia; ischemic injury; fibrosis of the lung, kidney or liver; T-cell mediated hypersensitivity disease; infantile hypertrophic pyloric stenosis; urinary obstructive syndrome; psoriatic arthritis; and Hasimoto's thyroiditis.
• An "autoimmune disease” herein is a disease or disorder arising from and directed against an individual's own tissues or a co-segregate or manifestation thereof or resulting condition therefrom.
• autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis,
• an "anti-angiogenesis agent” or “angiogenesis inhibitor” refers to a small molecular weight substance, a polynucleotide, a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly.
• an anti-angiogenesis agent is an antibody or other antagonist to an angiogenic agent as defined above, e.g., antibodies to VEGF (e.g., bevacizumab (AVASTIN®), bHl , bHl -44, bHl-81), antibodies to VEGF receptors, small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinib malate), AMG706).
• Anti- angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc.
• 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.
• cytotoxic agent refers to a substance that inhibits or prevents the function of a cell and/or causes destruction of a cell.
• the term is intended to include radioactive isotopes (e.g., At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , Ra 223 , 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 fragment
• 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 mo ⁇ holino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, i
• 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, LYl 17018, 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 either in vitro or in vivo.
• the growth inhibitory agent may be one which significantly reduces the percentage of 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 Gl arrest and M-phase arrest.
• Classical M-phase blockers include the vincas (e.g., vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
• the agents that arrest Gl also spill over into S-phase arrest, for example, 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.
• Anti-cancer therapy refers to a treatment that reduces or inhibits cancer in a subject.
• prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Harbor (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985).
• Prodrugs include, but are not limited to, phosphate- containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, beta- lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5- fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
• cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
• cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
• cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone (HGH), N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); epidermal growth factor (EGF); hepatic growth factor; fibroblast growth factor (FGF); prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (T)
• cytokine antagonist is meant a molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of of at least one cytokine.
• the cytokine antagonists may inhibit cytokine activity by inhibiting cytokine expression and/or secretion, or by binding to a cytokine or to a cytokine receptor.
• Cytokine antagonists include antibodies, synthetic or native-sequence peptides, immunoadhesins, and small-molecule antagonists that bind to a cytokine or cytokine receptor.
• the cytokine antagonist is optionally conjugated with or fused to a cytotoxic agent.
• Exemplary TNF antagonists are etanercept (ENBREL®), infliximab (REMIC ADE®), and adalimumab (HUMIRATM).
• immunosuppressive agent refers to substances that act to suppress or mask the immune system of the subject being treated. This includes substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens.
• immunosuppressive agents include 2-amino-6-aryl-5- substituted pyrimidines (see U.S. Pat. No. 4,665,077); mycophenolate mofetil such as CELLCEPT®; azathioprine (IMURAN®, AZASAN®/6-mercaptopurine; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No.
• anti-idiotypic antibodies for MHC antigens and MHC fragments include cyclosporin A; steroids such as corticosteroids and glucocorticosteroids, e.g., prednisone, prednisolone such as PEDIAPRED® (prednisolone sodium phosphate) or ORAPRED® (prednisolone sodium phosphate oral solution), methylprednisolone, and dexamethasone; methotrexate (oral or subcutaneous) (RHEUMATREX®, TREXALLTM); hydroxycloroquine/chloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antagonists including anti- interferon- ⁇ , - ⁇ , or - ⁇ antibodies, anti-tumor necrosis factor- ⁇ antibodies (infliximab or adalimumab), anti-TNF ⁇ immunoadhesin (ENBREL®,
• T cell receptor antibodies such as T10B9; cyclophosphamide (CYTOXAN®); dapsone; penicillamine (CUPRIMINE®); plasma exchange; or intravenous immunoglobulin (IVIG).
• T10B9 T10B9
• CYTOXAN® cyclophosphamide
• dapsone dapsone
• penicillamine CPRIMINE®
• plasma exchange or intravenous immunoglobulin (IVIG).
• IVIG intravenous immunoglobulin
• an “analgesic” refers to a drug that acts to inhibit or suppress pain in a subject.
• exemplary analgesics include non-steroidal anti-inflammatory drugs (NSAIDs) including ibuprofen (MOTRIN®), naproxen (NAPROSYN®), acetylsalicylic acid, indomethacin, sulindac, and tolmetin, including salts and derivatives thereof, as well as various other medications used to reduce the stabbing pains that may occur, including anticonvulsants (gabapentin, phenyloin, carbamazepine) or tricyclic antidepressants.
• NSAIDs non-steroidal anti-inflammatory drugs
• MOTRIN® ibuprofen
• NAPROSYN® naproxen
• acetylsalicylic acid indomethacin
• sulindac sulindac
• tolmetin including salts and derivatives thereof, including salts and derivatives thereof, as well as various
• acetaminophen aspirin, amitriptyline (ELAVIL®), carbamazepine (TEGRETOL®), phenyltoin (DILANTIN®), gabapentin (NEURONTIN®), (E)-N-Vanillyl-8-methyl-6- noneamid (CAPSAICIN®), or a nerve blocker.
• Corticosteroid refers to any one of several synthetic or naturally occurring substances with the general chemical structure of steroids that mimic or augment the effects of the naturally occurring corticosteroids.
• synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone triamcinolone, and betamethasone.
• a "cancer vaccine,” as used herein is a composition that stimulates an immune response in a subject against a cancer.
• Cancer vaccines typically consist of a source of cancer-associated material or cells (antigen) that may be autologous (from self) or allogenic (from others) to the subject, along with other components (e.g., adjuvants) to further stimulate and boost the immune response against the antigen.
• Cancer vaccines desirably result in stimulating the immune system of the subject to produce antibodies to one or several specific antigens, and/or to produce killer T cells to attack cancer cells that have those antigens.
• Radioactive radiotherapy refers to radiation therapy that inhibits or prevents the function of cells and/or causes destruction of cells. Radiation therapy may include, for example, external beam irradiation or therapy with a radioactive labeled agent, such as an antibody. The term is intended to include use of radioactive isotopes (e.g., At 211 , I 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , Ra 223 , P 32 , and radioactive isotopes of Lu).
• radioactive isotopes e.g., At 211 , I 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , Ra 223 , P 32 , and radioactive isotopes of Lu).
• Anti-emetic is a compound that reduces or prevents nausea in a subject.
• Antiemetic compounds include, for example, neurokinin-1 receptor antagonists, 5HT3 receptor antagonists (such as ondansetron, granisetron, tropisetron, and zatisetron), GABAB receptor agonists, such as baclofen, a corticosteroid such as dexamethasone, KENALOG®, ARISTOCORT®, or NASALIDE®, an antidopaminergic, phenothiazines (for example prochlorperazine, fluphenazine, thioridazine and mesoridazine), dronabinol, metroclopramide, domperidone, haloperidol, cyclizine, lorazepam, prochlo ⁇ erazine, and levoniepromazine.
• neurokinin-1 receptor antagonists such as ondansetron, granisetron, tropis
• a "subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs and horses), primates, mice, and rats.
• 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.
• constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.
• 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. Patent 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.-l 1 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 promo ter-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.
• An 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.
• a large number of promoters recognized by a variety of potential host cells are well known.
• 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. In some embodiments, 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 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, PeIB, OmpA, and MBP.
• STII heat-stable enterotoxin II
• LamB, PhoE, PeIB, OmpA, and MBP the signal sequences used in both cistrons of the expression system 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
• 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.
• strain W3110 Bactetidas, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis
• 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, p AC YC 177, or pKN410 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. ii. Antibody Production
• 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. Examples of 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.
• a selection agent chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector.
• 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 0 C to about 39°C, more preferably from about 25°C to about 37°C, even more preferably at about 3O 0 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, 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.
• 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., (1999) J. Biol. Chem.
• 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, JoIy et al., (1998), Proc. Natl. Acad. Sci. USA 95:2773-2777; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent 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.
• Standard protein purification methods known in the art can be employed.
• the following procedures are exemplary of suitable purification procedures: fractionation on immunoaff ⁇ nity 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 full length antibody products of the invention.
• Protein A is a 4IkD cell wall protein from Staphylococcus aureus which binds with a high affinity to the Fc region of antibodies.
• 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.
