WO2017118675A1 - Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies - Google Patents

Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies Download PDF

Info

Publication number
WO2017118675A1
WO2017118675A1 PCT/EP2017/050168 EP2017050168W WO2017118675A1 WO 2017118675 A1 WO2017118675 A1 WO 2017118675A1 EP 2017050168 W EP2017050168 W EP 2017050168W WO 2017118675 A1 WO2017118675 A1 WO 2017118675A1
Authority
WO
WIPO (PCT)
Prior art keywords
domain
antibody
cea
sequence
seq
Prior art date
Application number
PCT/EP2017/050168
Other languages
French (fr)
Inventor
Marina Bacac
Sara COLOMBETTI
Christian Klein
Johannes Sam
Jose SARO
Pablo Umaña
Original Assignee
F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP17700491.8A priority Critical patent/EP3400246B1/en
Priority to CA3006529A priority patent/CA3006529A1/en
Priority to ES17700491T priority patent/ES2837428T3/en
Priority to KR1020187019336A priority patent/KR20180097615A/en
Priority to CN201780004427.0A priority patent/CN108368179B/en
Priority to PL17700491T priority patent/PL3400246T3/en
Priority to BR112018011029A priority patent/BR112018011029A2/en
Priority to IL259588A priority patent/IL259588B2/en
Application filed by F. Hoffmann-La Roche Ag, Hoffmann-La Roche Inc. filed Critical F. Hoffmann-La Roche Ag
Priority to MX2018008347A priority patent/MX2018008347A/en
Priority to AU2017205089A priority patent/AU2017205089B2/en
Priority to JP2018535339A priority patent/JP6949030B2/en
Priority to EP20201657.2A priority patent/EP3862365A1/en
Publication of WO2017118675A1 publication Critical patent/WO2017118675A1/en
Priority to HK19101508.2A priority patent/HK1259020A1/en

Links

Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • 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
    • A61K2039/507—Comprising a combination of two or more separate 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • This invention relates to methods of treating CEA -positive cancers by administering a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
  • Cancer is one of the leading causes of death worldwide. Despite advances in treatment options, prognosis of patients with advanced cancer remains poor. Consequently, there is a persisting and urgent medical need for optimal therapies to increase survival of cancer patients without causing unacceptable toxicity.
  • P-Ll Programmed death-ligand 1
  • IFNy interferon gamma
  • PD-Ll programmed death-ligand 1
  • PD-L2 programmed death ligand 2
  • PD-Ll is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813) (Thompson RH et al., Cancer Res 2006, 66(7):3381).
  • the inhibition of PD-L1 signaling has been proposed as a means to enhance T cell immunity for the treatment of cancer (e.g., tumor immunity) and infection, including both acute and chronic (e.g., persistent) infection.
  • An optimal therapeutic treatment may combine blockade of PD-1 receptor/ligand interaction with an agent that activates T cells, particularly CD8+ T cells.
  • a method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a human PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
  • a method of enhancing immune function in an individual having cancer comprising administering an effective amount of a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
  • a human PD- 1 axis binding antagonist in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising an anti- CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier.
  • an anti-CEA/anti-CD3 bispecific antibody in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.
  • composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises
  • composition in combination with a second composition, wherein the second composition comprises an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier.
  • composition comprising an anti-CEA/anti- CD3 bispecific antibody and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises administration of said composition in combination with a second composition, wherein the second composition comprises a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.
  • kits comprising a medicament comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the medicament in combination with a composition comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.
  • kits comprising a first medicament comprising a PD- 1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a second medicament comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier.
  • the kit further comprises a package insert comprising instructions for administration of the first medicament and the second medicament for treating or delaying progression of cancer in an individual.
  • kits comprising a medicament comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the medicament in combination with a composition comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.
  • the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.
  • the PD-1 axis binding antagonist is an antibody.
  • the antibody is a humanized antibody, a chimeric antibody or a human antibody.
  • the antibody is an antigen binding fragment.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , and Fv.
  • the PD-1 axis binding antagonist is a PD-1 binding antagonist.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2.
  • the PD-1 binding antagonist is an antibody.
  • the PD-1 binding antagonist is selected from the group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108.
  • the PD-1 axis binding antagonist is a PD-L1 binding antagonist.
  • the PD-L1 binding antagonist inhibits the binding of PD- Ll to PD-1.
  • the PD-L1 binding antagonist inhibits the binding of PD- Ll to B7-1.
  • the PD-L1 binding antagonist inhibits the binding of PD- Ll to both PD-1 and B7-1.
  • the PD-L1 binding antagonist is an anti- PD-L1 antibody.
  • the PD-L1 binding antagonist is selected from the group consisting of: MPDL3280A (atezolizumab), YW243.55.S70, MDX- 1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab).
  • the anti-PD-Ll antibody is MPDL3280A (atezolizumab).
  • MPDL3280A is administered at a dose of about 800 mg to about 1500 mg every three weeks (e.g., about 1000 mg to about 1300 mg every three weeks, e.g., about 1100 mg to about 1200 mg every three weeks).
  • the anti-PD-Ll antibody comprises a heavy chain comprising HVR-Hl sequence of SEQ ID NO: 19, HVR-H2 sequence of SEQ ID NO:20, and HVR-H3 sequence of SEQ ID NO:21; and/or a light chain comprising HVR-L1 sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ ID NO:24.
  • the anti-PD-Ll antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 or 26 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:4.
  • the anti-PD-Ll antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4.
  • the anti-PD-Ll antibody comprises the three heavy chain HVR sequences of antibody YW243.55.S70 and/or the three light chain HVR sequences of antibody YW24355.S70 described in WO 2010/077634 and U.S. Patent No. 8,217,149, which are incorporated herein by reference.
  • the anti-PD-Ll antibody comprises the heavy chain variable region sequence of antibody YW243.55.S70 and/or the light chain variable region sequence of antibody YW24355.S70.
  • the PD-1 axis binding antagonist is a PD-L2 binding antagonist.
  • the PD-L2 binding antagonist is an antibody.
  • the PD-L2 binding antagonist is an immunoadhesin.
  • the PD-1 axis binding antagonist is an antibody (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-PD-L2 antibody) and comprises an aglycosylation site mutation.
  • the aglycosylation site mutation is a substitution mutation.
  • the substitution mutation is at amino acid residue N297, L234, L235, and/or D265 (EU numbering).
  • the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, and D265A.
  • the substitution mutation is a D265A mutation and an N297G mutation.
  • the aglycosylation site mutation reduces effector function of the antibody.
  • the PD-1 axis binding antagonist e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-PD-L2 antibody
  • the PD-1 axis binding antagonist is a human IgGi having Asn to Ala substitution at position 297 according to EU numbering.
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3, and a second antigen binding domain that binds to CEA.
  • the anti-CEA/anti-CD3 bispecific antibody further comprises a third antigen binding domain that binds to CEA.
  • the first antigen binding domain binds to a human CD3 polypeptide.
  • the CD3 polypeptide is a human CD3e polypeptide or a human CD3y polypeptide.
  • the CD3 polypeptide is a human CD3e polypeptide.
  • the first antigen binding domain binds to a human CD3e polypeptide or a human CD3 ⁇ polypeptide in native T-cell receptor (TCR) complex in association with other TCR subunits.
  • the first antigen binding domain comprises a heavy chain variable region (V H CD3) and a light chain variable region (V L CD3)
  • the second (and third, if present) antigen binding domain comprises a heavy chain variable region (V H CEA) and a light chain variable region
  • the first antigen binding domain comprises a heavy chain variable region (V H CD3) comprising HVR-H1 sequence of SEQ ID NO:44, HVR-H2 sequence of SEQ ID NO:45, and HVR-H3 sequence of SEQ ID NO:46; and/or a light chain variable region (V L CD3) comprising HVR-L1 sequence of SEQ ID NO:47, HVR-L2 sequence of SEQ ID NO:48, and HVR-L3 sequence of SEQ ID NO:49.
  • V H CD3 heavy chain variable region
  • V L CD3 light chain variable region
  • the first antigen binding domain comprises a heavy chain variable region (V H CD3) comprising the amino acid sequence of SEQ ID NO:50 and/or a light chain variable region (V L CD3) comprising the amino acid sequence of SEQ ID NO:51.
  • V H CD3 heavy chain variable region
  • V L CD3 light chain variable region
  • the second (and third, if present) antigen binding domain comprises a heavy chain variable region (V H CEA) comprising HVR-H1 sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3 sequence of SEQ ID NO:40; and/or a light chain variable region (V L CEA) comprising HVR-L1 sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ ID NO:42, and HVR-L3 sequence of SEQ ID NO:43.
  • V H CEA heavy chain variable region
  • V L CEA light chain variable region comprising HVR-L1 sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ ID NO:42, and HVR-L3 sequence of SEQ ID NO:43.
  • the second (and third, if present) antigen binding domain comprises a heavy chain variable region (V H CEA) comprising the amino acid sequence of SEQ ID NO:34 and/or a light chain variable region (V L CEA) comprising the amino acid sequence of SEQ ID NO:35.
  • V H CEA heavy chain variable region
  • V L CEA light chain variable region
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules.
  • the bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the first antigen binding domain is a crossover Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other) and the second (and third, if present) antigen binding domain is a conventional Fab molecule.
  • the first antigen binding domain is a crossover Fab molecule wherein the constant domains of the Fab heavy and light chain are exchanged.
  • the anti-CEA/anti-CD3 bispecific antibody comprises not more than one antigen binding domain that binds to CD3.
  • the first and the second antigen binding domain are fused to each other, optionally through a peptide linker.
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules and (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain.
  • the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding domain.
  • the anti-CEA/anti-CD3 bispecific antibody further comprises an Fc domain composed of a first and a second subunit capable of stable association.
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules and (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, and the second antigen binding domain is fused at the
  • the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain
  • the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules and (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, the second antigen binding domain is fused at the C-terminus of the C-terminus of the Fab heavy
  • the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain
  • the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain
  • the third antigen binding domain is fused at the C- terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
  • the anti-CEA/anti-CD3 bispecific antibody comprises
  • a second and a third antigen binding domain that bind to CEA comprising a heavy chain variable region (V H CEA) comprising HVR-H1 sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3 sequence of SEQ ID NO:40; and a light chain variable region (V L CEA) comprising HVR-L1 sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ ID NO:42, and HVR-L3 sequence of SEQ ID NO:43, wherein the second and third antigen binding moiety are each a Fab molecule, particularly a conventional Fab molecule;
  • an Fc domain composed of a first and a second subunit capable of stable association, wherein the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
  • the Fc domain comprised in the anti-CEA/anti-CD3 bispecific antibody is an IgG, specifically an IgGi or IgG 4 , Fc domain.
  • the Fc domain is a human Fc domain.
  • the Fc domain is a human IgGi Fc domain.
  • the Fc domain comprised in the anti-CEA/anti-CD3 bispecific antibody comprises a modification promoting the association of the first and the second subunit of the Fc domain.
  • an amino acid residue in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
  • the Fc domain comprised in the anti-CEA/anti-CD3 bispecific antibody exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain.
  • the Fc receptor is an Fey receptor and/or the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
  • said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (EU numbering).
  • each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function wherein said amino acid substitutions are L234A, L235A and P329G (EU numbering).
  • the anti-CEA/anti-CD3 bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 52, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 53, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 54, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55.
  • the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 52, a polypeptide comprising the sequence of SEQ ID NO: 53, a polypeptide comprising the sequence of SEQ ID NO: 54, and a polypeptide comprising the sequence of SEQ ID NO: 55.
  • CEA TCB CEA TCB
  • the anti-CEA/anti-CD3 bispecific antibody is CEA TCB.
  • CEA TCB is administered at a dose of about 5 mg to about 400 mg every week (e.g., about 10 mg to about 60 mg every week, e.g., about 10 mg to about 40 mg every week, e.g. about 80 mg to about 200 mg every week, e.g. about 80 mg to about 400 mg every week, or e.g. about 160 mg to 400 mg every week).
  • CEA TCB is administered at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg about 160 mg, particular at least about 80 mg, every week.
  • the cancer is a CEA-positive cancer.
  • the cancer is colon cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, breast cancer, kidney cancer, esophageal cancer, prostate cancer, or other cancers described herein.
  • the individual has cancer or has been diagnosed with cancer.
  • the individual has locally advanced or metastatic cancer or has been diagnosed with locally advanced or metastatic cancer.
  • the cancer cells in the individual express PD-L1.
  • the expression of PD-L1 may be determined by an immunohistochemistry (IHC) assay.
  • the treatment or administration of the human PD- 1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may result in a response in the individual.
  • the response is a complete response.
  • the response is a sustained response after cessation of the treatment.
  • the response is a complete response that is sustained after cessation of the treatment.
  • the response is a partial response. In some embodiments, the response is a partial response that is sustained after cessation of the treatment.
  • the anti-CEA/anti-CD3 bispecific antibody is administered before the PD-1 axis binding antagonist, simultaneous with the PD-1 axis binding antagonist, or after the PD-1 axis binding antagonist. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody is administered after the PD- 1 axis binding antagonist, particularly at least one hour after the PD-1 axis binding antagonist when they are both administered on the same day.
  • the treatment or administration of the human PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody comprises a dosing regimen comprising treatment cycles, wherein the individual is administered, on day 1 of each cycle, a human PD-1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 antibody at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg about 160 mg, particular at least about 80 mg, each cycle being repeated every 21 days.
  • the cycle is repeated until there is loss of clinical benefit and/or unacceptable toxicity.
  • cycles are repeated in which only the PD- 1 axis binding antagonist or only the anti-CEA/anti-CD3 antibody is administered.
  • the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody are in the same composition. In some embodiments, the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody are in separate composition.
  • the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibody is administered intravenously.
  • the treatment further comprises administering a chemotherapeutic agent for treating or delaying progression of cancer in an individual.
  • the individual has been treated with a chemotherapeutic agent before the combination treatment with the PD- 1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody.
  • the individual treated with the combination of the PD- 1 axis binding antagonist and/or the anti- CEA/anti-CD3 bispecific antibody is refractory to a chemotherapeutic agent treatment.
  • the individual treated with the combination of the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibody is intolerant to a
  • chemotherapeutic agent treatment Some embodiments of the methods, uses, compositions, and kits described throughout the application, further comprise administering a
  • chemotherapeutic agent for treating or delaying progression of cancer.
  • CD8 T cells in the individual have enhanced priming, activation, proliferation and/or cytolytic activity relative to prior to the administration of the combination.
  • the number of CD8 T cells is elevated relative to prior to administration of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 antibody.
  • the CD8 T cell is an antigen-specific CD8 T cell.
  • Treg function is suppressed relative to prior to the administration of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 antibody.
  • T cell exhaustion is decreased relative to prior to the administration of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 antibody.
  • FIG. 1 CEA TCB-mediated lysis of MKN45 cells.