• 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-specif ⁇ cally 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.
• 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.
• 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).
• 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. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978.
• the Rous Sarcoma Virus long terminal repeat can be used as the promoter.
• 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 CVl 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.
• mice Sertoli cells TM4, Mather, Biol. Reprod. 23:243- 251 (1980)
• monkey kidney cells CVl 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 FlO (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, hi addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem.102:255 (1980), U.S. Pat. Nos.
• 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 ⁇ l, ⁇ 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 CH3 domain
• the Bakerbond ABXTMresin J. T. Baker, Phillipsburg, NJ is useful for purification.
• 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 invention also provides immunoconjugates (interchangeably termed "antibody- drug conjugates” or "ADC”), comprising any of the anti-Notchl NRR antibodies described herein 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).
• 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.
• 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 IgGl 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.
• MYLOT ARGTM (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; US Patent Nos. 4,970,198;
• Cantuzumab mertansine (Immunogen, Inc.), an antibody drug conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the maytansinoid drug moiety, DMl, is advancing into Phase II trials for the treatment of cancers that express CanAg, such as colon, pancreatic, gastric, and others.
• MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.), an antibody drug conjugate composed of the anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DMl, 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 cAClO (specific to CD30 on hematological malignancies) (Doronina et al., (2003) Nature Biotechnology 21(7):778-784) and are under therapeutic development.
• 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.
• 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 ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
• Carbon- 14-labeled l-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, dolastatins, aurostatins, a trichothecene, and CC 1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein. i. Maytansine and maytansinoids
• the immunoconjugate comprises an antibody (full length or fragments) of the invention 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. Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Patent Nos.
• Maytansinoid drug moieties are attractive drug moieties in antibody drug conjugates because they are: (i) relatively accessible to prepare by fermentation or chemical modification, derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation through the non-disulfide linkers to antibodies, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.
• Immunoconjugates containing maytansinoids, methods of making same, and their therapeutic use are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent EP 0 425 235 Bl, the disclosures of which are hereby expressly incorporated by reference. Liu et al., Proc.
• 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. See, e.g., U.S. Patent No. 5,208,020 (the disclosure of which is hereby expressly incorporated by reference). 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. Patent No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove.
• 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.
• Antibody-maytansinoid conjugates comprising the linker component SMCC may be prepared as disclosed in U.S. Patent Application No. 10/960,602, filed Oct. 8, 2004.
• 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. Additional linking groups are described and exemplified herein.
• 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 (SMCC), 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
• 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.
• the immunoconjugate comprises an antibody of the invention conjugated to dolastatins or dolostatin peptidic analogs and derivatives, the auristatins (U.S. Patent Nos. 5,635,483 and 5,780,588).
• Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents Chemother. 42:2961-2965).
• the dolastatin or auristatin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172).
• peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments.
• Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. L ⁇ bke, "The Peptides,” volume 1, pp. 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
• the auristatin/dolastatin drug moieties may be prepared according to the methods of: U.S. Patent Nos. 5,635,483 and 5,780,588; Pettit et al., (1989) J. Am. Chem. Soc.
• the immunoconjugate comprises an antibody of the invention 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. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all to American Cyanamid Company).
• Structural analogues of calicheamicin which may be used include, but are not limited to, ⁇ /, ⁇ 2 ', ⁇ 3 r , N-acetyl- ⁇ /, PSAG and ⁇ (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. iv. Other cytotoxic agents
• the antibody may comprise a highly radioactive atom.
• radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At 211 , 1 131 , 1 125 , 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" 111 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 (SMCC), 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 l
• a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987).
• Carbon- 14-labeled l-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. Patent 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, IL., U.S.A). See pages 467-498, 2003-2004 Applications Handbook and Catalog. v. Preparation of antibody drug conjugates
• 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. Additional methods for preparing ADC are described herein.
• the linker may be composed of one or more linker components.
• exemplary linker components include 6-maleimidocaproyl ("MC”), maleimidopropanoyl ("MP”), valine- citrulline (“val-cit”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl 4-(N- maleimidomethyl) cyclohexane-1 carboxylate (“SMCC), and N-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”).
• MC 6-maleimidocaproyl
• MP maleimidopropanoyl
• val-cit valine- citrulline
• alanine-phenylalanine ala-phe
• PAB p-amin
• the linker may comprise amino acid residues.
• exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
• Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine- phenylalanine (af or ala-phe).
• Exemplary tripeptides include: glycine-valine-citrulline (gly- val-cit) and glycine-glycine-glycine (gly-gly-gly)- Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
• 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.
• Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).
• Antibody drug conjugates of the invention may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents 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. Patent No. 5,362,852).
• Such aldehyde can be reacted with a drug moiety or linker nucleophile.
• 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 individual, 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
• sustained-release preparations may be prepared.
• suitable examples of sustained- release preparations include semi-permeable 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.
• sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No.
• copolymers of L-glutamic acid and ⁇ 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.
• cancer embraces a collection of proliferative disorders, including but not limited to pre-cancerous growths, benign tumors, and malignant tumors.
• Benign tumors remain localized at the site of origin and do not have the capacity to infiltrate, invade, or metastasize to distant sites.
• Malignant tumors will invade and damage other tissues around them. They can also gain the ability to break off from where they started and spread to other parts of the body (metastasize), usually through the bloodstream or through the lymphatic system where the lymph nodes are located.
• Primary tumors are classified by the type of tissue from which they arise; metastatic tumors are classified by the tissue type from which the cancer cells are derived. Over time, the cells of a malignant tumor become more abnormal and appear less like normal cells.
• cancer cells This change in the appearance of cancer cells is called the tumor grade and cancer cells are described as being well-differentiated, moderately- differentiated, poorly-differentiated, or undifferentiated.
• Well-differentiated cells are quite normal appearing and resemble the normal cells from which they originated.
• Undifferentiated cells are cells that have become so abnormal that it is no longer possible to determine the origin of the cells.
• the tumor can be a solid tumor or a non-solid or soft tissue tumor.
• soft tissue tumors include leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, adult acute lymphoblastic leukemia, acute myelogenous leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, polymphocytic leukemia, or hairy cell leukemia), or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease).
• a solid tumor includes any cancer of body tissues other than blood, bone marrow, or the lymphatic system.
• Solid tumors can be further separated into those of epithelial cell origin and those of non-epithelial cell origin.
• epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, labium, nasopharynx, skin, uterus, male genital organ, urinary organs, bladder, and skin.
• Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors.
• angiogenesis is implicated in the pathogenesis of a variety of disorders. These include solid tumors and metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular diseases such as proliferative retinopathies, e.g., diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis.
• proliferative retinopathies e.g., diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma
• AMD age-related macular degeneration
• neovascular glaucoma immune rejection of transplanted corneal tissue and other tissues
• rheumatoid arthritis rheumatoid arthritis
• psoriasis psorias
• Excessive, inappropriate or uncontrolled angiogenesis occurs when there is new blood vessel growth that contributes to the worsening of the diseased state or causes a diseased state, such as in cancer, especially vascularized solid tumors and metastatic tumors (including colon, lung cancer (especially small-cell lung cancer), or prostate cancer), diseases caused by ocular neovascularization, especially diabetic blindness, retinopathies, primarily diabetic retinopathy or age-related macular degeneration (AMD), diabetic macular edema, cerebral edema (e.g., associated with acute stroke/closed head injury/trauma), synovial inflammation, pannus formation in rheumatoid arthritis, myositis ossificans, hypertropic bone formation, refractory ascites, polycystic ovarian disease, 3rd spacing of fluid diseases (pancreatitis, compartment syndrome, burns, bowel disease), uterine fibroids, premature labor, neovascularization of the angle (rub
• compositions of this invention have, or are at risk for developing, abnormal proliferation of fibrovascular tissue, acne rosacea, acquired immune deficiency syndrome, artery occlusion, atopic keratitis, bacterial ulcers, Bechets disease, blood borne tumors, carotid obstructive disease, choroidal neovascularization, chronic inflammation, chronic retinal detachment, chronic uveitis, chronic vitritis, contact lens overwear, corneal graft rejection, corneal neovascularization, corneal graft neovascularization, Crohn's disease, Eales disease, epidemic keratoconjunctivitis, fungal ulcers, Herpes simplex infections, Herpes zoster infections, hyperviscosity syndromes,
• Anti-angiogenesis therapies are useful in the general treatment of graft rejection, lung inflammation, primary pulmonary hypertension, nephrotic syndrome, preeclampsia, and pleural effusion, diseases and disorders characterized by undesirable vascular permeability, e.g., edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardial effusion (such as associated with pericarditis), permeability associated with cardiovascular diseases such as the condition following myocardial infarctions and strokes and the like, and sepsis.
• undesirable vascular permeability e.g., edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardial effusion (such as associated with pericarditis), permeability associated with cardiovascular diseases such as the condition following myocardial infarctions and strokes and the like, and sepsis.
• the antibody or antibody fragment need not be, but is optionally, formulated with one or more agents currently used to prevent or treat cancer or an autoimmune disorder or a risk of developing cancer or an autoimmune disorder.
• the effective amount of such other agents depends on the amount of antibody or antibody fragment 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.
• alleviation or treatment of a cancer involves the lessening of one or more symptoms or medical problems associated with the cancer.
• the therapeutically effective amount of the drug can accomplish one or a combination of the following: reduce (by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) the number of cancer cells; reduce or inhibit the tumor size or tumor burden; inhibit (i.e., to decrease to some extent and/or stop) cancer cell infiltration into peripheral organs; reduce hormonal secretion in the case of adenomas; reduce vessel density; inhibit tumor metastasis; reduce or inhibit tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
• the antibody or antibody fragment is used to prevent the occurrence or reoccurrence of cancer or an autoimmune disorder in the subject.
• the present invention can be used for increasing the duration of survival of a human patient susceptible to or diagnosed with a cancer or autoimmune disorder. Duration of survival is defined as the time from first administration of the drug to death. Duration of survival can also be measured by stratified hazard ratio (HR) of the treatment group versus control group, which represents the risk of death for a patient during the treatment.
• HR stratified hazard ratio
• the treatment of the present invention significantly increases response rate in a group of human patients susceptible to or diagnosed with a cancer who are treated with various anti-cancer therapies.
• Response rate is defined as the percentage of treated patients who responded to the treatment.
• the combination treatment of the invention using an antibody or antibody fragment and surgery, radiation therapy, or one or more chemotherapeutic agents significantly increases response rate in the treated patient group compared to the group treated with surgery, radiation therapy, or chemotherapy alone, the increase having a Chi-square p-value of less than 0.005.
• Therapeutic formulations are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (20 th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA).
• the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride, and preferably at about physiological concentrations.
• the formulations of the invention can contain a pharmaceutically acceptable preservative.
• the preservative concentration ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben are preferred preservatives.
• the formulations of the invention can include a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.
• 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
• encapsulated antibodies When encapsulated antibodies 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-disulf ⁇ de 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.
• the antibodies and antibody fragments described herein are administered to a human subject, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
• Local administration may be particularly desired if extensive side effects or toxicity is associated with VEGF and/or HER2 antagonism.
• An ex vivo strategy can also be used for therapeutic applications.
• Ex vivo strategies involve transfecting or transducing cells obtained from the subject with a polynucleotide encoding an antibody or antibody fragment.
• the transfected or transduced cells are then returned to the subject.
• the cells can be any of a wide range of types including, without limitation, hemopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.
• the label or package insert will further comprise instructions for administering the antibody composition to the patient.
• Articles of manufacture and kits comprising combinatorial therapies described herein are also contemplated.
• Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
• the package insert indicates that the composition is used for treating breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma, or ovarian cancer.
• the article of manufacture may further comprise a second 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 from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
• a pharmaceutically-acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
• Kits are also provided that are useful for various purposes, e.g., for purification or immunoprecipitation of VEGF or HER2 from cells.
• the kit can contain a VEGF/HER2 antibody (e.g., bHl-44 or bHl-81) coupled to beads (e.g., sepharose beads).
• Kits can be provided which contain the antibodies for detection and quantitation of VEGF or HER2 in vitro, e.g., in an ELISA or a Western blot.
• the kit comprises a container and a label or package insert on or associated with the container.
• the container holds a composition comprising at least one multispecific antibody or antibody fragment of the invention. Additional containers may be included that contain, e.g., diluents and buffers or control antibodies.
• the label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.
• the antigen-binding site of antibody is formed by the association of the variable domain (V H , V L ) of heavy chain (HC) and light chain (LC), each containing three CDR loops for antigen recognition. In many cases one of the two variable domains, often V H , determines the antigen specificity. Mice with transgenic HC but intact LC repertoire generate neutralizing antibody titers (Senn et al., Eur. J. Immunol. 33:950-961, 2003). We set out to investigate how bi-specificity of an antibody can occur and whether different utilization of the V H and the V L domains can enable dual antigen binding specificity.
• the heavy chain template (variable domain) sequence is set forth below (SEQ ID NO: 11).
• the LC library is a productive naive repertoire (Table 1). Listed are results from the screening of 95 random clones at the end of four rounds of selection. In particular, selection for new binding specificity was performed as described on immobilized targets (VEGF, DR5, and human Fc) (Sidhu et al., J. MoI. Biol. 338:299, 2004). After four rounds of selection 95 phage clones were assayed using ELISA for binding to the target, HER2, and a non-target protein, BSA, to ensure specific binding. To enrich for target binding clones that maintained HER2 binding, a final round of selection on HER2 was performed. The positive clones were sequenced.
• the IC 50 for antigen binding was determined by competitive ELISA (Sidhu et al., J. MoI. Biol. 338:299, 2004). The number of unique clones as determined by sequence analysis and the number of unique clones that maintain HER2 binding (bispecific clones) are shown. These clones show minimum background binding signals to irrelevant antigens, such as BSA. Table 1. Light chain library selection summary
• the number of mutations ranged from 3-17.
• the clones that retained HER2 binding (the bi-specific clones) contained fewer mutations on average than those that lost HER2 binding.
• Retaining the Herceptin® antibody CDR-L3 sequence was preferred but not sufficient to conserve HER2 binding. This is consistent with the report that the Herceptin® antibody CDR-L3 is the most important LC CDR for HER2 binding (Kelley and O'Connell, Biochemistry 32:6828. 1993).
• Representative VEGF-binding clones were expressed as Fab and IgG proteins (Table 2).
• Equilibrium binding affinities (K D ) of the LC library-derived mono-specific antibodies ranged from 15-150 nM.
• the bi-specific antibodies bound the new antigens (i.e., VEGF) with high nM to low ⁇ M affinity and HER2 with low nM affinity (Table 2).
• VEGF new antigens
• Enzymes and Ml 3-KO7 helper phage were from New England Biolabs.
• E. coli XLl - Blue was from Stratagene.
• Bovine serum albumin (BSA), ovalbumin, and Tween 20 were from Sigma.
• Neutravidin, casein, and Superblock were from Pierce.
• Immobilized protein G and anti-M13 conjugated horse-radish peroxidase (HRP) were from GE Healthcare (Piscataway, NJ).
• Maxisorp immunoplates were from NUNC (Roskilde, Denmark).
• Tetramethylbenzidine (TMB) substrate was from Kirkegaard and Perry Laboratories (Gaithersburg, MD). All protein antigens were generated by research groups at Genentech, Inc.
• the XYZ codon refers to a codon with unequal nucleotide ratios at each position of the codon triplet. X contained 38% G, 19% A, 26% T and 17% C; Y contained 31% G, 34% A, 17% T and 18% C; and Z contained 24% G and 76% C.
• the 2C4 Fab-C template phagemid pJB0290 was constructed by cloning the 2C4 heavy chain variable domain into a pV0354-Fab-C vector containing the alkaline phosphatase promoter (Lowman et al., 1991) and stll secretion signal for both light and heavy chain of Fab. It is engineered to contain a single cysteine at the C-terminus of the heavy chain variable domain 1 to allow bivalent Ml 3 phage display of the 2C4 Fab as previously described (Lee et al., 2004b).
• the 2C4 light chain CDRs were incorporated into the Fab-C vector by site- directed mutagenesis using the method of Kunkel et al (Kunkel et al., 1987).
• An epitope tag (gD tag) (Lasky and Dowbenko, 1984) was added at the C-terminus of the light chain to enable determination of the level of display as described (Sidhu et al., 2004).