  • the EC50 values of tumor cell killing are: 615 pM (24 h), 362 pM (48 h).
  • Target cell killing was assessed by quantification of LDH released into cell supernatants.
  • FIG. 1 CEA TCB-mediated up-regulation of PD-1 on T cells, and of PD-Ll on surviving tumor cells after tumor cell lysis.
  • PD-1 and PD-Ll expression was analyzed by flow cytometry.
  • CEA TCB was administered IV at doses of 0.5 and 2.5 mg/kg, starting one day (early treatment, panel A) or seven days (late treatment, panel B) after tumor/PBMCs co-grafting and SC injection.
  • Control groups received phosphate-buffer saline (PBS, vehicle), MCSP TCB, or DP47 TCB (as untargeted controls, respectively). In all studies, TCBs were administered twice a week (2q7d). Arrows indicate the day of start of therapy.
  • Figure 4 Histological analysis of tumors after treatment with CEA TCB (co- grafting, E:T 5: 1).
  • Panel B and E correspond to the marked area of panel A and D, respectively.
  • Panel C and F correspond to the marked area of panel B and E, respectively.
  • FIG. 5 Tumor weight after treatment with CEA TCB.
  • LS174T-fluc2 cells injected SC and grown until a volume of 100 mm was reached in NOG mice, followed by IP transfer of human PBMCs (10 x 10 6 cells).
  • mice received bi-weekly IV injections of CEA TCB (2.5 mg/kg), PBS (vehicle), or MCSP TCB (untargeted control, 2.5 mg/kg).
  • Tumor mass was determined at study termination by weighing the explanted tumors.
  • Graphs show average tumor weight and standard error of mean (SEM, 3 ⁇ n ⁇ 6 mice). ** p ⁇ 0.005 using unpaired, two-tailed t-test.
  • Figure 6 Flow cytometry analysis of tumor- infiltrating leukocytes after treatment with CEA TCB. Flow cytometry analysis of tumor tissues collected at termination in the human colon carcinoma xenograft model with IP transfer of human effector cells. The bar plot shows the number of intra-tumor human CD45+/CD3+ T-cells as assessed by flow cytometry in all treated groups. * p ⁇ 0.05 using unpaired, two-tailed t-test.
  • FIG. 7 Immuno PD analysis after treatment with CEA TCB (IP transfer of human PBMCs). Flow cytometry analysis of blood (A-F) and tumor tissues (G-L) collected at Day 18 (corresponding to three days after the third CEA TCB administration) after tumor cell inoculation in the human colon carcinoma xenograft model (IP transfer of human effector cells). Dot plots show the expression of CD69 (B, E, H, K) and PD-1 (C, F, I, L) on CD3 T- cells in both compartments. Representative mice for each group are shown. A-C, G-I:
  • FIG. 8 CEA TCB-induced tumor growth inhibition in fully humanized mice. Tumor growth inhibition of MKN45 in humanized mice. Tumor volumes at study termination (at day 32 after tumor cell injection) are shown. Each point represents an individual animal. CEA TCB was injected IV (2.5 mg/kg twice weekly), and tumor growth was measured by caliper.* p ⁇ 0.05 (unpaired t-test).
  • Figure 9 Increase of intra- tumoral human T-cell frequencies and T-cell activation by CEA TCB.
  • A, B Total human T-cell frequencies (A) and the ratio of CD8+ to CD4+ T- cells (B) in the tumor tissue.
  • C, D The expression of activation markers in tumor is shown as percent of human CD8+ (C) or CD4+ (D) T-cells. Each point represents an individual animal.
  • FIG. 10 Histological analyses of tumor tissues at study termination. Hematoxylin and eosin (H&E, panel A and D) and anti-PD-Ll (panel B, C, E and F) staining (Mab SP142) of vehicle (panel A-C) and CEA TCB (panel D-F) -treated tumors exhibiting strong induction of intra- tumoral PD-L1 expression upon CEA TCB treatment. All sections were
  • Panel B and E correspond to the marked area of panel A and D, respectively.
  • Panel C and F correspond to the marked area of panel B and E, respectively.
  • FIG. 11 In vivo anti-tumor activity upon combination of CEA TCB with anti- human PD-L1 blocking antibody in MKN45 tumor model in fully humanized mice.
  • B Time-to-event statistical analysis. The event was defined as reaching 500 mm tumor volume.
  • FIG. 12 Anti-tumor activity of CEA TCB in the MKN45 model in fully humanized NOG mice.
  • mice were administered either with vehicle, as control, or with CEA TCB (2.5 mg/kg, administered twice a week for 4 weeks, 7
  • mice in the CEA TCB-treated group were randomized into two sub-groups: one continued being administered with CEA TCB (2.5 mg/kg twice a week) and the other was administered with CEA TCB (2.5 mg/kg twice a week) in combination with anti-mouse PD-L1 antibody (10 mg/kg, administered once a week) for 5 weeks (11 administrations in total for CEA TCB and 6 administrations in total for anti-PD-Ll).
  • FIG. 13 Anti-tumor activity of CEA TCB in the MC38-huCEA model in fully immunocompetent huCEA Tg mice.
  • mice were administered either with vehicle, as control, with CEA TCB (2.5 mg/kg, administered twice a week for 4 weeks, 7 administrations in total), with anti-mouse PD-L1 antibody (10 mg/kg, administered once a week for 4 weeks, 4 administrations in total), and with a combination of CEA TCB and anti-mouse PD-L1, administered concomitantly with the respective dose and schedules used in the single therapeutic groups. Treatment was administered for 4 weeks.
  • CEA T cell bispecific antibody and anti-PD-Ll immune therapy act synergistically in their anti-cancer properties and their combination could provide meaningful clinical benefit in patients with cancer.
  • the data in the application show that the combination of a CEA T cell bispecific antibody (CEA TCB) with anti-PD-Ll immune therapy resulted in enhanced inhibition of tumor growth.
  • kits for treating or delaying progression of cancer in an individual comprising administering an effective amount of a human PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
  • compositions and uses for enhancing immune function in an individual having cancer comprising administering an effective amount of a human PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
  • PD-1 axis binding antagonist refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partner, so as to remove T-cell dysfunction resulting from signaling on the PD-1 signaling axis - with a result being to restore or enhance T-cell function (e.g., proliferation, cytokine production, target cell killing).
  • a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-Ll binding antagonist and a PD-L2 binding antagonist.
  • a "human" PD-1 axis binding antagonist refers to a PD- 1 axis binding antagonist which has the above- described effects on the human PD-1 signaling axis.
  • PD-1 binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- 1 with one or more of its binding partners, such as PD-Ll, PD-L2.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to PD-Ll and/or PD-L2.
  • PD-1 binding antagonists include anti-PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins,
  • a PD-1 binding antagonist reduces the negative co- stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • the PD-1 binding antagonist is an anti-PD-1 antibody.
  • a PD-1 binding antagonist is MDX-1106
  • a PD-1 binding antagonist is MK- 3475 (pembrolizumab) described herein.
  • a PD-1 binding antagonist is CT-011 (pidilizumab) described herein.
  • a PD-1 binding antagonist is MEDI-0680 (AMP-514) described herein.
  • a PD-1 binding antagonist is PDR001 described herein.
  • a PD-1 binding antagonist is REGN2810 described herein.
  • a PD-1 binding antagonist is BGB-108 described herein.
  • PD-Ll binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- Ll with either one or more of its binding partners, such as PD-1, B7-1.
  • a PD-Ll binding antagonist is a molecule that inhibits the binding of PD-Ll to its binding partners.
  • the PD-Ll binding antagonist inhibits binding of PD-Ll to PD-1 and/or B7-1.
  • the PD-Ll binding antagonists include anti-PD-Ll antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-Ll with one or more of its binding partners, such as PD-1, B7-1.
  • a PD-Ll binding antagonist reduces the negative co- stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-Ll so as to render a dysfunctional T-cell less dysfunctional ⁇ e.g., enhancing effector responses to antigen recognition).
  • a PD-Ll binding antagonist is an anti-PD-Ll antibody.
  • an anti-PD-Ll antibody is YW243.55.S70 described herein.
  • an anti- PD-Ll antibody is MDX-1105 described herein.
  • an anti-PD- Ll antibody is MPDL3280A (atezolizumab) described herein.
  • an anti-PD-Ll antibody is MDX-1105 described herein.
  • an anti-PD-Ll antibody is YW243.55.S70 described herein.
  • an anti-PD-Ll antibody is MEDI4736 (durvalumab) described herein.
  • an anti-PD-Ll antibody is MSB0010718C (avelumab) described herein.
  • PD-Ll binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- L2 with either one or more of its binding partners, such as PD-1.
  • a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners.
  • the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1.
  • the PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins,
  • a PD-L2 binding antagonist reduces the negative co- stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • a PD-L2 binding antagonist is an immunoadhesin.
  • the term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation.
  • the term includes the common elements of both exhaustion and/or anergy in which antigen recognition may occur, but the ensuing immune response is ineffective to control infection or tumor growth.
  • disfunctional also includes refractory or unresponsive to antigen recognition, specifically, impaired capacity to translate antigen recognition into down-stream T-cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing.
  • the term "anergy” refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T-cell receptor (e.g. increase in intracellular Ca 2+ in the absence of ras-activation). T cell anergy can also result upon stimulation with antigen in the absence of co-stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of costimulation.
  • the unresponsive state can often be overriden by the presence of interleukin-2. Anergic T-cells do not undergo clonal expansion and/or acquire effector functions.
  • exhaustion refers to T cell exhaustion as a state of T cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Exhaustion can result from both extrinsic negative regulatory pathways (e.g.,
  • immunoregulatory cytokines as well as cell intrinsic negative regulatory (co stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).
  • Enhancing T-cell function means to induce, cause or stimulate a T-cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T- cells.
  • enhancing T-cell function include: increased secretion of ⁇ -interferon from CD8 + T-cells, increased proliferation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) relative to such levels before the intervention.
  • the level of enhancement is as least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to one of ordinary skill in the art.
  • a "T cell dysfunctional disorder” is a disorder or condition of T-cells characterized by decreased responsiveness to antigenic stimulation.
  • a T-cell dysfunctional disorder is a disorder that is specifically associated with inappropriate increased signaling through PD-1.
  • a T-cell dysfunctional disorder is one in which T-cells are anergic or have decreased ability to secrete cytokines, proliferate, or execute cytolytic activity.
  • the decreased responsiveness results in ineffective control of a pathogen or tumor expressing an immunogen.
  • T cell dysfunctional disorders characterized by T-cell dysfunction include unresolved acute infection, chronic infection and tumor immunity.
  • Tumor immunity refers to the process in which tumors evade immune
  • tumor immunity is "treated” when such evasion is attenuated, and the tumors are recognized and attacked by the immune system.
  • tumor recognition include tumor binding, tumor shrinkage and tumor clearance.
  • Immunogenicity refers to the ability of a particular substance to provoke an immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the clearance of the tumor cells by the immune response. Examples of enhancing tumor immunogenicity include treatment with a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody.
  • sustained response refers to the sustained effect on reducing tumor growth after cessation of a treatment.
  • the tumor size may remain to be the same or smaller as compared to the size at the beginning of the administration phase.
  • the sustained response has a duration at least the same as the treatment duration, at least 1.5X, 2. OX, 2.5X, or 3. OX length of the treatment duration.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. "Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed. [0076] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.
  • an individual is successfully "treated” if one or more symptoms associated with cancer are mitigated or eliminated, including, but are not limited to, reducing the proliferation of (or destroying) cancerous cells, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging survival of individuals.
  • delay progression of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
  • an "effective amount” is at least the minimum amount required to effect a measurable improvement or prevention of a particular disorder.
  • An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual.
  • An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects.
  • beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival.
  • an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting ⁇ i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit ⁇ i.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • in conjunction with refers to administration of one treatment modality in addition to another treatment modality.
  • in conjunction with refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the individual.
  • a “disorder” is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • cell proliferative disorder and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation.
  • the cell proliferative disorder is cancer.
  • the cell proliferative disorder is cancer.
  • proliferative disorder is a tumor.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include, but not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non- small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, nodular melan
  • intermediate grade/follicular NHL intermediate grade diffuse NHL
  • high grade follicular NHL intermediate grade diffuse NHL
  • immunoblastic NHL high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and
  • ALL lymphoblastic leukemia
  • PTLD post-transplant lymphoproliferative disorder
  • cancers that are amenable to treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma.
  • breast cancer colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma.
  • the cancer is selected from: small cell lung cancer, glioblastoma,
  • the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast carcinoma, including metastatic forms of those cancers.
  • the cancer is a CEA-positive cancer.
  • cytotoxic agent refers to any agent that is detrimental to cells (e.g., causes cell death, inhibits proliferation, or otherwise hinders a cellular function).
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , 1131 , 1125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • radioactive isotopes e.g., At 211 , 1131 , 1125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu
  • chemotherapeutic agents e.g., At 211 , 1131 , 1125 , Y 90
  • Exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid biosynthesis, cell cycle signalling inhibitors, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism.
  • the cytotoxic agent is a taxane.
  • the taxane is paclitaxel or docetaxel.
  • the cytotoxic agent is a platinum agent. In one embodiment the cytotoxic agent is an antagonist of EGFR. In one embodiment the antagonist of EGFR is N-(3-ethynylphenyl)-6,7-bis(2- methoxyethoxy)quinazolin-4-amine (e.g., erlotinib). In one embodiment the cytotoxic agent is a RAF inhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or CRAF inhibitor. In one embodiment the RAF inhibitor is vemurafenib. In one embodiment the cytotoxic agent is a PI3K inhibitor.
  • Chemotherapeutic agent includes compounds useful in the treatment of cancer.
  • chemotherapeutic agents include erlotinib (TARCEVA ® , Genentech/OSI Pharm.), bortezomib (VELCADE ® , Millennium Pharm.), disulfiram, epigallocatechin gallate , salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX ® , AstraZeneca), sunitib (SUTENT ® , Pfizer/Sugen), letrozole (FEMARA ® , Novartis), imatinib mesylate (GLEEVEC ® , Novartis), finasunate (VATALANIB ® , Novartis), oxaliplatin (ELOXATIN ® , Sanofi), 5-FU (5-
  • 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, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine;
  • acetogenins especially bullatacin and bullatacinone
  • a camptothecin including topotecan and irinotecan
  • bryostatin callystatin
  • CC-1065 including its adozelesin, carzelesin and bizelesin synthetic analogs
  • cryptophycins particularly cryptophycin 1 and cryptophycin 8
  • adrenocorticosteroids including prednisone and prednisolone
  • cyproterone acetate 5a- reductases including finasteride and dutasteride
  • vorinostat romidepsin, panobinostat, valproic acid, mocetinostat dolastatin
  • aldesleukin, talc duocarmycin including the synthetic analogs, KW-2189 and CB1-TM1
  • pancratistatin a sarcodictyin
  • spongistatin nitrogen mustards such as chloride
  • neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, ADRIAMYCIN ® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puro
  • methotrexate, pteropterin, trimetrexate purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; dia
  • TAXOL paclitaxel
  • ABRAXANE Cyremophor-free
  • albumin-engineered nanoparticle formulations of paclitaxel American Pharmaceutical Partners, Schaumberg, 111.