• the Fabl2-G library template pV1283 was created by cloning a highly displayed heavy chain variable domain into pV0354-Fab-C, and the light chain variable domain was modified to contain CDR-L3 of Fab-12 (humanized A4.6.1, an anti-VEGF antibody).
• the highly-displayed V 11 was selected from a Fab library that randomized heavy chain CDR residues of G6 Fab using shotgun alanine scanning mutagenesis (Liang et al., 2006; Vajdos et al., 2002) with CDR-L3 converted to Fab-12 (Y 91 STVPW 96 ; SEQ ID NO:24) by panning on immobilized anti-gD antibody.
• the design and construction of the phagemid pV1384, displaying 4d5 (LC-R66G) scFv bivalently on the surface of Ml 3 phage particles was modified from the template pS2018 described previously (Sidhu et al., 2004).
• the scFv fragment contained a gD epitope tag in the linker region between light chain and heavy chain.
• LC framework residue Arg66 was mutated to Gly66, which is the prevalent residue in this position in over 95% of natural kappa light chains.
• the mutation R66G reduces Herceptin® antibody binding affinity to HER2 only slightly ( ⁇ 2 fold) as described in Kelley and Connell (Biochemistry 32:6828, 1993).
• the CDR sequences of the library templates are summarized in Figure 3.
• Phage-displayed libraries were created using oligonucelotide-directed mutagenesis as described (Sidhu et al., 2004).
• the library template vectors contained a stop codon (TAA) embedded in CDR-Ll , which was repaired during the mutagenesis reaction using degenerate oligonucleotides that annealed over the sequences encoding CDR-Ll , CDR-L3 (all libraries), CDR-L2 (L1/L2/L3-A, -B, -C, +L4-D) and the light chain framework 3 (L1/L4 and L1/L2/L3+L4-D).
• TAA stop codon
• the library mutagenesis reactions were performed according to the method of Kunkel et al (Kunkel et al., 1987).
• the light chain CDR designs for the libraries are described in Figure 1, which summarizes the degenerate codons used at each position for the different libraries.
• Three or four oligonucleotides were mixed at certain ratios for each CDR to encode the desired frequency of amino acid types at each position targeted for randomization ( Figure 4).
• the oligonucleotides were combined in different ratios to fine- tune the diversity to reflect the amino acid frequency in natural light chain kappa sequences at selected positions.
• CAT NNK NNK RST SEQ ID NO:25
• KMT XYZ XYZ RST SEQ ID NO:26
• DGG XYZ XYZ RST SEQ ID NO:27
• XYZ is a variation of NNK that has equal proportions of the A/G/T/C for each site to reduce the coverage of aliphatic hydrophobic amino acids (Lee et al, J. MoI. Biol. 340: 1073, 2004).
• NNK GST TCC NNK SEQ ID NO:28
• TGG GST TCC NNK SEQ ID NO:29
• KGG GST TCC TMT SEQ ID NO:30
• NNK GST TCC TMT SEQ ID NO:31
• each length was a mixture of three oligonucleotides containing codons for position 28-33: G 70 A 70 C 70 RTT NNK NNK TAC STA (SEQ ID NO:32), G 70 A 70 C 70 RTT NNK NNK DGG STA (SEQ ID NO:33), or G 70 A 70 C 70 RTT NNK NNK NMT STA (SEQ ID NO:34) at 1 : 1 :2 ratios.
• G 70 A 70 C 70 is a "soft" codon that allows 70% of the designated nucleotide and 10% each of the other three, encoding ⁇ 50% of GIu and -50% of the other amino acids.
• Figure 1 shows the comparison of the light chain natural diversity and the actual library designs.
• the mutagenesis products were pooled into one reaction per library and electroporated into E. CoIi SS320 cells supplemented with KO7 helper phage and were grown overnight at 30 0 C (Lee et al., J. MoI. Biol. 340: 1073, 2004). ⁇ l ⁇ " cells and -5-10 ⁇ g DNA were used in each electroporation reaction.
• the library phage were purified (Sidhu et al., J. MoI. Biol. 338:299, 2004). The number of transformants ranged from 10 9 -10 10 .
• the display level of intact Fabs or scFv on the surface of phage was determined in an ELISA binding assay where 96 randomly selected clones from each library were tested for their ability to bind an anti-gD antibody. The display level ranged from 5-25% ( Figure 2). 25% of the clones displaying antibody retained HER2 binding. Approximately 150 displaying clones were sequenced to examine the actual library diversity as compared to the design diversity. A portion (-30%) of the functionally displayed library members retained the Herceptin® antibody CDR-L2 and/or CDR-L3 sequence due to incomplete mutagenesis (a template stop codon in CDR-I ensured 100% mutation of this CDR in expressed scFvs). These were excluded from the sequence analysis of the actual library diversity.
• NUNC 96-well Maxiso ⁇ plates were coated overnight with antigen (5 ⁇ g/ml) and blocked for 1 hour with alternating blocking agents (Figure 7). Phage solutions of 10 13 phage/ml were added to the coated immunoplates in the first selection cycle. The phage concentration was decreased in each round of selection. Following incubation of the phage solutions on the immunoplates to allow binding to the immobilized antigen, the plates were washed with PBS, 0.5 % Tween 20, repeatedly.
• random clones from round four were assayed using phage ELISA where binding of individually amplified clones to the target and HER2 was compared to binding of a non-target protein (BSA) to test binding specificity.
• BSA non-target protein
• the eluted phage from the third and fourth round of VEGF or DR5 selection were amplified and subjected to another round of selection on HER2 coated wells.
• the V L and V H regions of the positive clones were amplified by PCR and sequenced.
• the hit rate for hFC, hVEGF, and hDR5-lf was 63, 77, and 85% respectively.
• the V L regions of the positive clones were amplified by PCR and sequenced as described (Sidhu et al., 2004).
• the DNA sequence analysis of the positive specific binders revealed a percentage of unique clones of 51% (hFC), 55% (hVEGF), and 6.1% (hDR5-lf).
• the sequences of unique hVEGF binding clones are summarized in Figure 8.
• this method generated 94 Her2/hVEGF bi-specific clones out of the 94 unique clones tested (100%) (Figure 8).
• the sequences of all isolated unique hVEGF/Her2 bi- specific antibodies from both isolation strategies are summarized in Figures 1OA and 1OB, The sequences of isolated clones that lost all detectable binding to Her2 are shown in Figure 11.
• the sequences of isolated clones that have dual specificity nearly all retained the Herceptin® antibody CDR-L3, making it likely that maintaining CDR-L3 is important for maintaining HER2 binding.
• hDR5 2 out of the 7 unique Her2 -binding clones were bi-specific (29%, 12 clones sequenced).
• One of the dual specific clones had some homologous changes in CDR-L3.
• a high-throughput single spot competitive ELISA in a 96-well format was used to screen for high affinity clones for hVEGF and to study the VEGFRl- blocking profiles. Briefly, Maxisorp Immunoplates were coated with 2 ⁇ g/ml hVEGFi O9 , overnight at 4°C and blocked with 1% (w/v) BSA for 1 hour. Phagemid clones in E. coli XLl -Blue were grown in 150 ⁇ l of 2YT broth supplemented with carbenicillin and M13-KO7 helper phage; the cultures were grown with shaking overnight at 37°C in a 96-well format.
• PBST PBS with 0.05% Tween 20 and 0.5% (w/v) BSA
• hVEGF coated wells were incubated with or without VEGFRl Domain 1-3 (Dl-3) and VEGFRl Domain 2 (D2) before adding five-fold diluted phage supernatant (Liang et al., 2006; Wiesmann et al., 1997). After incubation for 1 hour at room temperature (RT), the mixtures were transferred to the coated plates with hVEGF
• RT room temperature
• the plate was washed with PBT (PBS with 0.05% Tween 20) and incubated for 30 minutes with anti-M13 antibody horseradish peroxidase conjugate diluted 5000-fold to 1 nM in PBST.
• the plates were washed, developed with TMB substrate for approximately five minutes, quenched with 1.0 M H 3 PO 4 , and read spectrophotometrically at 450 nm.
• 09 to that in the absence of solution-phase hVEGFur ⁇ was used as an indication of the affinity.
• hVEGF-specific and hVEGF/Her2 bi-specific phage clones that gave rise to the lowest signal ratios in the single spot competitive ELISA were selected for affinity measurement by competitive ELISA as well as the DR5- binding and DR5/Her2 bi-specific phage clones from the initial single spot ELISA screen and VEGF binding clones from the combined plate and solution selection.
• Phage clones were propagated from a single colony by growing in 25 ml of 2YT culture supplemented with carbenicillin and KO7 helper phage overnight at 30 0 C i Phage purified by precipitation in PEG/NaCI were first diluted serially in PBST and tested for binding to an antigen-coated plate. The dilution that gave 50-70% saturating signal was used in the solution binding assay in which phage were first incubated with increasing concentration of antigen for one to two hours and then transferred to antigen-coated plates for 10-15 minutes to capture the unbound phage.
• IC 50 was calculated as the concentration of antigen in solution-binding stage that inhibited 50% of the phage from binding to immobilized antigen (Lee et al., 2004a).
• Figure 14B depicts the curves from which the IC 50 was calculated for the analyzed hVEGF binding clones from the plate sorting strategy.
• the IC 50 values ranged from 22 nM to >1 ⁇ M ( Figure 14B).
• the IC 50 values for the hVEGF binders isolated by combined plate and solution based selection ranged from 41 nM-226 nM ( Figure 9).
• IC 50 values of DR5-binding clones ranged from 20 nM to >1 ⁇ M.
• the IC 50 values for hVEGF/Her2 bi-specific clones are summarized in Figure 15.
• the digested DNA fragment was ligated into a similarly digested vector (pAP2009) designed for the phage display of Fab hu4D5 by fusion to the C-terminal domain of the M13 gene-3 minor coat protein (Lee et al., 2004b).
• the resulting bi-cistronic phagemid contains the light chain fused to an epitope (gD) tag at the C- terminus and heavy chain (V H and C H I ) fused to the gene for M13 minor coat protein (p3) C- terminally under the control of the alkaline phosphatase promoter.
• the first open reading frame encoded a polypeptide consisting of the stll secretion signal followed by the Fab4D5 light chain, with the CDRs replaced by those of 3-7 anti-hVEGF and 4-1 anti-hDR5 scFv'2, followed by a gD-tag epitope.
• the second open reading frame encoded a fusion polypeptide consisting of the following: the stll secretion signal, the Fab4D5 heavy chain, an amber (TAG) stop codon, a Gly/Ser linker sequence and c-terminal domain of g3 protein (cP3).
• coli XL-I Blue co-infected with M13 ⁇ CO7 resulted in the production of M 13 bacteriophage displaying Fab versions of 3-7 and 4-1 scFv'2.
• Competitive phage ELISAs were used to estimate the affinities of the phage-displayed scFvs and Fabs for hVEGF and hDR5 as IC 5O values. The data from the two different formats were in good agreement (data not shown).
• plasmid pAP2009 was modified to encode bHIFab.
• Versions of the bHl Fab were used as library templates containing stop codons (TAA) in either the three LC CDRs or the three HC CDRs for the LC and HC library, respectively.
• TAA stop codons
• Separate heavy chain and light chain alanine and homolog scanning libraries were constructed as previously described (Vajdos et al., J. MoI. Biol. 320:415, 2002). The degeneracy ranged from I xIO 5 to IxIO 8 and the actual library size from 6xlO 9 to 4xlO 10 .
• the libraries were constructed as described above.
• VEGF vascular endothelial growth factor
• HER2-ECD protein L
• anti-gD mlgG anti-gD mlgG
• Target binding clones were screened by phage ELISA for target binding followed by DNA sequencing and sequence alignment to calculate the wild-type/mutation ratios at each position.
• the ratios from sequence analysis of approximately 100 unique sequences of VEGF and HER2 binding clones were corrected for display and protein folding effect by dividing with ratios calculated from the sequences of more than 100 anti-gD binding clones to yield the F ⁇ ,TM,, values.
• the phage display of the gD tag is indicative of proper folding and association of light chain and heavy chain.
• protein L binding to a non-linear epitope on the light chain of the Fab also resulted in similar wild-type/mutation ratios as gD tag selections.
• the hlgGs were expressed by transient transfection of 293 cells and hlgG was purified with protein A affinity chromatography (Fuh et al., J. Biol. Chem. 273: 1 1 197, 1998).
• the 1 L E. coli cultures were purified with protein G affinity chromatography. The columns were washed with PBS and Fab protein was eluted with 100 raM acetic acid and dialyzed against PBS.
• the 4 L E. coli cultures were purified on a protein A affinity column followed by cation exchange chromatography as previously described (Muller et al., 1998). Protein concentrations were determined spectrophotometrically.
• the final yield for Fab was typically 0.8-15 mg/1 purified from a small-scale shake flask growth. IgG production yield was medium to high at 6.7-60 mg/1 in small-scale culture ( Figure 17).
• the purified proteins were first characterized using size exclusion chromatography and light scattering to ensure that the proteins did not exhibit significant levels of protein aggregation ( ⁇ 5%).
• hVEGF and hDR5 were immobilized at a concentration of 2 ⁇ g/ml.
• a fixed concentration of hlgG was incubated with serial dilutions of Her2-ECD followed by capture of the hlgG on the immobilized antigen.
• Her2-ECD binding was found competitive with binding to the other antigens ( Figure 20).
• the affinity of the hVEGF binding antibodies 3- 1, 3-6 and 3-7 was found to be in the nano molar range.
• the bi-specific antibodies analyzed (bHl , H3, H4_N, H4_D) showed low tnicromolar to micromolar affinities for hVEGF.
• the affinities for Her2 ranged from 8-59 nM (Fab).
• the light chain variable domains were grafted onto the anti-Her2 2C4 Fab by cloning the light chain variable domains into a 2C4 Fab expression vector pJB0524, thus replacing 2C4 light chain variable domain.
• the Fabs were expressed as previously described.
• the bHl/2C4 and H3/2C4 chimeric Fabs did not express at detectable levels.
• the H4_N/2C4 chimeric Fab protein was isolated and tested for binding to hVEGF (bHl original specificity) and Her2 (bHl, 2C4 original specificity). No binding was detected to hVEGF and Her2 by a standard ELISA binding assay ( Figure 22). The results indicate that the heavy chain of bHl is required for antigen binding.
• the solution competition binding assay used biotinylated VEGF equilibrated with serial dilutions of purified IgG proteins, and the unbound biotin-VEGF was captured with immobilized Fab or IgG coated on Maxisorb plates and was detected with streptavidin-conjugated HRP (Lee et al., J. MoI. Biol. 340: 1073, 2004). Antibodies that block hVEGF from binding other hVEGF-binding antibodies or hVEGF- receptors are likely to share over-lapping epitopes.
• 3-1 in contrast, does not block hVEGF from binding VEGFRl, even at the highest concentration (0.5 ⁇ M) (Figure 23). Furthermore, we could not detect 3-1 hlgG blocking of the Avastin® antibody (Figure 25). However, 3-1 hlgG block hVEGF binding to VEGFR2 (KDR) ( Figure 23) as well as to B20-4.1 ( Figure 24). These results indicate that 3-1 has a unique epitope compared to the other antibodies.
• the Herceptin® antibody and bHl differ in CDR-Ll (V 29 NTA 32 vs. I 29 PRSISGY 32 ; SEQ ID NOS:35 and 36) and CDR-L2 (S 50 ASF 53 vs. W 50 GSY 53 ; SEQ ID NOS:37 and 38).
• the bHl anti-VEGF/Her2 was chosen as representative for structural characterization based on its dual specific nature and its relatively high affinity for VEGF and Her2.
• the receptor-binding portion of human VEGF consisting of residues 8-109, was expressed, refolded and purified as described previously (Christinger et al., 1996). Residue 1-624 of the extra cellular domain of Her2 was expressed and purified as previously described (Franklin et al., 2004; Hudziak and Ullrich, 1991 ).
• Crystals of bHl Fab-Her2( 1-624) was obtained by mixing protein solution (1 1 mg/ml, 25 mM Tris pH 8 and 150 mM sodium chloride) with crystallization buffer containing 25% w/v PEG 200O , 0.1 M MES pH 6.5. Crystals appeared after 12 hours that belonged to space group P2
• the data was processed using Denzo and Scalepack (Otwinowski, 1997).