  • TAXOTERE docetaxel, doxetaxel; Sanofi-Aventis
  • chlorambucil GEMZAR ® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mit
  • vincristine vincristine; NAVELBINE ® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA ® ); ibandronate; CPT-11 ; topoisomerase inhibitor RFS 2000; difluoromethylorni thine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.
  • NAVELBINE ® vinorelbine
  • novantrone teniposide
  • edatrexate daunomycin
  • aminopterin capecitabine
  • XELODA ® ibandronate
  • CPT-11 topoisomerase inhibitor
  • DMFO difluoromethylorni thine
  • retinoids such as retinoic acid
  • pharmaceutically acceptable salts, acids and derivatives of any of the above pharmaceutically acceptable salts, acids and derivative
  • Chemotherapeutic agent also includes (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX ® ;
  • SERMs selective estrogen receptor modulators
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE ® (megestrol acetate), AROMASIN ® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR ® (vorozole), FEMARA ® (letrozole; Novartis), and AREVIIDEX ® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide,
  • fluoxymesterone all transretionic acid, fenretinide, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
  • protein kinase inhibitors a 1,3-dioxolane nucleoside cytosine analog
  • lipid kinase inhibitors e.g., IL-12 kinase inhibitors
  • antisense oligonucleotides particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras
  • ribozymes such as VEGF expression inhibitors (e.g.
  • vaccines such as gene therapy vaccines, for example, ALLOVECTIN ® , LEUVECTIN ® , and VAXID ® ; PROLEUKIN ® , rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN ® ; ABARELIX ® rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.
  • Chemotherapeutic agent also includes antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen pie), pertuzumab (OMNrfARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin
  • Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, n
  • rovelizumab ruplizumab, sibrotuzumab, siplizumab, thankuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti- interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) which is a recombinant exclusively human- sequence, full-length IgGi ⁇ antibody genetically modified to recognize interleukin-12 p40 protein.
  • ABT-874/J695 Wyeth Research and Abbott Laboratories
  • Chemotherapeutic agent also includes "EGFR inhibitors,” which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an "EGFR antagonist.”
  • EGFR inhibitors refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity
  • Examples of such agents include antibodies and small molecules that bind to EGFR.
  • antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No.
  • EMD 55900 Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)
  • EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF- alpha for EGFR binding (EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as El. l, E2.4, E2.5, E6.2, E6.4, E2.l l, E6. 3 and E7.6.
  • the anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g.,
  • EGFR antagonists include small molecules such as compounds described in US Patent Nos: 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, as well as the following PCT publications: W098/14451,
  • EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA ® Genentech/OSI Pharmaceuticals); PD 183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4- morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca);
  • BIBX-1382 N8-(3-chloro-4-fluoro-phenyl)-N2-(l-methyl-piperidin-4-yl)-pyrimido[5,4- d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(l- phenylethyl)amino]-lH-pyrrolo[2,3-d]pyrimidin-6-yl] -phenol); (R)-6-(4-hydroxyphenyl)-4- [(l-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3- bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4- fluorophenyl)amino]-3-cyano-7-ethoxy-6-
  • Chemotherapeutic agents also include "tyrosine kinase inhibitors" including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan- HER inhibitors such as canertinib (CI- 1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling;
  • PTK787/ZK222584 available from Novartis/Schering AG
  • MAPK extracellular regulated kinase I inhibitor CI-1040 available from Pharmacia
  • quinazolines such as PD 153035,4- (3-chloroanilino) quinazoline
  • pyridopyrimidines such as PD 153035,4- (3-chloroanilino) quinazoline
  • pyridopyrimidines such as pyrimidopyrimidines
  • pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706
  • pyrazolopyrimidines 4-(phenylamino)-7H- pyrrolo[2,3-d] pyrimidines
  • curcumin diiferuloyl methane, 4,5-bis (4- fluoroanilino)phthalimide
  • tyrphostines containing nitrothiophene moieties PD-018380
  • Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprel
  • Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone- 17-butyrate, hydrocortisone- 17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate,
  • ImSAIDs immune selective anti-inflammatory peptides
  • FEG phenylalanine-glutamine-glycine
  • FeG D-isomeric form
  • anti-rheumatic drugs such as azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNFa) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia
  • miscellaneous investigational agents such as thioplatin, PS-341,
  • phenylbutyrate ET- 18- OCH 3 , or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone;
  • polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof
  • autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone;
  • lapachol lapachol
  • colchicines betulinic acid
  • acetylcamptothecin scopolectin
  • Targetedron® bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate
  • SKELID® or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone;
  • COX-2 inhibitor e.g. celecoxib or etoricoxib
  • proteosome inhibitor e.g. PS341
  • CCI-779 e.g. tipifarnib (R11577); orafenib, ABT510
  • Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone;
  • farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASARTM); and
  • CHOP an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone
  • FOLFOX an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin.
  • Chemotherapeutic agents also include non-steroidal anti-inflammatory drugswith analgesic, antipyretic and anti-inflammatory effects.
  • NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase.
  • Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumirac
  • NSAIDs can be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathies, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea, metastatic bone pain, headache and migraine, postoperative pain, mild-to-moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.
  • conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathies, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea, metastatic bone pain, headache and migraine, postoperative pain, mild-to-moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.
  • 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.
  • growth inhibitory agent is growth inhibitory antibody that prevents or reduces proliferation of a cell expressing an antigen to which the antibody binds.
  • the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase. Examples of 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 (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • Those 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.
  • Taxanes are anticancer drugs both derived from the yew tree.
  • 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.
  • radiation therapy is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one-time administration and typical dosages range from 10 to 200 units (Grays) per day.
  • a "subject” or an “individual” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human. An individual or subject may be a patient.
  • antibody herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies ⁇ e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
  • An "isolated' antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other substances.
  • an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site.
  • the constant domain contains the C H I , C H 2 and C H 3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
  • variable region or “variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody.
  • the variable domain of the heavy chain may be referred to as "V R .”
  • the variable domain of the light chain may be referred to as "V L .” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR).
  • HVRs hypervariable regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs 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, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • the "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (" ⁇ ") and lambda (“ ⁇ "), based on the amino acid sequences of their constant domains.
  • IgG immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • antibodies can be assigned to different classes.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
  • full length antibody “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.
  • a "naked antibody” for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.
  • Antibody fragments comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof.
  • the antibody fragment described herein is an antigen-binding fragment.
  • Examples of antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen-binding site.
  • a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer.
  • HVRs confer antigen-binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three HVRs specific for an antigen has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York, 1994), pp. 269-315.
  • diabodies refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
  • 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.
  • a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, 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 target binding sequence is also a monoclonal antibody of this invention.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • 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 invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14 (3): 253-260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to
  • Chimeric antibodies include PRIMATIZED ® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non- human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody.
  • a 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 will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et ah, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • a "species-dependent antibody” is one which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species.
  • the species-dependent antibody "binds specifically" to a human antigen (e.g., has a binding affinity (K D ) value of no more than about 1x10 " M, preferably no more than about 1x10 - " 8 M and preferably no more than about 1x10 - " 9 M) but has a binding affinity for a homologue of the antigen from a second nonhuman mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen.
  • the species-dependent antibody can be any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
  • hypervariable region when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops.
  • antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
  • H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies.
  • HVR delineations are in use and are encompassed herein.
  • the Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • the AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
  • the "contact" HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.
  • HVRs may comprise "extended HVRs” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (HI), 50-65 or 49-65 (H2) and 93- 102, 94-102, or 95-102 (H3) in the VH.
  • the variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.
  • variable domain residue numbering as in Kabat or “amino acid position numbering as in Kabat” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the "EU numbering system” or "EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et ah, supra).
  • the "EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.
  • linear antibodies refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CHl-VH-CHl) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
  • the term “binds”, “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an antibody that binds to or specifically binds to a target is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets.
  • the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA).
  • an antibody that specifically binds to a target has a dissociation constant (Kd) of ⁇ ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • Kd dissociation constant
  • an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species.
  • specific binding can include, but does not require exclusive binding.
  • bispecific means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants.
  • a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.
  • an antigen binding domain refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen.
  • An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions).
  • an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • a "Fab molecule” refers to a protein consisting of the VH and CHI domain of the heavy chain (the "Fab heavy chain”) and the VL and CL domain of the light chain (the "Fab light chain”) of an immunoglobulin.
  • a "crossover" Fab molecule (also termed “Crossfab”) is meant a Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (VL-CH1, in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in N- to C- terminal direction).
  • VL-CH1 variable chain variable domain
  • VH-CL variable chain constant domain
  • crossover Fab molecule Conversely, in a crossover Fab molecule wherein the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.
  • a "conventional" Fab molecule is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant domains (VH-CHl, in N- to C-terminal direction), and a light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction).
  • Fc domain or "Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). This may be the case where the final two C- terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU
  • the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above).
  • a "subunit" of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association.
  • a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
  • fused is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.
  • a "modification promoting heterodimerization” is a manipulation of the peptide backbone or the post-translational modifications of a polypeptide, e.g. an immunoglobulin heavy chain, that reduces or prevents the association of the polypeptide with an identical polypeptide to form a homodimer.
  • a modification promoting heterodimerization as used herein particularly includes separate modifications made to each of two polypeptides desired to form a dimer, wherein the modifications are complementary to each other so as to promote association of the two polypeptides. For example, a modification promoting
  • heterodimerization may alter the structure or charge of one or both of the polypeptides desired to form a dimer so as to make their association sterically or electrostatically favorable, respectively. Heterodimerization occurs between two non-identical polypeptides, e.g. two immunoglobulin heavy chains wherein the variable regions are not the same.
  • the modification promoting heterodimerization comprises an amino acid mutation, specifically an amino acid substitution.
  • the amino acid mutation specifically an amino acid substitution.
  • modification promoting heterodimerization comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two polypeptides desired to form a dimer.
  • an "activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcyRIIIa (CD 16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).
  • effector functions when used in reference to antibodies refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.
  • a method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody. Also provided herein is a method of enhancing immune function in an individual having cancer comprising
  • a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.
  • PD-1 programmeed death 1
  • PDCD1 programmeed cell death 1
  • CD279 a PD-L1 binding antagonist
  • PD-L1 programmeed death ligand 1
  • PDCD1LG1, CD274, B7-H and PDL1.
  • PD-L1 An exemplary human PD-L1 is shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1.
  • PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2", PDCD1LG2, CD273, B7-DC, Btdc, and PDL2.
  • An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot Accession No. Q9BQ51.
  • PD-1, PD- Ll, and PD-L2 are human PD-1, PD-L1 and PD-L2.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners.
  • the PD-1 ligand binding partners are PD-L1 and/or PD-L2.
  • a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners.
  • PD- Ll binding partners are PD-1 and/or B7-1.
  • the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners.
  • a PD-L2 binding partner is PD-1.
  • the antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
  • the anti-PD-1 antibody is selected from the group consisting of MDX 1106 (nivolumab), MK- 3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001,
  • the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PD-1 binding antagonist is AMP-224.
  • the PD-L1 binding antagonist is anti-PD-Ll antibody.
  • the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70,
  • MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab).
  • Antibody YW243.55.S70 is an anti-PD-Ll described in WO 2010/077634.
  • MDX-1105 also known as BMS-936559, is an anti-PD-Ll antibody described in
  • MEDI4736 is an anti-PD-Ll monoclonal antibody described in
  • Nivolumab also known as MDX-1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO ® , is an anti-PD-1 antibody described in WO2006/121168.
  • Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA ® , and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335.
  • CT-011 also known as hBAT, hBAT-1 or pidilizumab, is an anti-PD-1 antibody described in WO2009/101611.
  • AMP-224 also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.
  • the PD-1 axis binding antagonist is an anti-PD-Ll antibody.
  • the anti-PD-Ll antibody is capable of inhibiting binding between PD- Ll and PD-1 and/or between PD-L1 and B7-1.
  • the anti-PD-Ll antibody is a monoclonal antibody.
  • the anti-PD-Ll antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab') 2 fragments.
  • the anti-PD-Ll antibody is a humanized antibody. In some embodiments, the anti-PD-Ll antibody is a human antibody.
  • anti-PD-Ll antibodies useful for the methods, uses, compositions and kits of this invention, and methods for making thereof are described in PCT patent application WO 2010/077634, WO2007/005874, WO2011/066389, and US2013/034559, which are incorporated herein by reference.
  • the anti-PD-Ll antibodies useful in this invention, including compositions containing such antibodies, may be used in combination with an anti-CEA/anti-CD3 bispecific antibody to treat cancer.
  • the anti-PD-1 antibody is MDX-1106.
  • Alternative names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558 or nivolumab.
  • the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).
  • an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO: 1 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO:2.
  • an isolated anti-PD-1 antibody comprising a heavy chain and/or a light chain sequence, wherein:
  • the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence:
  • the light chain sequences has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the light chain sequence:
  • Anti-PD-Ll antibodies described in WO 2010/077634 Al and US 8,217,149 may be used in the methods, uses, compositions and kits described herein.
  • the anti-PD-Ll antibody comprises a heavy chain variable region sequence of SEQ ID NO:3 and/or a light chain variable region sequence of SEQ ID NO:4.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and/or a light chain sequence, wherein:
  • the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSA (SEQ ID NO:3), and
  • the light chain sequences has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the light chain sequence:
  • the anti-PD-Ll antibody comprises a heavy chain variable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3 sequence, wherein:
  • HVR-H1 sequence is GFTFSXiSWIH (SEQ ID NO:5)
  • HVR-H2 sequence is AWK 2 PYGGSX 3 YYADSVKG (SEQ ID NO:6)
  • the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:7) further wherein: Xi is D or G; X 2 is S or L; X 3 is T or S. In one specific aspect, Xi is
  • the polypeptide further comprises variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)- (HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4).
  • the framework sequences are derived from human consensus framework sequences.
  • the framework sequences are VH subgroup III consensus framework.
  • at least one of the framework sequences is the following:
  • HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8)
  • HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO:9)
  • HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 10)
  • HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 11).
  • the heavy chain polypeptide is further combined with a variable region light chain comprising an HVR-L1, HVR-L2 and HVR-L3, wherein:
  • the HVR-L1 sequence is R AS QX 4 X 5 X 6 TX 7 X 8 A (SEQ ID NO: 12)
  • HVR-L2 sequence is SASX 9 LX 10 S, (SEQ ID NO: 13)
  • the HVR-L3 sequence is QQXIIXI 2 XI 3 XI 4 PXI 5 T (SEQ ID NO: 14) wherein: X 4 is D or V; X5 is V or I; X 6 is S or N; X 7 is A or F; Xg is V or L; X9 is F or T; X10 is Y or A; X n is Y, G, F, or S; X i2 is L, Y, F or W; X i3 is Y, N, A, T, G, F or I; X 14 is H, V, P, T or I; X 15 is A, W, R, P or T.
  • the light chain further comprises variable region light chain framework sequences juxtaposed between the HVRs according to the formula: (LC-FR1)- (HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).
  • the framework sequences are derived from human consensus framework sequences.
  • the framework sequences are VL kappa I consensus framework.
  • at least one of the framework sequence is the following:
  • LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15)
  • LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 16)
  • LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17)
  • LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 18).
  • an isolated anti-PD-Ll antibody or antigen binding fragment comprising a heavy chain and a light chain variable region sequence, wherein:
  • the heavy chain comprises and HVR-H1, HVR-H2 and HVR-H3, wherein further:
  • HVR-H1 sequence is GFTFSXiSWIH; (SEQ ID NO:
  • HVR-H2 sequence is AWIX 2 PYGGSX 3 YYADSVKG (SEQ ID NO:
  • the HVR-H3 sequence is RHWPGGFDY, and (SEQ ID NO:
  • the light chain comprises and HVR-L1, HVR-L2 and HVR-L3, wherein further:
  • the HVR-L1 sequence is RASQX 4 X 5 X 6 TX 7 X 8 A (SEQ ID NO: 12)
  • the HVR-L2 sequence is SASX 9 LX 10 S; and (SEQ ID NO: 13)
  • the HVR-L3 sequence is QQXnXi 2 Xi 3 Xi 4 PXi 5 T; (SEQ ID NO: 14)
  • Xi is D
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC-FR1)-(HVR-H1)-(HC-FR2)- (HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FRl)-(HVR-Ll)- (LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10 and 11. In a still further aspect, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4.
  • the human constant region is IgGl.
  • the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3.
  • the murine constant region is IgG2A.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from an "effector- less Fc mutation" or aglycosylation.
  • the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • an anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein:
  • the heavy chain further comprises an HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO:20) and RHWPGGFDY (SEQ ID NO:21), respectively, or
  • the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3
  • sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC-FR1)-(HVR-H1)-(HC-FR2)- (HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FRl)-(HVR-Ll)- (LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10 and 11. In a still further aspect, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4.
  • the human constant region is IgGl.
  • the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3.
  • the murine constant region is IgG2A.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from an "effector- less Fc mutation" or aglycosylation.
  • the effector- less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein:
  • the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
  • the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC- FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)
  • the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence.
  • the heavy chain framework sequence is a VH subgroup III consensus framework.
  • one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10 and
  • the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4.
  • the human constant region is IgGl.
  • the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3.
  • the murine constant region is IgG2A.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from production in prokaryotic cells.
  • the minimal effector function results from an "effector-less Fc mutation" or aglycosylation.
  • the effector-less Fc mutation is an N297A or D265A/N297A
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC-FR1)-(HVR-H1)-(HC-FR2)- (HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FRl)-(HVR-Ll)- (LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences is the following:
  • the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences is the following:
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4.
  • the human constant region is IgGl.
  • the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3.
  • the murine constant region is IgG2A.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from an "effector- less Fc mutation" or aglycosylation.
  • the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • an anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein: (c) the heavy chain further comprises an HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO:20) and RHWPGGFDY (SEQ ID NO:21), respectively, and/or
  • the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3
  • sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC-FR1)-(HVR-H1)-(HC-FR2)- (HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FRl)-(HVR-Ll)- (LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10 and WGQGTLVTVSSASTK (SEQ ID NO:31).
  • the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence.
  • the light chain framework sequences are VL kappa I consensus framework.
  • one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4.
  • the human constant region is IgGl.
  • the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3.
  • the murine constant region is IgG2A.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from an "effector- less Fc mutation" or aglycosylation.
  • the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein:
  • the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:25.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4 and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein:
  • the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:4.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:26.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4 and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:26.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein:
  • the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein:
  • the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
  • the light chain sequences has at least 85% sequence identity to the light chain sequence:
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:33.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:32 or 56.
  • an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:33 and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:32 or 56.
  • an isolated anti- PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:33 and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:32.
  • the isolated anti-PD-Ll antibody is aglycosylated.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X- serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X is any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites form an antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) is removed. The alteration may be made by substitution of an asparagine, serine or threonine residue within the glycosylation site another amino acid residue (e.g., glycine, alanine or a conservative substitution).
  • the isolated anti-PD-Ll antibody can bind to a human PD-L1, for example a human PD-L1 as shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1, or a variant thereof.
  • nucleic acid encoding any of the antibodies described herein.
  • nucleic acid further comprises a vector suitable for expression of the nucleic acid encoding any of the previously described anti-PDLl antibodies.
  • the vector is in a host cell suitable for expression of the nucleic acid.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the eukaryotic cell is a mammalian cell, such as Chinese hamster ovary (CHO) cell.
  • the antibody or antigen binding fragment thereof may be made using methods known in the art, for example, by a process comprising culturing a host cell containing nucleic acid encoding any of the previously described anti-PDLl antibodies or antigen- binding fragment in a form suitable for expression, under conditions suitable to produce such antibody or fragment, and recovering the antibody or fragment.
  • a method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody. Also provided herein is a method of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
  • CEA human carcinoembryonic antigen
  • Alternative names for “CEA” include CEACAM5.
  • CEA refers to any native CEA from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the term encompasses "full-length” and unprocessed CEA as well as any form of CEA that results from processing in the cell (e.g., mature protein).
  • CEA is human CEA.
  • the amino acid sequence of human CEA is shown in UniProt (www.uniprot.org) accession no. P06731, or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004354.2.
  • the antigen binding domain of a bispecific antibody that binds to CEA comprises a heavy chain variable region (V H CEA) comprising the amino acid sequence:
  • V L CEA light chain variable region
  • the antigen binding domain of a bispecific antibody that binds to CEA comprises a heavy chain variable region comprising an HVR-H1 sequence of EFGMN (SEQ ID NO:38), an HVR-H2 sequence of WINTKTGEATYVEEFKG (SEQ ID NO:39), and an HVR-H3 sequence of WDFAYYVEAMDY (SEQ ID NO:40) and a light chain variable region comprising an HVR-L1 sequence of KASAAVGTYVA (SEQ ID NO:41), an HVR-L2 sequence of SASYRKR (SEQ ID NO:42), and an HVR-L3 sequence of HQYYTYPLFT (SEQ ID NO:43).
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds a human CD3 polypeptide, and a second antigen binding domain that binds CEA.
  • the anti-CEA/anti-CD3 bispecific antibody further comprises a third antigen binding domain that binds CEA.
  • the second and the third antigen binding domain are identical (i.e. have the same amino acid sequences)
  • CD3 cluster of differentiation 3
  • T-cell co-receptor is a protein complex and is composed of four distinct chains.
  • the complex contains a CD3y chain, a CD35 chain, and two CD3e chains. These chains associate with the T-cell receptor (TCR) and the ⁇ -chain to generate an activation signal in T lymphocytes.
  • TCR T-cell receptor
  • the TCR, ⁇ -chain, and CD3 molecules together form the TCR complex.
  • CD3 refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated.
  • the term encompasses "full-length” and unprocessed protein as well as any form of the protein or one or more of the CD3 chains (polypeptides) that result from processing in the cell (e.g., mature polypeptides).
  • the term also encompasses naturally occurring variants and isoforms of CD3, e.g., splice variants or allelic variants. For example, descriptions of CD3y, CD35, and CD3e chains and sequences are provided at
  • CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3s).
  • the amino acid sequence of human CD3s is shown in UniProt (www.uniprot.org) accession no. P07766 (version 144), or NCBI
  • the bispecific antibody binds to a human CD3 epsilon (CD3e) polypeptide. In some embodiments, the bispecific antibody binds to a human CD3 epsilon polypeptide in native T-cell receptor (TCR) complex in association with other TCR subunits. In some embodiments, the bispecific antibody binds to a human CD3 gamma (CD3y) polypeptide. In some embodiments, the bispecific antibody binds a human CD3 gamma polypeptide in native T-cell receptor (TCR) complex in association with other TCR subunits.
  • assays are provided for identifying anti-CD3 antibodies thereof having biological activity.
  • Biological activity may include, for example, binding to a CD3 polypeptide (e.g., CD3 on the surface of a T cell), or a peptide fragment thereof, either in vivo, in vitro, or ex vivo.
  • biological activity may also include, for example, effector cell activation (e.g., T cell (e.g., CD8+ and/or CD4+ T cell) activation), effector cell population expansion (i.e., an increase in T cell count), target cell population reduction (i.e., a decrease in the population of cells expressing CEA on their cell surfaces), and/or target cell killing.
  • effector cell activation e.g., T cell (e.g., CD8+ and/or CD4+ T cell) activation
  • effector cell population expansion i.e., an increase in T cell count
  • target cell population reduction i.e., a decrease in the population of cells expressing CEA on their cell surfaces
  • target cell killing i.e., a decrease in the population of cells expressing CEA on their cell surfaces
  • Antibodies having such biological activity in vivo and/or in vitro are provided. In certain embodiments, an antibody of the invention is tested for such biological activity.
  • the antigen binding domain(s) of a bispecific antibody that binds to a CEA comprises a heavy chain variable region (V H CEA) comprising the amino acid sequence of SEQ ID NO:34, and a light chain variable region (V L CEA) comprising the amino acid sequence of SEQ ID NO:35.
  • the antigen binding domain(s) that binds to a CEA comprises a heavy chain variable region comprising an HVR-H1 sequence of EFGMN (SEQ ID NO:38), an HVR-H2 sequence of WINTKTGEATYVEEFKG (SEQ ID NO:39), and an HVR-H3 sequence of WDFAYYVEAMDY (SEQ ID NO:40) and comprises a light chain variable region comprising an HVR-L1 sequence of KASAAVGTYVA (SEQ ID NO:41), an HVR-L2 sequence of SASYRKR (SEQ ID NO:42), and an HVR-L3 sequence of HQYYTYPLFT (SEQ ID NO:43).
  • the antigen binding domain of a bispecific antibody that binds to a CD3 comprises a heavy chain variable region (V H CD3) comprising the amino acid sequence of SEQ ID NO:50, and a light chain variable region (V L CD3) comprising the amino acid sequence of SEQ ID NO:51.
  • V H CD3 heavy chain variable region
  • V L CD3 light chain variable region
  • the antigen binding domain that binds to a CD3 comprises a heavy chain variable region comprising an HVR-H1 sequence of TYAMN (SEQ ID NO:44), an HVR-H2 sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO:45), and an HVR-H3 sequence of HGNFGNSYVSWFAY (SEQ ID NO:46) and comprises a light chain comprising an HVR-L1 sequence of GSSTGAVTTSNYAN (SEQ ID NO:47), an HVR-L2 sequence of GTNKRAP (SEQ ID NO:48), and an HVR-L3 sequence of ALWYSNLWV (SEQ ID NO:49).
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules (e.g. conventional or crossover Fab molecules).
  • the bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the first antigen binding domain is a crossover Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e.
  • the second (and third, if present) antigen binding domain is a conventional Fab molecule.
  • the first antigen binding domain is a crossover Fab molecule wherein the constant domains of the Fab heavy and light chain are exchanged.
  • the anti-CEA/anti-CD3 bispecific antibody comprises not more than one antigen binding domain that binds to CD3, i.e. provides monovalent binding to CD3.
  • the first and the second antigen binding domain are fused to each other, optionally through a peptide linker.
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules and (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain.
  • the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding domain.
  • the Fab light chain of the first antigen binding domain and the Fab light chain of the second antigen binding domain may additionally be fused to each other.
  • the anti-CEA/anti-CD3 bispecific antibody further comprises an Fc domain composed of a first and a second subunit capable of stable association.
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3, a second antigen binding domain that binds to CEA, and an Fc domain composed of a first and a second subunit capable of stable association, wherein the antigen binding domains are Fab molecules and wherein (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second anti
  • the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding domain
  • the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
  • the anti-CEA/anti-CD3 bispecific antibody essentially consists of a first antigen binding domain that binds to CD3, a second antigen binding domain that binds to CEA, an Fc domain composed of a first and a second subunit capable of stable association, and optionally one or more peptide linkers, wherein the antigen binding domains are Fab molecules and wherein (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, and the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N
  • the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3, a second and a third antigen binding domain that binds to CEA, and an Fc domain composed of a first and a second subunit capable of stable association, wherein the antigen binding domains are Fab molecules and wherein (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain
  • the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain
  • the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain
  • the third antigen binding domain is fused at the C- terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
  • the anti-CEA/anti-CD3 bispecific antibody essentially consists of a first antigen binding domain that binds to CD3, a second and a third antigen binding domain that binds to CEA, an Fc domain composed of a first and a second subunit capable of stable association, and optionally one or more peptide linkers, wherein the antigen binding domains are Fab molecules and wherein (i) the second antigen binding domain is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus
  • the anti-CEA/anti-CD3 bispecific antibody comprises
  • a second and a third antigen binding domain that bind to CEA comprising a heavy chain variable region (V H CEA) comprising HVR-H1 sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3 sequence of SEQ ID NO:40; and a light chain variable region (V L CEA) comprising HVR-L1 sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ ID NO:42, and HVR-L3 sequence of SEQ ID NO:43, wherein the second and third antigen binding moiety are each a Fab molecule, particularly a conventional Fab molecule;
  • an Fc domain composed of a first and a second subunit capable of stable association, wherein the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
  • the Fab molecules may be fused to the Fc domain or to each other directly or through a peptide linker, comprising one or more amino acids, typically about 2-20 amino acids.
  • Peptide linkers are known in the art and are described herein.
  • Suitable, non- immunogenic peptide linkers include, for example, (G 4 S) n , (SG 4 ) n , (G 4 S) n or G 4 (SG 4 ) n peptide linkers, "n" is generally an integer from 1 to 10, typically from 2 to 4.
  • said peptide linker has a length of at least 5 amino acids, in one embodiment a length of 5 to 100, in a further embodiment of 10 to 50 amino acids.
  • said peptide linker is (G 4 S) 2 .
  • a particularly suitable peptide linker for fusing the Fab light chains of the first and the second antigen binding domain to each other is (G 4 S) 2 .
  • An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and the second antigen binding domain comprises the sequence (D)-(G 4 S) 2 .
  • Another suitable such linker comprises the sequence (G 4 S) 4 .
  • linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where a Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.
  • the anti-CEA/anti-CD3 bispecific antibody comprises (i) a polypeptide wherein the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e.