• the structures of bHl Fab complexes was solved by Phaser (L. C. Storoni, 2004; Read, 2001).
• the bHl-Fab-VEGF(8-109) complex was solved using coordinates of VEGF from a previously described VEGF-Fab complex (2FJG) and Fab fragments containing either the variable domains V L /V H or the constant domains C H i/C L of the Herceptin® antibody Fab-Her2 complex (1N8Z). Fragments of Her2 and the variable domain of the Herceptin® antibody Fab from the Her2-Fab complex 1N8Z were used as search models when solving bHl-Her2 structure.
• the constant domain of the bH l Fab could not be found using the Herceptin® antibody Fab constant portion as a search model (1N8Z) and had to be docked manually guided by the Herceptin® antibody Fab-Her2 complex structure.
• Model building and refinement were performed using the programs Refmac (Collaborative Computational Project, 1994) and Coot (Emsley and Cowtan, 2004), respectively.
• the Fab binds to domain IV of HER2 in a manner similar to the Herceptin® antibody (Cho et al., Nature 421 :756, 2003); the two complexes superimpose with a root mean square deviation (r.m.s.d.) of Ca positions of 2.3 A.
• bHl recognizes an epitope that overlaps with the binding sites of the VEGF receptors VEGFRl and VEGFR2 and of other VEGF antibodies (Wiesmann et al., Cell 91 :695, 1997; Muller et al., Proc. Natl. Acad. Sci. USA 94:7192, 1997).
• the CDR-Ll is an exception and differs significantly in the two complex structures; the deviation is 4.6 A (Ca of residues 27-32).
• Figure 30 shows that the CDR conformations of bHl Fab bound to VEGF are markedly similar to HER2-bound bHl and to the Herceptin® antibody, with exception of the CDR-Ll .
• Figure 30 is a superposition of the CDR loops as tubes of VEGF-bound bHl (dark shading), HER2-bound bHl (white) and HER2-bound the Herceptin® antibody (light shading).
• the CDR-Ll loop exhibits significantly different conformations in the two bHl structures (r.m.s.d.c ⁇ ⁇ . ⁇ for bHl residues 27-32) ( Figure 31).
• the CDR-Ll is minimally involved in antigen interaction and part of the loop (residues 28-30b) appears flexible.
• the entire loop is well structured and contributes 26% of surface area buried by VEGF.
• the CDR-Ll conformation is further stabilized by hydrogen bonds between Tyr32 and the LC framework residue Gly72.
• the structural analysis confirms that Tyr32 is critical for VEGF binding as mutation to either alanine or phenylalanine is not tolerated. Contrary to VEGF binding, mutation of Tyr32 to alanine (back to the Herceptin® antibody residue) is preferred for HER2 binding. Superposition of the two complexes reveals that VEGF would clash with Tyr32 of CDR-Ll in its HER2 bound state ( Figure 31).
• the side chains of residues Tyr32, Ile30c, Ile29, and Gly72 are shown as sticks. Residues with temperature factors higher than average are shown in darker shading (residues 28-3Ob). Hydrogen bonding between Tyr32 and Gly72 is illustrated by a dotted line.
• Solvent exposed residues in the CDRs were scanned using phage-displayed libraries in which the wild type residues were allowed to vary as either alanine or wild type (Alanine Scan) or as a homolog residue or wild type (Homolog Scan).
• the nature of the genetic code required some other substitutions to be included in the library in addition to Wt/Alanine or Wt/Homlog residues ( Figure 33).
• Separate heavy chain and light chain alanine and homolog scanning libraries were constructed. The libraries are described in Figure 34. The degeneracy ranged from 1.3xlO 5 to 1.3xlO 8 and the actual library size from 6x10 9 to 4xlO 10 .
• a previously described plasmid AP2009 designed to display hu4D5Fab on phage fused to the C- terminal domain of the M13 gene-3 minor coat protein was modified to encode bHIFab using standard molecular biology techniques.
• the C-terminus of the light chain contained an epitope (gD) tag.
• “Stop template” versions of the bHl Fab was used as library template (Sidhu et al., 2004).
• the light chain alanine and homolog scanning library had stop codons in CDR-Ll , CDR-L2 and CDR-L3 and the heavy chain alanine and homolog libraries contained stop codons in each heavy chain CDR.
• the libraries were constructed by previously described methods (Sidhu et al., 2004) using Kunkel mutagenesis (Kunkel et al., 1987) on the respective stop templates.
• NUNC 96-well Maxisorp immunoplates were coated with 5 ⁇ g/ml capture target (hVEGF, 09 , Her2-ECD or anti-gD mlgG) and blocked with 1 % BSA (w/v) in PBS.
• Phage from the above-described libraries were propagated with KO7 helper phage (NEB) as described (Lee et al., 2004a).
• the library phage solutions were added to the coated plates at a concentration of 10 13 phage particles/ml and incubated for 1-2 hours at RT.
• the plates were washed 8 times with PBST and followed by elution of bound phage with 0.1 M HCI for 30 minutes. Enrichment after each round of selection was determined as described previously.
• Figure 39A and Figure 39B show shotgun alanine- and homolog scanning of bHl Fab for binding to VEGF and HER2.
• the effects of mutation of alanine (m l ), or additional mutations (m2, m3; due to limitations of shotgun-alanine codons), or to a homologous amino acid (m4) are calculated as the ratio of occurrence of wild-type and mutants (wt/mut) among the clones binding to human VEGF ( Figure 39A) or HER2 ( Figure 39B). In cases where only the wild-type residue appeared, the ratios are shown as larger than ">" the wild-type count.
• the identity of the amino acid substitutions (ml-m4) is shown as superscripts on the F values.
• VEGF binding interaction is mediated primarily by the LC CDRs with Tyr32 of CDR-Ll and His91 of CDR-L3 as the core hot spot ( ⁇ G wt/a i a >1.5 kcal/mol).
• HER2 binding is mainly contributed by HC CDRs.
• Figure 32 shows crystal structures where the bHl and the Herceptin® antibody residues are shaded on the Fab surface based on their functional importance (dark shading and white lettering, ⁇ G > 1.5 kcal/mol; intermediate shading and black lettering, 1 ⁇ ⁇ G ⁇ 1.5 kcal/mol; light shading and black lettering, 0.5 ⁇ ⁇ G ⁇ 1 kcal/mol of alanine mutation).
• the black dotted line outlines the contact area as in Figure 28.
• the white dotted line depicts the divide of light and heavy chain.
• Trp95 of CDR-H3 is the only common hot spot residue for the two interactions ( ⁇ G wt/a i ⁇ l .5 kcal/mol).
• Trp95 of CDR-H3 is the only common hot spot residue for the two interactions ( ⁇ G wt/a i ⁇ l .5 kcal/mol).
• the VEGF binding interaction is mediated primarily by the LC CDRs while HER2 binding is dominated by HC CDRs.
• bHl with weaker HER2 binding affinity maintains the same core hot spot residues for HER2 binding (Arg50, Trp95, and TyrlOOa) while the importance of peripheral residues is redistributed (Figure 32).
• the stringency was increased in each round of selection by decreasing the concentration of biotinylated VEGF from 50 nM in the first round to 20 nM in the second round. 138 clones were sequenced from the last round of selection. Most clones were found to be unique.
• a high-throughput ELISA assay with immobilized VEGF (8-109), anti-gD antibody, and Her2-ECD was used to identify clones that bound to VEGF, Her2-ECD, and anti-gD mlgG but not to BSA.
• the VEGF- ELISA binding signals were normalized by the anti-gD ELISA signals to estimate the relative affinity of the VEGF binding clones.
• the K 0 for human VEGF was increased from 250 (bHl; IgG) to 41 (bHl -81 ; IgG) or 16 nM (bHl -44; IgG) and the K D for HER2 was increased from 21 (bHl ; IgG) to 7 (bHl -81 ; IgG) or 1 nM (bHl -44; IgG).
• Table 5 shows the randomized positions in bold and summarizes the CDR sequences (SEQ ID NOS: 1-9 and 39-41 ) of bHl, bHl -81 , and bHl -44 and their affinities (as determined by surface plasmon resonance).