  • the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with a first Fc domain subunit (VH(i ) -CHl(i)- VH(2 ) -CL(2 ) -CH2-CH3(-CH4)), (ii) a crossover Fab light chain polypeptide of the second Fab molecule, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ), (iii) the Fab light chain polypeptide of the first Fab molecule (VL(i)- CL(i)), (iv) a polypeptide wherein the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with a second Fc domain subunit (VH (3) -
  • the Fab light chain polypeptide of the first and the third Fab molecule are identical. In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond. In certain embodiments, the first and the third Fab molecule bind to CEA, and the second Fab molecule binds to CD3.
  • the anti-CEA/anti-CD3 bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 52, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 53, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 54, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55.
  • the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 52, a polypeptide comprising the sequence of SEQ ID NO: 53, a polypeptide comprising the sequence of SEQ ID NO: 54, and a polypeptide comprising the sequence of SEQ ID NO: 55.
  • the anti-CEA/anti-CD3 bispecific antibody used in the present invention may comprise an Fc domain which consists of a pair of polypeptide chains comprising heavy chain domains of an antibody molecule.
  • Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains.
  • the two subunits of the Fc domain are capable of stable association with each other.
  • the Fc domain is an IgG Fc domain.
  • the Fc domain is an IgGi Fc domain.
  • the Fc domain is an IgG 4 Fc domain.
  • the Fc domain is an IgG 4 Fc domain comprising an amino acid substitution at position S228 (EU numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG 4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).
  • the Fc domain is human. An exemplary sequence of a human IgGi Fc region is given in SEQ ID NO: 57.
  • the anti-CEA/anti-CD3 bispecific antibody used in the present invention may comprise different components (e.g. antigen binding domains) fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of such antibodies in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antibody a modification promoting the association of the desired polypeptides.
  • different components e.g. antigen binding domains
  • the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain.
  • the site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.
  • said modification is in the CH3 domain of the Fc domain.
  • the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed).
  • said modification promoting the association of the first and the second subunit of the Fc domain is a so-called "knob-into-hole” modification, comprising a "knob” modification in one of the two subunits of the Fc domain and a "hole” modification in the other one of the two subunits of the Fc domain.
  • knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
  • the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • an amino acid residue in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
  • amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).
  • amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the "hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V).
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (EU numbering).
  • the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (EU numbering).
  • S354C cysteine residue
  • E356C glutamic acid residue at position 356
  • the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (EU numbering).
  • the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W
  • the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (EU numbering).
  • the antigen binding domain that binds to CD3 in the anti-CEA/anti-CD3 bispecific antibody described herein is fused to the first subunit of the Fc domain (comprising the "knob" modification).
  • fusion of the CD3 binding domain to the knob-containing subunit of the Fc domain will (further) minimize the generation of bispecific antibodies comprising two CD3 binding domains (steric clash of two knob-containing polypeptides).
  • the heterodimerization approach described in EP 1870459 Al is used alternatively.
  • This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain.
  • One preferred embodiment are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (EU numbering).
  • the antibody comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (EU numbering).
  • the antibody comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to EU numbering).
  • a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (EU numbering).
  • the first CH3 domain comprises further amino acid mutation L351K.
  • the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (EU numbering).
  • the heterodimerization approach described in WO 2012/058768 is used alternatively.
  • a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F.
  • the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g.
  • T411N, T411R, T411Q, T41 IK, T41 ID, T41 IE or T411W b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (EU numbering).
  • a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F.
  • a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F.
  • the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (EU numbering).
  • heterodimerization approach described in WO 2011/143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (EU numbering).
  • a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A.
  • a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (EU numbering).
  • the antibody or its Fc domain is of IgG 2 subclass and the heterodimerization approach described in WO 2010/129304 is used alternatively.
  • a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004.
  • this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
  • a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g.
  • the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K409D or R409D).
  • the first CH3 domain further or alternatively comprises amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to EU numbering).
  • a negatively charged amino acid e.g. glutamic acid (E), or aspartic acid (D)
  • EU numbering all numberings according to EU numbering.
  • a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (EU numbering).
  • the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D
  • the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (EU numbering).
  • the Fc domain confers to an antibody, such as a bispecific antibody, favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the antibody to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with other immuno stimulatory properties the antibody may have and the long half-life of the antibody, results in excessive activation of cytokine receptors and severe side effects upon systemic administration.
  • the Fc domain of the anti-CEA/anti-CD3 bispecific antibody exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain.
  • the Fc domain or the molecule, e.g.
  • antibody comprising said Fc domain
  • the Fc domain or the molecule, e.g.
  • the Fc receptor is an Fey receptor.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
  • the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC.
  • the Fc domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc domain domain.
  • FcRn neonatal Fc receptor
  • Substantially similar binding to FcRn is achieved when the Fc domain (or the molecule, e.g. antibody, comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or the corresponding molecule comprising a native IgGi Fc domain) to FcRn.
  • the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain.
  • the Fc domain comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function.
  • the same one or more amino acid mutation is present in each of the two subunits of the Fc domain.
  • the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor.
  • the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5- fold, or at least 10-fold.
  • the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold.
  • the molecule, e.g. antibody, comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a corresponding molecule comprising a non-engineered Fc domain.
  • the Fc receptor is an Fey receptor.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
  • binding to each of these receptors is reduced.
  • binding affinity to a complement component, specifically binding affinity to Clq is also reduced.
  • binding affinity to neonatal Fc receptor (FcRn) is not reduced.
  • Substantially similar binding to FcRn i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the molecule, e.g. antibody, comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or a corresponding molecule comprising said non-engineered form of the Fc domain) to FcRn.
  • the Fc domain, or molecule e.g.
  • the Fc domain is engineered to have reduced effector function, as compared to a non-engineered Fc domain.
  • the reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody- dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming.
  • the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a corresponding molecule comprising a non-engineered Fc domain).
  • the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution.
  • the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (EU numbering).
  • the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (EU numbering).
  • the Fc domain comprises the amino acid substitutions L234A and L235A (EU numbering).
  • the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain.
  • the Fc domain comprises an amino acid substitution at position P329.
  • the amino acid substitution is P329A or P329G, particularly P329G (EU numbering).
  • the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (EU numbering).
  • the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S.
  • the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 EU numbering).
  • the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA").
  • the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain.
  • the "P329G LALA" combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
  • WO 2012/130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.
  • the Fc domain is an IgG 4 Fc domain, particularly a human IgG 4 Fc domain.
  • the IgG 4 Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P (EU numbering).
  • the IgG 4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (EU numbering).
  • the IgG 4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (EU
  • the IgG 4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (EU numbering).
  • IgG 4 Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
  • the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain is a human IgGi Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG 4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (EU numbering).
  • N-glycosylation of the Fc domain has been eliminated.
  • the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) or glycine (N297G) (EU numbering).
  • Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (EU numbering).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.
  • Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression.
  • SPR Surface Plasmon Resonance
  • binding affinity of Fc domains or molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.
  • Effector function of an Fc domain, or a molecule (e.g. an antibody) comprising an Fc domain can be measured by methods known in the art.
  • a suitable assay for measuring ADCC is described herein.
  • Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499- 1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g. in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).
  • binding of the Fc domain to a complement component, specifically to Clq is reduced.
  • said reduced effector function includes reduced CDC.
  • Clq binding assays may be carried out to determine whether the Fc domain, or molecule (e.g. antibody) comprising the Fc domain, is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045- 1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).
  • the bispecific antibody is a single-chain bispecific antibody comprising the first antigen binding domain and the second antigen binding domain.
  • the single-chain bispecific antibody comprises variable regions, as arranged from N-terminus to C-terminus, selected from the group consisting of (1) V H CEA-V L CEA- V H CD3-V L CD3, (2) V H CD3-V L CD3-V H CEA-V L CEA, (3) V H CD3-V L CD3-V L CEA-V H CEA, (4) V H CEA-V L CEA-V L CD3-V H CD3, (5) V L CEA-V H CEA-V H CD3-V L CD3, and (6) V L CD3- V H CD3-V H CEA-V L CEA.
  • Antibodies described herein are prepared using techniques available in the art for generating antibodies, exemplary methods of which are described in more detail in the following sections.
  • the antibody is directed against an antigen of interest (i.e., PD-L1 (such as a human PD-L1), CEA, or CD3 (such as a human CD3)).
  • an antigen of interest i.e., PD-L1 (such as a human PD-L1), CEA, or CD3 (such as a human CD3).
  • the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disorder can result in a therapeutic benefit in that mammal.
  • an antibody provided herein has a dissociation constant (K D ) of ⁇ ⁇ , ⁇ 150 nM, ⁇ 100 nM, ⁇ 50 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 "8 M or less, e.g. from 10 "8 M to 10 "13 M, e.g., from 10 "9 M to 10 "13 M).
  • K D dissociation constant
  • K D is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay.
  • RIA radiolabeled antigen binding assay
  • Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).
  • MICROTITER ® multi-well plates (Thermo Scientific) are coated overnight with 5 ⁇ g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C).
  • a non-adsorbent plate (Nunc #269620)
  • pM or 26 pM [ I] -antigen are mixed with serial dilutions of a Fab of interest.
  • the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20
  • K D is measured using surface plasmon resonance assays using a BIACORE ® -2000 or a BIACORE ® -3000 (BIAcore, Inc.,
  • CM5 chips Piscataway, NJ
  • immobilized antigen CM5 chips at -10 response units (RU).
  • carboxymethylated dextran biosensor chips CM5, BIACORE, Inc.
  • EDC iV-ethyl-iV'- (3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N- hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml (-0.2 ⁇ ) before injection at a flow rate of 5 ⁇ /minute to achieve approximately 10 response units (RU) of coupled protein.
  • Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for generating antibodies.
  • immunogens for transmembrane molecules, such as receptors, fragments of these (e.g. the extracellular domain of a receptor) can be used as the immunogen.
  • transmembrane molecules such as receptors
  • fragments of these e.g. the extracellular domain of a receptor
  • cells expressing the transmembrane molecule can be used as the immunogen.
  • Such cells can be derived from a natural source (e.g. cancer cell lines) or may be cells which have been transformed by recombinant techniques to express the transmembrane molecule.
  • Other antigens and forms thereof useful for preparing antibodies will be apparent to those in the art.
  • Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example,
  • Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions.
  • aggregating agents such as alum are suitably used to enhance the immune response.
  • Monoclonal antibodies can be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), and further described, e.g., in Hongo et al, Hybridoma, 14 (3): 253-260 (1995), Harlow et ah, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et ah, in: Monoclonal
  • Human hybridoma technology (Trioma technology) is described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).
  • a mouse or other appropriate host animal such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • Antibodies are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of the invention or a fragment thereof, and an adjuvant, such as monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.).
  • MPL monophosphoryl lipid A
  • TDM trehalose dicrynomycolate
  • polypeptide of the invention e.g., antigen
  • polypeptide of the invention may be prepared using methods well known in the art, such as recombinant methods, some of which are further described herein. Serum from immunized animals is assayed for anti-antigen antibodies, and booster immunizations are optionally administered. Lymphocytes from animals producing anti-antigen antibodies are isolated. Alternatively, lymphocytes may be immunized in vitro.
  • Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
  • a suitable fusing agent such as polyethylene glycol
  • Myeloma cells may be used that fuse efficiently, support stable high-level production of antibody by the selected antibody- producing cells, and are sensitive to a medium such as HAT medium.
  • Exemplary myeloma cells include, but are not limited to, murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif.
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium, e.g., a medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium e.g., a medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • serum-free hybridoma cell culture methods are used to reduce use of animal- derived serum such as fetal bovine serum, as described, for example, in Even et al. , Trends in Biotechnology, 24(3), 105-108 (2006).
  • Oligopeptides as tools for improving productivity of hybridoma cell cultures are described in Franek, Trends in Monoclonal Antibody Research, 111-122 (2005). Specifically, standard culture media are enriched with certain amino acids (alanine, serine, asparagine, proline), or with protein hydrolyzate fractions, and apoptosis may be significantly suppressed by synthetic oligopeptides, constituted of three to six amino acid residues. The peptides are present at millimolar or higher concentrations.
  • Culture medium in which hybridoma cells are growing may be assayed for production of monoclonal antibodies that bind to an antibody of the invention.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoadsorbent assay
  • the binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis. See, e.g., Munson et al., Anal. Biochem., 107:220 (1980).
  • hybridoma cells After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. See, e.g., Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • One procedure for isolation of proteins from hybridoma cells is described in US 2005/176122 and U.S. Pat. No. 6,919,436.
  • the method includes using minimal salts, such as lyotropic salts, in the binding process and preferably also using small amounts of organic solvents in the elution process.
  • Antibodies useful in the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Additional methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.
  • phage display methods repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol, 12: 433-455 (1994). Phage typically display antibody fragments, either as single- chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • PCR polymerase chain reaction
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993).
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
  • Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,
  • Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
  • an antibody provided herein is a chimeric antibody.
  • chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • HVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol, 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci.
  • an antibody provided herein is a human antibody.
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous
  • Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol, 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci.
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
  • Antibody fragments may be generated by traditional means, such as enzymatic digestion, or by recombinant techniques. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9: 129-134.
  • F(ab') 2 fragments can be isolated directly from recombinant host cell culture.
  • Fab and F(ab') 2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No.
  • an antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458.
  • Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use.
  • scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See Antibody Engineering, ed. Borrebaeck, supra.
  • the antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibodies may be monospecific or bispecific.
  • Multispecific antibodies have binding specificities for at least two different epitopes, where the epitopes are usually from different antigens. While such molecules normally will only bind two different epitopes (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies).
  • immunoglobulin heavy chain-light chain pairs where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J, 10:3655-3659 (1991).
  • immunoglobulin polypeptides to associate may be engineered such that a "knob” or “protuberance” (these terms may be used interchangeably herein) located in the interface of one immunoglobulin polypeptide corresponds with a "hole” or “cavity” (these terms may be used interchangeably herein) located in the interface of the other
  • the hole is of identical or similar size to the knob and suitably positioned such that when the two interfaces interact, the knob of one interface is positionable in the corresponding hole of the other interface. Without wishing to be bound to theory, this is thought to stabilize the heteromultimer and favor formation of the heteromultimer over other species, for example homomultimers. In some embodiments, this approach may be used to promote the heteromultimerization of two different immunoglobulin polypeptides, creating a bispecific antibody comprising two immunoglobulin polypeptides with binding specificities for different epitopes.
  • a knob may be constructed by replacing a small amino acid side chain with a larger side chain.
  • a hole may be constructed by replacing a large amino acid side chain with a smaller side chain.
  • Knobs or holes may exist in the original interface, or they may be introduced synthetically.
  • knobs or holes may be introduced synthetically by altering the nucleic acid sequence encoding the interface to replace at least one "original" amino acid residue with at least one "import” amino acid residue. Methods for altering nucleic acid sequences may include standard molecular biology techniques well known in the art. The side chain volumes of various amino acid residues are shown in the following table.
  • original residues have a small side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and import residues for forming a knob are naturally occurring amino acids and may include arginine, phenylalanine, tyrosine, and tryptophan.
  • original residues have a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), and import residues for forming a hole are naturally occurring amino acids and may include alanine, serine, threonine, and valine.
  • Trp Tryptophan
  • original residues for forming a knob or hole are identified based on the three-dimensional structure of the heteromultimer.
  • Techniques known in the art for obtaining a three-dimensional structure may include X-ray crystallography and NMR.
  • the interface is the CH3 domain of an immunoglobulin constant domain.
  • the CH3/CH3 interface of human IgGi involves sixteen residues on each domain located on four anti-parallel ⁇ -strands.
  • mutated residues are preferably located on the two central anti-parallel ⁇ -strands to minimize the risk that knobs can be accommodated by the surrounding solvent, rather than the compensatory holes in the partner CH3 domain.
  • the mutations forming corresponding knobs and holes in two immunoglobulin polypeptides correspond to one or more pairs provided in the following table.
  • F405W T394S Mutations are denoted by the original residue, followed by the position using the EU numbering system, and then the import residue (all residues are given in single-letter amino acid code). Multiple mutations are separated by a colon.
  • an immunoglobulin polypeptide comprises a CH3 domain comprising one or more amino acid substitutions listed in Table 2 above.
  • a bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising one or more amino acid substitutions listed in the left column of Table 2, and a second immunoglobulin polypeptide comprising a CH3 domain comprising one or more corresponding amino acid substitutions listed in the right column of Table 2.
  • polynucleotides encoding modified immunoglobulin polypeptides with one or more corresponding knob- or hole- forming mutations may be expressed and purified using standard recombinant techniques and cell systems known in the art. See, e.g., U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; U.S. Pub. No. 2013/0089553; and Spiess et al., Nature Biotechnology 31: 753-758, 2013.
  • Modified immunoglobulin polypeptides may be produced using prokaryotic host cells, such as E.
  • knob- and hole-bearing immunoglobulin polypeptides may be expressed in host cells in co-culture and purified together as a heteromultimer, or they may be expressed in single cultures, separately purified, and assembled in vitro.
  • two strains of bacterial host cells one expressing an immunoglobulin polypeptide with a knob, and the other expressing an immunoglobulin polypeptide with a hole
  • the two strains may be mixed in a specific ratio, e.g., so as to achieve equal expression levels in culture.
  • the two strains may be mixed in a 50:50, 60:40, or 70:30 ratio.
  • the cells may be lysed together, and protein may be extracted.
  • Standard techniques known in the art that allow for measuring the abundance of homo-multimeric vs. hetero-multimeric species may include size exclusion chromatography.
  • each modified immunoglobulin polypeptide is expressed separately using standard recombinant techniques, and they may be assembled together in vitro.
  • Assembly may be achieved, for example, by purifying each modified immunoglobulin polypeptide, mixing and incubating them together in equal mass, reducing disulfides (e.g., by treating with dithiothreitol), concentrating, and reoxidizing the polypeptides.
  • Formed bispecific antibodies may be purified using standard techniques including cation-exchange chromatography and measured using standard techniques including size exclusion chromatography. For a more detailed description of these methods, see Spiess et al., Nat Biotechnol 31:753-8, 2013.
  • modified immunoglobulin polypeptides may be expressed separately in CHO cells and assembled in vitro using the methods described above.
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is typical to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions.
  • CHI first heavy-chain constant region
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • One interface comprises at least a part of the C H 3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end- products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytic ally cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light- chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • bispecific T cell engager or BiTE® approach (see, e.g., WO2004/ 106381, WO2005/061547,
  • a single polypeptide chain includes two single chain Fv (scFv) fragments, each having a variable heavy chain (V H ) and a variable light chain (V L ) domain separated by a polypeptide linker of a length sufficient to allow intramolecular association between the two domains.
  • This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments.
  • Each scFv recognizes a different epitope, and these epitopes may be specific for different cell types, such that cells of two different cell types are brought into close proximity or tethered when each scFv is engaged with its cognate epitope.
  • One particular embodiment of this approach includes a scFv recognizing a cell-surface antigen expressed by an immune cell, e.g., a CD3 polypeptide on a T cell, linked to another scFv that recognizes a cell-surface antigen expressed by a target cell, such as a malignant or tumor cell.
  • the bispecific T cell engager may be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., a CHO cell line.
  • specific purification techniques see, e.g., EP1691833 may be necessary to separate monomeric bispecific T cell engagers from other multimeric species, which may have biological activities other than the intended activity of the monomer.
  • a solution containing secreted polypeptides is first subjected to a metal affinity chromatography, and polypeptides are eluted with a gradient of imidazole
  • This eluate is further purified using anion exchange chromatography, and polypeptides are eluted using with a gradient of sodium chloride concentrations. Finally, this eluate is subjected to size exclusion chromatography to separate monomers from multimeric species.
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60 (1991).
  • a single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 Bl).
  • a single-domain antibody consists of all or a portion of the heavy chain variable domain of an antibody.
  • amino acid sequence modification(s) of the antibodies described herein are contemplated.
  • Amino acid sequence variants of the antibody may be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made.
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the HVRs and FRs.
  • Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions.” More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Table 3. Exemplary Substitutions.
  • Amino acids may be grouped according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody.
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
  • Alterations may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • HVR "hotspots” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)
  • SDRs a-CDRs
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR "hotspots" or SDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
  • Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
  • oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants comprising an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function.
  • antibodies are contemplated herein that have reduced fucose relative to the amount of fucose on the same antibody produced in a wild-type CHO cell. That is, they are characterized by having a lower amount of fucose than they would otherwise have if produced by native CHO cells (e.g., a CHO cell that produce a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene).
  • the antibody is one wherein less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon comprise fucose.
  • the amount of fucose in such an antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the antibody is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the antibody is completely without fucose, or has no fucose or is afucosylated.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about + 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function.
  • Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys.
  • knockout cell lines such as alpha-1,6- fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al.
  • Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by
  • Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean- Mairet et al.); US Patent No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5): 851-861 (2006). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • the antibody variants comprising an Fc region described herein are capable of binding to an FcyRIII. In certain embodiments, the antibody variants comprising an Fc region described herein have ADCC activity in the presence of human effector cells or have increased ADCC activity in the presence of human effector cells compared to the otherwise same antibody comprising a human wild-type IgGlFc region,
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 ® non-radioactive cytotoxicity assay (Promega, Madison, WI).
  • 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 may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'lAcad. Sci.
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101: 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759- 1769 (2006)).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • the antibody comprising the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.
  • alterations are made in the Fc region that result in altered ⁇ i.e., either improved or diminished) Clq binding and/or Complement Dependent
  • Cytotoxicity e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
  • the antibodies used in the invention can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody are water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol,
  • polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • Antibodies may also be produced using recombinant methods.
  • nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the antibody may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • 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.
  • An antibody may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (e.g., 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 of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, a factor leader (including Saccharomyces and Kluyveromyces a-factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646.
  • mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, are available.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria, the 2 ⁇ , plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter.
  • Selection genes may contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • 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 antibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • cells transformed with the DHFR gene are identified by culturing the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other co- transformed nucleic acid.
  • Mtx methotrexate
  • a Chinese hamster ovary (CHO) cell line deficient in endogenous DHFR activity e.g., ATCC CRL-9096 may be used.
  • cells transformed with the GS gene are identified by culturing the transformants in a culture medium containing L-methionine sulfoximine (Msx), an inhibitor of GS. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acid.
  • the GS selection/amplification system may be used in combination with the DHFR selection/amplification system described above.
  • host cells particularly wild-type hosts that contain endogenous DHFR transformed or co-transformed with DNA sequences encoding an antibody of interest, wild-type DHFR gene, and another selectable marker such as aminoglycoside 3'- phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.
  • APH aminoglycoside 3'- phosphotransferase
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et ah, Nature, 282:39 (1979)).
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting
  • Lew2-deficient yeast strains ATCC 20,622 or 38,626 are complemented by known plasmids bearing the Leul gene.
  • vectors derived from the 1.6 ⁇ circular plasmid pKDl can be used for transformation of Kluyveromyces yeasts.
  • an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8: 135 (1990).
  • Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
  • Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding an antibody.
  • Promoters suitable for use with prokaryotic hosts include the phoA promoter, ⁇ -lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter.
  • trp tryptophan
  • Other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding an antibody.
  • S.D. Shine-Dalgarno
  • Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde- 3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • Yeast enhancers also are advantageously used with yeast promoters.
  • Antibody transcription from vectors in mammalian host cells can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), or from hetero
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment.
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978.
  • Enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a- fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100- 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will 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 antibody is polyadenylated fragments in the untranslated portion of the mRNA encoding antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See W094/11026 and the expression vector disclosed therein.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B.
  • Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g., Salmonella typhimurium
  • Serratia e.g., Serratia marcesc
  • subtilis and B. licheniformis e.g., B. licheniformis 4 IP disclosed in DD 266,710 published 12 Apr. 1989
  • Pseudomonas such as P. aeruginosa
  • Streptomyces One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • Full length antibody, antibody fusion proteins, and antibody fragments can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) that by itself shows effectiveness in tumor cell destruction.
  • a cytotoxic agent e.g., a toxin
  • Full length antibodies have greater half-life in circulation. Production in E. coli is faster and more cost efficient.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., U.S. Pat. No.
  • coli cell paste in a soluble fraction can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g., in CHO cells.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K.
  • thermotolerans ⁇ ⁇ . marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • yeasts and filamentous fungi for the production of therapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22: 1409-1414 (2004).
  • Certain fungi and yeast strains may be selected in which glycosylation pathways have been "humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describing humanization of the glycosylation pathway in Pichia pastoris); and Gerngross et al., supra.
  • Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa calif ornica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed ⁇ Leninaceae), alfalfa (M. truncatula), and tobacco can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing
  • PLANTIBODIESTM technology for producing antibodies in transgenic plants
  • Vertebrate cells may be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals NY. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • CHO Chinese hamster ovary
  • DHFR " CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and Sp2/0.
  • CHO Chinese hamster ovary
  • myeloma cell lines such as NS0 and Sp2/0.
  • Yazaki and Wu Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268.
  • Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce an antibody of this invention may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody can be produced intracellularly, in the periplasmic space, 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. Carter et ah, Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • 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.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being among one of the typically preferred purification steps.
  • affinity chromatography is among one of the typically preferred purification steps.
  • 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 ⁇ , ⁇ 2, or ⁇ 4 heavy chains (Lindmark et ah, J. Immunol. Meth. 62: 1-13 (1983)).
  • Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et ah, EMBO J. 5: 15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a C H 3 domain
  • the Bakerbond ABXTM resin J. T. Baker, Phillipsburg, N.J.
  • Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin
  • polyaspartic acid column chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
  • Antibodies produced as described above may be subjected to one or more
  • biological activity assays to select an antibody with beneficial properties from a therapeutic perspective or selecting formulations and conditions that retain biological activity of the antibody.
  • the antibody may be tested for its ability to bind the antigen against which it was raised.
  • methods known in the art such as ELISA, Western Blot, etc.
  • the antigen binding properties of the antibody can be evaluated in an assay that detects the ability to bind to PD-L1.
  • the binding of the antibody may be determined by saturation binding; ELISA; and/or competition assays (e.g. RIA's), for example.
  • the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic.
  • biological activity assays are known in the art and depend on the target antigen and intended use for the antibody.
  • the biological effects of PD-L1 blockade by the antibody can be assessed in CD8+T cells, a lymphocytic choriomeningitis virus (LCMV) mouse model and/or a syngeneic tumor model e.g., as described in US Patent 8,217,149.
  • LCMV lymphocytic choriomeningitis virus
  • epitope mapping e.g. as described in Champe et al., J. Biol. Chem. 270: 1388- 1394 (1995), can be performed to determine whether the antibody binds an epitope of interest.
  • compositions and formulations comprising a PD-1 axis binding antagonist and/or an antibody described herein (such as an anti-PD-Ll antibody, or a bispecific antibody that binds CEA and CD3) and a pharmaceutically acceptable carrier.
  • an antibody described herein such as an anti-PD-Ll antibody, or a bispecific antibody that binds CEA and CD3
  • a pharmaceutically acceptable carrier such as an anti-PD-Ll antibody, or a bispecific antibody that binds CEA and CD3
  • compositions and formulations as described herein can be prepared by mixing the active ingredients (e.g. a PD-1 axis binding antagonist and/or an anti- CEA/anti-CD3 bispecific antibody) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine;
  • preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol
  • Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20
  • insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20
  • sHASEGPs and methods of use including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • Aqueous antibody formulations include those described in US Patent No.
  • composition and formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example,
  • microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in
  • Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. IV. Methods of Treatment
  • kits for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
  • the treatment results in a response in the individual after treatment.
  • the response is a partial response.
  • the response is a complete response.
  • the treatment results in a sustained response (e.g., a sustained partial response or complete response) in the individual after cessation of the treatment.
  • the methods described herein may find use in treating conditions where enhanced
  • immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer.
  • methods of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of a PD-1 axis binding antagonist (e.g., MPDL3280A) and an anti-CEA/anti-CD3 bispecific antibody (e.g. CEA TCB).
  • a PD-1 axis binding antagonist e.g., MPDL3280A
  • an anti-CEA/anti-CD3 bispecific antibody e.g. CEA TCB
  • the methods provided herein include administration of an effective amount of a PD- 1 axis binding antagonist selected from the group consisting of a PD-1 binding antagonist, a PD-Ll binding antagonist, and a PD-L2 binding antagonist.
  • the PD-Ll binding antagonist is an antibody, such as an antibody that is capable of inhibiting PD-Ll binding to PD-1 and B7.1, but does not disrupt binding of PD-1 to PD-L2.
  • the PD-Ll binding antagonist antibody is MPDL3280A, which may be administered at a dose of about 800 mg to about 1500 mg every three weeks (e.g., about 1000 mg to about 1300 mg every three weeks, e.g., about 1100 mg to about 1200 mg every three weeks). In some embodiments, MPDL3280A is administered at a dose of about 1200 mg every three weeks.
  • the therapeutically effective amount of a PD-1 axis binding antagonist may be administered to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight whether by one or more administrations.
  • the antagonist e.g., anti-PD-Ll antibody, e.g., MPDL3280A
  • the antagonist is administered in a dose of about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example.
  • the antagonist e.g., anti-PD- Ll antibody, e.g., MPDL3280A
  • a PD-1 axis binding antagonist e.g., anti-PD- Ll antibody, e.g., MPDL3280A
  • a PD-1 axis binding antagonist is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg.