• bHl and 3-1 antibodies could inhibit hVEGF
• HUVECs were plated in each well of the 96-well cell culture plate and incubated in Dulbecco's modified Eagle's/F-12 medium (1 : 1) supplemented with 1.0% (v/v) fetal bovine serum (assay medium) for 18 hours.
• Dulbecco's modified Eagle's/F-12 medium (1 : 1) supplemented with 1.0% (v/v) fetal bovine serum (assay medium) for 18 hours.
• Fresh assay medium with fixed amounts of VEGF 0.2 nM final concentration
• bHl and bH3 antibodies to NR6 fibroblast cells over- expressing Her2 was studied by Flow Cytometry.
• One million NR6-Her2 cells were incubated with 100 ⁇ g/ml Fab and IgG for 1 hour, followed by incubation with an Alexa488-conjugated murine anti-human IgG antibody for 1 hour.
• Fab and IgG binding to non-expressing NR6 cells was studied.
• bHl and bH3 bind specifically to Her2 on NR6 cells as Fab and as IgG.
• bHl-81 and bHl-44 antibodies inhibit VEGF-induced proliferation of HUVEC cells and growth of BT474 cells to a greater extent than bHl .
• the increased potencies of the bHl variants correlate with their relative affinities.
• the highest affinity variant, bHl-44 inhibits growth of HUVEC and BT474 cells with a potency similar to bevacizumab or Herceptin® antibody, respectively.
• the antibody-dependent inhibition of proliferation of the HER2 expressing cells was determined by measuring the fluorescent signal after 6 hours.
• the binding specificity of the antibodies derived from the LC library was determined. IgGs binding to various immobilized purified proteins or cell lysates including the cognate antigens was assayed by ELISA. The antigens were immobilized and incubated with hlgG at a concentration of 15 ⁇ g/mL for an hour. Bound IgG were detected spectrophotometrically (optical density at 450 nm; y-axis; Figure 46).
• vascular endothelial growth factor A vascular endothelial growth factor A
• murine vascular endothelial growth factor murine vascular endothelial growth factor
• vascular endothelial growth factor C vascular endothelial growth factor C
• hVEGF-C murine vascular endothelial growth factor C
• hVEGF-D vascular endothelial growth factor D
• HER2 extracellular domain HER2 ECD
• epidermal growth factor receptor extracellular domain hEGFR
• ErbB3/HER3 extracellular domain HER3 ECD
• human death receptor 5 hDR5
• BSA bovine serum albumin
• FBS Fetal Bovine Serum
• Neutravidin Neutravidin
• 5% milk mouse fibroblast cell lysate
• mouse fibroblast cell lysate mouse fibroblast cell lysate spiked with hVEGF-A or HER2 ECD.
• VEGF and HER2 were shown to compete for binding to bHl-44 bispecific IgG antibody in solution ( Figure 48).
• Human bHl-44 IgG antibody at a concentration of 0.1 nM was incubated with 0.1 nM biotinylated human VEGF
• bHl-44 was captured by immobilized anti-human Fc and bHl-44-bound biotin-VEGF detected with streptavidin-HRP.
• HER2 ECD bound to captured bHl-44 was detected using a murine anti-HER2 antibody binding a non- overlapping epitope on HER2 followed by an HRP-conjugated goat anti-mouse IgG (Figure 48A).
• Human bHl-44 IgG at a concentration of 0.2 nM was incubated with 0.6 nM biotinylated HER2 in the presence of increasing concentrations of human VEGF] 65 .
• bHl -44 was captured by immobilized anti-human Fc and bHl-44-bound biotin-HER2 detected with streptavidin-HRP ( Figure 48B).
• NR6 cells were non-specifically biotinylated, lysed, and cell membrane proteins detergent solubilized. Cell lysates corresponding to 5-10 million cells/mL of NR6 cells, NR6 cells spiked with 0.1 ⁇ g/mL biotinylated VEGF 165 ,or HER2 over expressing NR6 cells were incubated with 15 ⁇ g/mL antibody.
• the antibody was captured using proteinA-coated sepharose beads and bound proteins eluted.
• the eluted proteins were separated by SDS-PAGE.
• Cell lysates corresponding to approximately 25- 50,000 cells and immunopreciptate from approximately 0.12-0.25 million cells were loaded onto the gel.
• Captured biotinylated proteins were detected by Western blotting using streptavidin-HRP.
• Example 7 Analysis of IgG activity in in vivo assays
• mice xenograft tumor models known to be responsive to treatment by anti-VEGF antibody Cold-VEGF antibody
• Herceptin® antibody BT474M1, breast cancer cell line
• Colo205 xenografts were used in nu/nu mice and BT474M1 xenografts were used in beige nude XID mice. All animal studies were in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care and the Genentech Institutional Animal Care and Use Committee.
• BT474M 1 in-house
• Colo205 ATCC, Manassas, VA cells were cultured in RPMI media/10% fetal bovine serum.
• HBSS Hank's Buffered Salt Solution
• matrigel (1 : 1) mixure were injected into the mammary fat pad of Harlan beige nude XID mice (Indianapolis, IN) implanted with an estradiol pellet subcutaneously.
• 5x10 6 Colo205 cells in HBSS were subcutaneously injected into Charles River nu/nu mice (Hollister, CA).
• PR Partial responses
• the concentration of human antibody was determined using ELISA.
• Donkey anti-human IgG Fc was immobilized on an immuno plate. Dilutions of serum and an antibody standard were incubated on the plate for 2 hours. Bound antibody was detected by Horseradish Peroxidase conjugated goat anti-human IgG Fc followed by TMB Substrate/1 M Phosphoric Acid. The plates were read at 450/620 nm. Sample concentrations were determined using a 4-parameter algorithm. The bHl-44 treated groups were compared with groups treated with anti-VEGF
• the combination treatment showed similar efficacy as anti-VEGF alone.
• bHl-44 antibody administered at 10 and 20 mg/kg/week yielded dose-dependent responses.
• VEGF and HER2 binding interfaces with these antibodies were identified.
• the structural contacts listed in Table 7 were identified based on the crystal structure coordinates 3BDY (bHl /VEGF) and 3BEl (bHl/HER2).
• the binding interface was calculated using the program XSAE. This program defined the interface as polar, hydrophobic, and mixed.
• Table 7 lists the bHl residues with >25% of the total surface area buried upon HER2 or VEGF binding.
• Table 7 also lists the VEGF and HER2 residues within 4.5 A of the bHl residues.
• the surface area of each residue that is buried upon complex formation was calculated using IMOL based on the coordinates of the crystal structures 3BDY, 3BEl , and 1N8Z (PDB).
• the polar and hydrophobic interface areas reported in Table 11 reflect the polar interface area and half of the mixed.
• the hydrophobic interface area reported consists of the hydrophobic areas and half of the mixed.
• Herceptin® antibody residues that contribute more than 10% of the total binding energy based on alanine scanning mutagenesis are conserved, and many of them are also part of the binding hotspots of bHl and bHl -44 (Bostrom et al., 2009; Kelley and O'Connell, 1993) (Table 14, Figure 62).
• the interfaces between bHl/VEGF and bHl/HER2 bury 1506 A 2 and 1579 A 2 are mainly hydrophobic (60% and 63%, respectively).
• the Herceptin®/HER2 binding interface has similar size and composition as the bHl/HER2 interface (1524 A 2 , 60% hydrophobic, Table 11), and is also characterized by high shape complementarity (Table 8) (Bostrom et al., 2009).
• Table 7 List of structural contacts of the complex of bHl Fab/HER2 ECD and bHl/Fab/VEGF] 09 .
• the table lists residues with >25% of the total surface area buried upon HER2 and VEGF binding.
• the VEGF and HER2 residues within 4.5 A of the bHl residues are listed.
• the surface area of each residue that is buried upon complex formation was calculated using IMOL based on the coordinates of the crystal structures 3BDY, 3BEl, and 1N8Z (PDB).
• the shape complementarity (represented as Sc in Table 8) between the antibody and the antigen was determined as described (Lawrence et al., 1993).
• the high shape complementarity in the bHl/VEGF and bHl/HER2 complexes similar to the complementarity between the Herceptin® antibody and HER2, are in the range of reported antibody-antigen complexes (Sc ⁇ 0.64-0.68; Lawrence et al., 1993).