  • a PD-1 axis binding antagonist e.g., anti-PD-Ll antibody, e.g., MPDL3280A
  • a PD-1 axis binding antagonist e.g., anti-PD-Ll antibody, e.g., MPDL3280A
  • the dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions.
  • the dose of the antibody administered in a combination treatment may be reduced as compared to a single treatment.
  • the method for treating or delaying progression of cancer in an individual comprises a dosing regimen comprising treatment cycles, wherein the individual is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist (e.g., anti-PD-Ll antibody, e.g.,
  • MPDL3280A at a dose of about 1200 mg, wherein each cycle is 21 days (i.e., each cycle is repeated every 21 days). The progress of this therapy is easily monitored by conventional techniques.
  • the methods provided herein include administration of an effective amount of an anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB).
  • the anti-CEA/anti-CD3 bispecific antibody e.g., CEA TCB
  • the anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) is administered at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg or about 160 mg, every week.
  • the therapeutically effective amount of an anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) administered to a human will be in the range of about 5 to about 400 mg (e.g., about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg, about 160 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg), whether by one or more administrations.
  • an anti-CEA/anti-CD3 bispecific antibody e.g., CEA TCB
  • the therapeutically effective amount of an anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) administered to a human will be in the range of about 5 to about 400 mg (e.g., about 5 mg
  • CEA TCB is administered at 5 mg, 10 mg, 20 mg, about 40 mg, about 80 mg or about 160 mg, of CEA TCB.
  • CEA TCB is administered at 5 mg, 10 mg, 20 mg, 40 mg, 80 mg or 160 mg, once a week.
  • at least about 80 mg of CEA TCB is administered.
  • CEA TCB is administered at 80 mg once a week.
  • CEA TCB is administered at 80-200 mg, 80-400 mg or 160-400 mg once a week.
  • the anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) may be administered weekly, every 2 weeks, every 3 weeks, every 4 weeks, on days 1, 8 and 15 of each 21-day cycle, or on days 1, 8, and 15 of each 28-day cycle.
  • the PD-1 axis binding antagonist e.g., an anti-PD-Ll antibody, e.g., MPDL3280A
  • the anti-CEA/anti-CD3 bispecific antibody e.g., CEA TCB
  • the administration of these agents may be concurrent or separate within the context of the dosing regimen.
  • the methods provided herein include a dosing regimen comprising treatment cycles, wherein the individual is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecific antibody at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg or about 160 mg, each cycle being repeated every 21 days.
  • the individual is administered, on days 1 of each cycle, a human PD- 1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecific antibody at a dose of about 80 mg, each cycle being repeated every 21 days.
  • the individual is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecific antibody at a dose of at least about 80 mg, each cycle being repeated every 21 days.
  • the individual is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecific antibody at a dose of about 80 mg to about 200 mg. about 80 mg to about 400 mg, or about 160 mg to about 400 mg, each cycle being repeated every 21 days.
  • the individual is a human. In some embodiments, the individual is suffering from locally advanced or metastatic breast cancer. In some embodiments, the individual has CEA positive cancer. In some embodiments, CEA positive cancer is colon cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, breast cancer, kidney cancer, esophageal cancer, or prostate cancer. In a particular embodiment, the cancer is colorectal carcinoma. In some
  • the breast cancer is a breast carcinoma or a breast adenocarcinoma.
  • the breast carcinoma is an invasive ductal carcinoma.
  • the lung cancer is a lung adenocarcinoma.
  • the colon cancer is a colorectal adenocarcinoma.
  • the cancer cells in the individual express PD-L1.
  • the cancer cells in the individual express CEA protein at a level that is detectable (e.g., detectable using methods known in the art).
  • the individual has been treated with a cancer therapy before the combination treatment with a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
  • the individual has cancer that is resistant to one or more cancer therapies.
  • resistance to cancer therapy includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment.
  • resistance to a cancer therapy includes progression of the cancer during treatment with the anti-cancer therapy.
  • resistance to a cancer therapy includes cancer that does not response to treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, the cancer is at early stage or at late stage.
  • the combination therapy of the invention comprises administration of a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
  • the PD- 1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may be administered in any suitable manner known in the art.
  • the PD- 1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may be administered sequentially (at different times) or concurrently (at the same time).
  • the PD-1 axis binding antagonist is in a separate composition as the anti-CEA/anti-CD3 bispecific antibody.
  • the PD-1 axis binding antagonist is in the same composition as the anti-CEA/anti-CD3 bispecific antibody.
  • the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may be administered by the same route of administration or by different routes of administration.
  • the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally,
  • the anti-CEA/anti-CD3 bispecific antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • An effective amount of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may be administered for prevention or treatment of disease.
  • the appropriate dosage of the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibody may be determined based on the type of disease to be treated, the type of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
  • the methods may further comprise an additional therapy.
  • the additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a
  • the additional therapy may be in the form of adjuvant or neoadjuvant therapy.
  • the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent.
  • the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.).
  • the additional therapy is radiation therapy.
  • the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting PI3K/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent. The additional therapy may be one or more of the chemotherapeutic agents described herein.
  • Also provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual a human PD-1 axis binding antagonist (e.g., anti-PD-Ll antibody, e.g., MPDL3280A) and an anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) in conjunction with another anti-cancer agent or cancer therapy.
  • a human PD-1 axis binding antagonist e.g., anti-PD-Ll antibody, e.g., MPDL3280A
  • an anti-CEA/anti-CD3 bispecific antibody e.g., CEA TCB
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a chemotherapy or
  • a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with a radiation therapy or radiotherapeutic agent.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a targeted therapy or targeted therapeutic agent.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an immunotherapy or immunotherapeutic agent, for example a monoclonal antibody.
  • enhancing T cell stimulation by promoting an activating co- stimulatory molecule or by inhibiting a negative co-stimulatory molecule, may promote tumor cell death thereby treating or delaying progression of cancer.
  • a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against an activating co- stimulatory molecule.
  • an activating co-stimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127.
  • the agonist directed against an activating co- stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against an inhibitory co-stimulatory molecule.
  • an inhibitory co- stimulatory molecule may include CTLA-4 (also known as CD 152), PD-1, TIM-3, BTLA, VISTA, LAG- 3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.
  • the antagonist directed against an inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, PD-1, TEVI-3, BTLA, VISTA, LAG- 3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against CTLA-4 (also known as CD152), e.g., a blocking antibody.
  • a PD-1 axis binding antagonist may be administered in conjunction with ipilimumab (also known as MDX-010, MDX-101, or YERVOY®).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with tremelimumab (also known as ticilimumab or CP-675,206).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against B7-H3 (also known as CD276), e.g., a blocking antibody.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with MGA271.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against a TGF beta, e.g., metelimumab (also known as CAT- 192), fresolimumab (also known as GC1008), or LY2157299.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment comprising adoptive transfer of a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment comprising adoptive transfer of a T cell comprising a dominant- negative TGF beta receptor, e.g, a dominant- negative TGF beta type II receptor.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment comprising a HERCREEM protocol (see, e.g., ClinicalTrials.gov Identifier NCT00889954).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or ILA), e.g., an activating antibody.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with urelumab (also known as BMS-663513).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against CD40, e.g., an activating antibody.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with CP-870893.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against OX40 (also known as CD134), e.g., an activating antibody.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an anti-OX40 antibody (e.g., AgonOX).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against CD27, e.g., an activating antibody.
  • a PD-1 axis binding antagonist may be administered in conjunction with CDX-1127.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against indoleamine-2,3- dioxygenase (IDO).
  • IDO indoleamine-2,3- dioxygenase
  • with the IDO antagonist is 1-methyl-D- tryptophan (also known as 1-D-MT).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody-drug conjugate.
  • the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE).
  • MMAE monomethyl auristatin E
  • a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with and anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599).
  • DNIB0600A or RG7599 anti-NaPi2b antibody-MMAE conjugate
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYLA®, Genentech).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with DMUC5754A.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), e.g., an antibody directed against EDNBR conjugated with MMAE.
  • EDNBR endothelin B receptor
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an angiogenesis inhibitor.
  • a PD-1 axis binding antagonist may be administered in conjunction with an antibody directed against a VEGF, e.g., VEGF-A.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with bevacizumab (also known as AVASTIN®, Genentech).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody directed against angiopoietin 2 (also known as Ang2).
  • angiopoietin 2 also known as Ang2
  • a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with MED 13617.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antineoplastic agent.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agent targeting CSF-IR (also known as M-CSFR or CD115).
  • a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody may be administered in conjunction with anti-CSF-lR (also known as IMC-CS4).
  • a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody may be administered in conjunction with an interferon, for example interferon alpha or interferon gamma.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with Roferon-A (also known as recombinant Interferon alpha-2a).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or Leukine®).
  • GM-CSF also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or Leukine®.
  • a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with IL-2 (also known as aldesleukin or Proleukin®).
  • IL-12 also known as aldesleukin or Proleukin®
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody targeting CD20.
  • the antibody targeting CD20 is obinutuzumab (also known as GA101 or Gazyva®) or rituximab.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody targeting GITR.
  • the antibody targeting GITR is TRX518.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a cancer vaccine.
  • the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine.
  • the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide cocktail, a hybrid peptide, or a peptide- pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104: 14-21, 2013).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an adjuvant.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment comprising a TLR agonist, e.g., Poly-ICLC (also known as Hiltonol®), LPS, MPL, or CpG ODN.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with tumor necrosis factor (TNF) alpha.
  • TNF tumor necrosis factor
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with IL-1.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with HMGB1.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an IL-10 antagonist.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an IL-4 antagonist.
  • a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with an IL-13 antagonist.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an HVEM antagonist.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an ICOS agonist, e.g., by administration of ICOS-L, or an agonistic antibody directed against ICOS.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment targeting CX3CL1. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment targeting CXCL9. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment targeting CXCL10. In some embodiments, a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment targeting CCL5.
  • a PD-1 axis binding antagonist may be administered in conjunction with an LFA-1 or ICAMl agonist.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a Selectin agonist.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a targeted therapy.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of B-Raf.
  • a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with vemurafenib (also known as Zelboraf®).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with dabrafenib (also known as Tafinlar®).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with erlotinib (also known as Tarceva®).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of a MEK, such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with cobimetinib (also known as GDC-0973 or XL-518).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with trametinib (also known as Mekinist®).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of K-Ras.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of c-Met.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with onartuzumab (also known as MetMAb).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of Alk.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with AF802 (also known as CH5424802 or alectinib).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of a phosphatidylinositol 3- kinase (PI3K).
  • PI3K phosphatidylinositol 3- kinase
  • a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody may be administered in conjunction with BKM120.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with idelalisib (also known as GS-1101 or CAL-101).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with perifosine (also known as KRX-0401).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of an Akt.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with MK2206.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with GSK690693.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with GDC-0941. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of mTOR. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with sirolimus (also known as rapamycin). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with temsirolimus (also known as CCI-779 or Torisel®).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with everolimus (also known as RAD001).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with ridaforolimus (also known as AP-23573, MK-8669, or deforolimus).
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with OSI-027.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with AZD8055.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with INK128. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a dual PBK/mTOR inhibitor. In some embodiments, a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with XL765. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with GDC-0980.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with BEZ235 (also known as NVP-BEZ235).
  • BEZ235 also known as NVP-BEZ235
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with BGT226.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with
  • a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody may be administered in conjunction with PF-04691502.
  • a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with PF-05212384 (also known as PKI-587).
  • an article of manufacture or a kit comprising a PD-1 axis binding antagonist and/or an anti-CEA/anti-CD3 bispecific antibody.
  • the article of manufacture or kit further comprises package insert comprising instructions for suing the PD- 1 axis binding antagonist in conjunction with an anti-CEA/anti-CD3 bispecific antibody to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer.
  • Any of the PD- 1 axis binding antagonist and/or an anti-CEA/anti-CD3 bispecific antibodies described herein may be included in the article of manufacture or kits.
  • the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody are in the same container or separate containers.
  • Suitable containers include, for example, bottles, vials, bags and syringes.
  • the container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy).
  • the container holds the formulation and the label on, or associated with, the container may indicate directions for use.
  • the article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent).
  • another agent e.g., a chemotherapeutic agent, and anti-neoplastic agent.
  • Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
  • Example 1 CEA TCB-mediated tumor lysis leads to up-regulation of PD-1 on human T cells and of PD-L1 on surviving tumor cells in vitro
  • CEA TCB is a novel T cell bispecific antibody targeting the carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells. It is currently being investigated as a single agent in a Phase I study in patients with advanced and /or metastatic CEA expressing tumors.
  • PBMCs were isolated from a buffy coat obtained from the "Blutspende Zurich”. Blood was diluted 3: 1 with PBS. About 30 ml of the blood/PBS mixture was layered on 15 ml of Histopaque and centrifuged for 30 min at 450 g without brake at room temperature (RT).
  • Lymphocytes were collected with a 10 ml pipette into 50 ml tubes containing PBS and filled up to 50 ml with PBS and centrifuged 10 min at 350 g. The supernatant was discarded, the pellet re-suspended in 50 ml PBS and centrifuged for 10 min at 300 g. The washing step was repeated once. Cells were re-suspended in RPMI containing 10% FCS and 1% GlutaMax and stored at 37°C, 5% C0 2 in the incubator until the initiation of the assay.
  • Adherent MKN-45 target cells were harvested with Trypsin/EDTA one day before the assay, washed and plated at density of 1.4 mio cells/well using flat-bottom 24- well plates. Cells were left to adhere overnight in a humidified incubator. On the day of the assay, plates were centrifuged at 350 g for 5 min and the medium was aspirated. 550 ⁇ per well of assay medium (RPMI1640, 2% FCS, 1% GlutaMax) were added.
  • CEA TCB or untargeted TCB were added at indicated concentrations (range of 6.4 pM - 100 nM, in triplicates).
  • PBMCs were added to target cells at the final EffectonTarget ratio E:T 10: 1, final volume per well was 1.1 ml.
  • Figure 2 displays a dose-dependent up-regulation of PD-1 receptor on CD8+ (A) and on CD4+ (B) T cells as well as of PD-L1 on tumor cells that resisted to killing (C) and were harvested after 48 h of incubation.
  • Example 2 CEA TCB-mediated tumor lysis leads to up-regulation of PD-1 on human T cells and of PD-L1 on surviving tumor cells in vivo
  • Nonclinical in vivo pharmacology studies were conducted using a human colon carcinoma xenograft tumor model (LS174T-fluc2) in immunodeficient NOG mice. Two different experimental settings were used: 1) tumor cells were co-grafted with human PBMCs (subcutaneous [SC]) at different E:T ratios, and 2) tumor cells were injected SC followed by intraperitoneal (IP) transfer of PBMCs once tumors were established (reached a palpable mass).