• Superposition of HER2 with bHl in its VEGF-bound conformation or VEGF with bHl in its HER2-bound form reveals little shape complementarity observed when juxtaposing an antibody with an unrelated antigen. (Sc ⁇ 0.35; Lawrence et al., 1993).
• the results demonstrate the extent to which bHl rearranges to accommodate the two different antigens.
• the affinity of bHl was improved by selecting the high affinity variant bHl -44 from phage-displayed antibody libraries of bHl .
• Shotgun alanine scanning mutagensis demonstrated that bHl -44 conserved the hotspot for antigen binding of bHl (Tables 9A-B, 10, and 14).
• Shotgun alanine scanning mutagenis of bHl -44 was performed using the techniques described above for the shotgun alanine scanning mutagenesis of bHl .
• HER2 in the crystal structure are listed.
• the energetic hotspots for binding are defined by the antibody residues that result in greater than approximately 10% of the total binding energy of the interaction.
• Table 11 The data in Table 11 indicate that the polarity and size of the binding interfaces are similar between bHl /VEGF, bHl/HER2, and the Herceptin®/HER2 complex.
• the polarity of each interface was analyzed using XSAE. All the numbers depicted in Table 11 represent the area in A 2 , unless otherwise indicated.
• Table 9A Shotgun alanine- and homolog-scanning of bHl-44 Fab for binding to VEGF.
• Table 9B Shotgun alanine- and homolog-scanning of bH 1-44 Fab for binding to HER2.
• Table 10 The structural and functional paratopes for VEGF and HER2.
• VEGF I09 and HER2 extracellular domains were immobilized on CM5 chips at a density that allowed for an Rmax in the range of 50-150 RU.
• Serial dilutions of Fabs in PBS with 0.05% Tween20 were injected at 30 ⁇ l/min.
• the binding responses were corrected by subtracting responses from a blank flow cell and by normalizing for buffer effects.
• a 1 : 1 Langmuir fitting model was used to estimate the k a (onrate) and kj (offrate). The K 0 values were determined from the ratios of k a and k d .
• Table 12 depicts the kinetic profiles of the bHl variants and the Herceptin® antibody determined by surface plasmon resonance measurement using BIAcore at 3O 0 C.
• Fabs were bound to immobilized VEGF or HER2, and the on-rate (k a ), off-rate (k d ), and dissociation constant (KD) were determined using a 1 : 1 Langmuir binding fitting model.
• the bHl-44 antibody has a similar kinetic profile and affinity for HER2 as the Herceptin® antibody.
• the two double mutants (bHl-44 I29A + Y32A and bHl-44 R50A + R58A) that lost binding to VEGF or HER2 retained the kinetic profile and affinity for the other antigen.
• ⁇ H enthalpy
• ⁇ S entropy
• ITC isothermal titration calorimetry
• 09 and the extracellular domain of HER2 were performed on a VP-ITC titration calorimeter (Microcal Inc.) as described (Starovasnik et al., 1999). Protein solutions were extensively dialyzed into phosphate-buffered saline. The antigen and Fabs were dialyzed in the same vessel to minimize mixing heat effects due to differences in buffer composition. Fabs at a concentration of 100-220 ⁇ M were titrated into antigen solutions (HER2-ECD or VEGF, 09 ) at a concentration of 10-22 ⁇ M.
• ⁇ S ( ⁇ H- ⁇ G)/T, where T is the temperature (K).
• microcalorimetric measurements were performed as described above at different temperatures ranging from 20 to 37 0 C.
• the ⁇ Cp was determined by linear regression by plotting ⁇ H as a function of the temperature ( Figure 62).
• Table 13 depicts the ⁇ G (binding free energy), ⁇ S (entropy change), and ⁇ H (enthalpy change) in kcal/mol.
• the affinities shown were measured in at least two independent experiments using kinetic analysis by BIAcore at 30 0 C.
• the ⁇ H was measured using ITC and represents the average of two or three independent measurements followed by the standard deviations.
• the ⁇ G and ⁇ S were calculated as described above.
• the high affinity variants bHl-81 and bHl-44 displayed similar thermodynamic profiles as bHl .
• Their interactions with VEGF and HER2 were also characterized by favorable enthalpy and entropy (Table 13, Figure 60).
• thermodynamic properties contrast the many similarities in HER2 binding characteristics between Herceptin® and bHl-44, which include affinity, kinetics, and many of the residues of the energetic hotspots.
• hot spot residues of Herceptin® that contribute more than 10% of the total binding energy for HER2 are similar to those of bHl and bHl-44, there are some clear differences.
• Table 14 shows the bHl, bHl -44, and the Herceptin® antibody hotspots for HER2 binding determined by alanine scanning mutagenesis.
• the mutagenesis was performed as described in Kelley et al., 1993.
• the numbers in Table 14 represent the change in binding free energy ( ⁇ Gw t . m m) when the residue is mutated to alanine.
• the hotspot residues in Table 14 are shaded and are defined as ⁇ G greater than or equal to 10% of the total binding free energy ( ⁇ G).
• Residues LC-Thr94, HC-Tyr33, HC-Asp98 arc conserved in sequence in bHl but have different functions in HER2 binding (Table 14, Figure 61).
• the mutations in the antigen-binding site of Herceptin® that recruited VEGF binding appear to have made some fundamental changes to the antigen-binding site that affect the interaction with HER2.
• the dual specific antibodies accommodate the introduced mutations by utilizing a different HER2 recognition strategy that results in equally high affinity for HER2 as Herceptin®. It is interesting to note that except for LC-Ser94 of bHl-44 the mutations that improved the affinity for HER2 more than 100-fold compared to bHl are not parts of the binding hotspot, but appear to optimize the existing interactions.
• ⁇ Cp was estimated from the slope of the temperature dependence of ⁇ H by linear regression ( Figure 62, Table 15).
• ⁇ Cp of bHl-44 was determined to -400 cal/molK for the interaction with VEGF, and -440 cal/molK for the interaction with HER2.
• the large negative heat capacities indicate the importance of the hydrophobic effect as previously described (Kauzmann, 1959), which is consistent with the hydrophobic nature of the structural interfaces in the two complexes (Table 11).
• the ⁇ Cp for Herceptin®/HER2 which was previously determined to -370 cal/molK in a similar temperature interval (Kelley et al., 1992), is smaller than the ⁇ Cp of bHl-44/HER2, but still indicates the important role of the hydrophobic effect in HER2 binding.
• the total entropy change ( ⁇ S) of binding free energy is a sum of entropy changes from three sources (Murphy et al., 1994): entropy changes associated with desolvation of the binding surfaces ( ⁇ S SOLV X entropy changes from the loss of rotational and translational degree of freedom ( ⁇ S RT ), and entropy changes due to the changes in configurational and conformational dynamics of the interacting molecules ( ⁇ S CONF )-
• ⁇ S CONF can thus be estimated as:
• ⁇ S SOLV is estimated to 96 calmor'K. "1 for bHl-44/VEGF, 105 calmor'K “ ' for bHl-44/HER2, and 89 calmol ' 'K “ ' for Herceptin®/HER2 (Table 15). This translates to ⁇ S CONF of -72 calmol " 'K " ' for bHl-44/VEGF, -70 calmor'lC 1 for bHl- 44/HER2, and -80 calmor'K "1 for Herceptin®/HER2 (Table 15).
• T M of the dual specific variants (77.2°C, 75.6°C, 74.3 0 C for bHl, bHl -81, and bHl-44, respectively, Table 16) are slightly lower than that of Herceptin® (82.5 0 C), but high and within the range of what has been reported for other therapeutic antibodies (Garber and Demarest, 2007).
• each residue was mutated to alanine in the bHl-44 (LC-Ile29, LC-Tyr32, HC- Arg50, HC-Arg58) or the Herceptin® (HC-Arg50, HC-Arg58) scaffolds, individually or in combination, and expressed the mutants as Fabs and IgGs.
• HC-R50A, HC-R58A in the Herceptin® scaffold also disrupted binding to HER2 to various extents, while the double mutant HC R50A+R58A showed no detectable HER2 binding (Table 12).
• thermodynamic parameters of the double mutants were next analyzed and compared to the values for bHl-44.
• the double mutants displayed the same thermodynamic and kinetic profiles as bHl-44.

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