  • SC subcutaneous
  • IP intraperitoneal
  • CEA TCB The in vivo efficacy of CEA TCB was also evaluated in xenograft models, in which tumor cells were injected SC followed by IP transfer of PBMCs once tumors reached a palpable mass.
  • CEA TCB (2.5 mg/kg IV bi-weekly) led to significant tumor regression (reduction of tumor weight, Figure 5) and to an increase in T-cell infiltration into tumors (flow cytometry analysis ( Figure 6) and histology (not shown)), with T-cells having activated phenotype (as shown by flow cytometry analysis indicating upregulation of CD25 and CD69 activation markers, not shown). This was in contrast to the vehicle (PBS) and MCSP TCB (untargeted control), where only a low number of T-cells infiltrated tumors and showed a resting phenotype.
  • PBS vehicle
  • MCSP TCB untargeted control
  • CEA TCB The antitumor activity of CEA TCB was assessed in vivo in a humanized mouse model bearing the CEA-expressing MKN45 cancer cell line subcutaneously.
  • CEA TCB was injected IV (2.5 mg/kg, twice/week) on Day 11 after tumor cell injection, and tumor growth was measured twice weekly by caliper.
  • Figure 8 shows individual tumor volumes at study termination; a statistically significant (p ⁇ 0.05) difference in tumor volume was found.
  • CD8+ T-cells revealed an up-regulation of the intracellular Granzyme B (GZB) expression as well as a higher proliferation rate (Ki-67 marker).
  • GZB Granzyme B
  • Ki-67 marker a higher proliferation rate
  • the immunological changes triggered by CEA TCB treatment were tumor- specific indicating that the cross-linking and activation of human T-cells occurs exclusively in CEA expressing tumors and not in other areas that are negative for CEA like blood and spleen.
  • Example 3 Assessment of in vivo anti-tumor activity upon combination of CEA TCB with anti-PD-Ll blocking antibody in gastric carcinoma mouse model
  • An anti-mouse PD-Ll surrogate antibody based on the YW243.55.S70 PD-Ll antibody described in WO 2010/077634 was generated for use in vivo tumor models.
  • This antibody contained a DAPG mutation to abolish FcyR interaction.
  • the variable region of YW243.55.S70 was attached to a murine IgGl constant domain with DAPG Fc mutations.
  • polypeptide sequences of YW243.55.S70 PD-Ll muIgGl are as follows: YW243.55.S70 PD-Ll muIgGl DAPG HC (SEQ ID NO:36):
  • mice were randomized in four groups: the first group received phosphate-buffer saline (PBS, vehicle) as control, the second group received CEA TCB (at the dose of 2.5 mg/kg, administered twice a week for 7 weeks, 15 administrations in total), the third group received anti-PD-Ll antibody (at the dose of 10 mg/kg, administered once a week for 7 weeks, 8 administrations in total), and the fourth group received a combination of CEA TCB and anti-PD-Ll antibody, administered concomitantly with the respective dose and schedules used in the single therapeutic groups for 7 weeks (8 administrations of anti-PD-Ll antibody 10 mg/kg given once a week and 15 administrations of CEA TCB 2.5 mg/kg given twice a week).
  • PBS phosphate-buffer saline
  • CEA TCB at the dose of 2.5 mg/kg, administered twice a week for 7 weeks, 15 administrations in total
  • anti-PD-Ll antibody at the dose of 10 mg/kg, administered once a week for
  • a second study was designed to assess whether, after a first line treatment with CEA TCB monotherapy, the combination of CEA TCB with a-PD-Ll could exert superior anti-tumor activity than continuing treatment with CEA TCB as monotherapy.
  • mice 1 xlO 6 MKN45 tumor cells were injected sub-cutaneously in fully humanized NOG mice. 10 days after tumor cell injection, mice received either phosphate- buffer saline (PBS, vehicle) as control, or CEA TCB (at the dose of 2.5 mg/kg, administered twice a week for 4 weeks, 7 administrations in total).
  • PBS phosphate- buffer saline
  • CEA TCB at the dose of 2.5 mg/kg, administered twice a week for 4 weeks, 7 administrations in total.
  • CEA TCB- treated mice were randomized into two sub-groups: one continued receiving CEA TCB administration (2.5 mg/kg, twice a week), the other received CEA TCB (2.5 mg/kg, twice a week) in combination with anti-PD-Ll antibody (10 mg/kg, once a week) for 5 weeks (11 administrations in total for CEA TCB, and 6 administrations in total for anti-PD-Ll antibody).
  • Example 4 Assessment of in vivo anti-tumor activity upon combination of CEA TCB with anti-PD-Ll blocking antibody in colorectal carcinoma mouse model
  • mice were randomized in four groups for first line treatment: the first group received phosphate-buffer saline (PBS, vehicle) as control, the second group received CEA TCB (at the dose of 2.5 mg/kg, administered twice a week for 4 weeks, 7 administrations in total), the third group received an anti-mouse PD-L1 antibody (at the dose of 10 mg/kg, administered once a week for 4 weeks, 4 administrations in total), and the fourth group received a combination of CEA TCB and anti- PD-L1 antibody, administered concomitantly with the respective dose and schedules used in the single therapeutic groups for 4 weeks (4 administrations of anti-PD-Ll 10 mg/kg given once a week and 7
  • mice were left untreated for 5 weeks.
  • first line- treated mice plus two groups of naive huCEA Tg mice were injected sub- cutaneously with 0.5 xlO 6 MC38-huCEA tumor cell lines on the opposite flank.
  • No therapy injection was applied to any of the groups, while tumor growth was monitored. The results show that in all first line treated mice, tumor protection upon tumor rechallenge was observed, with significant tumor control as compared to naive mice.
  • Example 5 An open-label, multi-center, dose escalation and expansion Phase IB study to evaluate the safety, pharmacokinetics and therapeutic activity of CEA TCB in combination with atezolizumab in patients with locally advanced and/or metastatic CEA-positive solid tumors
  • the starting dose is 5 mg administered on a QW schedule, to be administered at least one hour after the administration of 1200 mg of atezolizumab when they are both administered on the same day (Day 1 of each cycle).
  • Key eligibility criteria include confirmed locally advanced and/or metastatic solid tumor in patients who have progressed on a standard therapy, are intolerant to standard therapy, and/or are non-amenable to standard therapy; Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) 0-1; and locally confirmed CEA expression in tumor tissue ( > 50% of tumor cells staining with at least moderate to high intensity of CEA expression for all solid tumors with exception of NSCLC > 20% of tumor cells staining with at least moderate to high intensity of CEA expression) or centrally confirmed CEA expression.
  • EOG Eastern Cooperative Oncology Group
  • PS Performance Status
  • Tumor response is evaluated according to RECIST vl .1 and modified RECIST criteria using unidimensional measurement such as computed tomography (CT) scan or magnetic resonance imaging.
  • CT computed tomography
  • the combination treatment was at current dose levels (up to 160 mg CEA TCB QW + 1200 mg atezolizumab Q3W) safe and well tolerated. No patients were discontinued due to a treatment related safety event.
  • Atezolizumab act synergistically in their anti-cancer properties and collectively their combination could provide meaningful clinical benefit in patients with cancer.

Abstract

The invention provides compositions and methods for treating CEA-positive cancers. The method comprising administering a PD-1 axis binding antagonist and a bispecific antibody that targets CEA and CD3.

Description

METHODS OF TREATING CEA-POSITIVE CANCERS USING PD-1 AXIS BINDING ANTAGONISTS AND ANTI- CEA/ANTI- CD3 BISPECIFIC ANTIBODIES
FIELD OF THE INVENTION
[0001] This invention relates to methods of treating CEA -positive cancers by administering a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
BACKGROUND OF THE INVENTION
[0002] Cancer is one of the leading causes of death worldwide. Despite advances in treatment options, prognosis of patients with advanced cancer remains poor. Consequently, there is a persisting and urgent medical need for optimal therapies to increase survival of cancer patients without causing unacceptable toxicity.
[0003] Recent results from clinical trials have shown that immune therapies, particularly immune checkpoint inhibitors, can extend the overall survival of cancer patients and lead to durable responses. Despite these promising results, current immune-based therapies are only effective in a proportion of patients and combination strategies are needed to improve therapeutic benefit.
[0004] Programmed death-ligand 1 (PD-Ll) is found on the surface of immune and tumor cells and its expression is induced by interferon gamma (IFNy). It prevents the immune system from destroying cancer cells by interacting with the inhibitory programmed death- 1 (PD-1) and B7.1 receptors on activated T cells, which results in a T-cell inhibitory signal.
[0005] As a result, therapeutic targeting of PD- 1 and other molecules which signal through interactions with PD-1, such as programmed death-ligand 1 (PD-Ll) and programmed death ligand 2 (PD-L2) are an area of intense interest. PD-Ll is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813) (Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, the majority of tumor infiltrating T lymphocytes predominantly express PD-1, in contrast to T lymphocytes in normal tissues and peripheral blood T lymphocytes indicating that up-regulation of PD-1 on tumor-reactive T cells can contribute to impaired antitumor immune responses (Blood 2009 114(8): 1537). This may be due to exploitation of PD-Ll signaling mediated by PD-Ll expressing tumor cells interacting with PD-1 expressing T cells to result in attenuation of T cell activation and evasion of immune surveillance (Sharpe et al., Nat Rev 2002) (Keir ME et al., 2008 Annu. Rev. Immunol. 26:677). Therefore, inhibition of the PD-Ll/PD-1 interaction may enhance CD8+ T cell-mediated killing of tumors.
[0006] The inhibition of PD-L1 signaling has been proposed as a means to enhance T cell immunity for the treatment of cancer (e.g., tumor immunity) and infection, including both acute and chronic (e.g., persistent) infection. An optimal therapeutic treatment may combine blockade of PD-1 receptor/ligand interaction with an agent that activates T cells, particularly CD8+ T cells.
[0007] Dual blockade of PD-1 and PD-L1 has been reported to improve T cell killing of tumor directed by CEA BiTE in an in vitro model (Osada et al., Cancer Immunol
Immunother (2015) 64(6):677-88).
[0008] There remains a need for optimal therapies for treating, stabilizing, preventing, and/or delaying development of various cancers in patients.
[0009] All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot Accession numbers are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.
SUMMARY OF THE INVENTION
[0010] In one aspect, provided herein is a method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a human PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
[0011] In another aspect, provided herein is a method of enhancing immune function in an individual having cancer comprising administering an effective amount of a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
[0012] In another aspect, provided herein is use of a human PD- 1 axis binding antagonist in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising an anti- CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier.
[0013] In another aspect, provided herein is use of an anti-CEA/anti-CD3 bispecific antibody in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.
[0014] In another aspect, provided herein is a composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises
administration of said composition in combination with a second composition, wherein the second composition comprises an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier.
[0015] In another aspect, provided herein is a composition comprising an anti-CEA/anti- CD3 bispecific antibody and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises administration of said composition in combination with a second composition, wherein the second composition comprises a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.
[0016] In another aspect, provided herein is a kit comprising a medicament comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the medicament in combination with a composition comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.
[0017] In another aspect, provided herein is a kit comprising a first medicament comprising a PD- 1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a second medicament comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier. In some embodiments, the kit further comprises a package insert comprising instructions for administration of the first medicament and the second medicament for treating or delaying progression of cancer in an individual.
[0018] In another aspect, provided herein is a kit comprising a medicament comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the medicament in combination with a composition comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.
[0019] In some embodiments of the methods, uses, compositions, and kits described above and herein, the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist. In some embodiments, the PD-1 axis binding antagonist is an antibody. In some embodiments, the antibody is a humanized antibody, a chimeric antibody or a human antibody. In some embodiments, the antibody is an antigen binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, and Fv.
[0020] In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is selected from the group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108.
[0021] In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD- Ll to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD- Ll to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD- Ll to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an anti- PD-L1 antibody. In some embodiments, the PD-L1 binding antagonist is selected from the group consisting of: MPDL3280A (atezolizumab), YW243.55.S70, MDX- 1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). In some embodiments, the anti-PD-Ll antibody is MPDL3280A (atezolizumab). In some embodiments, MPDL3280A is administered at a dose of about 800 mg to about 1500 mg every three weeks (e.g., about 1000 mg to about 1300 mg every three weeks, e.g., about 1100 mg to about 1200 mg every three weeks). In some embodiments, MPDL3280A is administered at a dose of about 1200 mg every three weeks. In some embodiments, the anti-PD-Ll antibody comprises a heavy chain comprising HVR-Hl sequence of SEQ ID NO: 19, HVR-H2 sequence of SEQ ID NO:20, and HVR-H3 sequence of SEQ ID NO:21; and/or a light chain comprising HVR-L1 sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ ID NO:24. In some embodiments, the anti-PD-Ll antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 or 26 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:4. In some
embodiments, the anti-PD-Ll antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the anti-PD-Ll antibody comprises the three heavy chain HVR sequences of antibody YW243.55.S70 and/or the three light chain HVR sequences of antibody YW24355.S70 described in WO 2010/077634 and U.S. Patent No. 8,217,149, which are incorporated herein by reference. In some
embodiments, the anti-PD-Ll antibody comprises the heavy chain variable region sequence of antibody YW243.55.S70 and/or the light chain variable region sequence of antibody YW24355.S70.
[0022] In some embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2 binding antagonist is an antibody. In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.
[0023] In some embodiments, the PD-1 axis binding antagonist is an antibody (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-PD-L2 antibody) and comprises an aglycosylation site mutation. In some embodiments, the aglycosylation site mutation is a substitution mutation. In some embodiments, the substitution mutation is at amino acid residue N297, L234, L235, and/or D265 (EU numbering). In some embodiments, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, and D265A. In some embodiments, the substitution mutation is a D265A mutation and an N297G mutation. In some embodiments, the aglycosylation site mutation reduces effector function of the antibody. In some embodiments, the PD-1 axis binding antagonist (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-PD-L2 antibody) is a human IgGi having Asn to Ala substitution at position 297 according to EU numbering.
[0024] In some embodiments of the methods, uses, compositions, and kits described above and herein, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3, and a second antigen binding domain that binds to CEA. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody further comprises a third antigen binding domain that binds to CEA. In some embodiments, the first antigen binding domain binds to a human CD3 polypeptide. In some embodiments, the CD3 polypeptide is a human CD3e polypeptide or a human CD3y polypeptide. In some embodiments, the CD3 polypeptide is a human CD3e polypeptide. In some embodiments, the first antigen binding domain binds to a human CD3e polypeptide or a human CD3 γ polypeptide in native T-cell receptor (TCR) complex in association with other TCR subunits. In some embodiments, the first antigen binding domain comprises a heavy chain variable region (VHCD3) and a light chain variable region (VLCD3), and the second (and third, if present) antigen binding domain comprises a heavy chain variable region (VHCEA) and a light chain variable region
(VLCEA). In some embodiments, the first antigen binding domain comprises a heavy chain variable region (VHCD3) comprising HVR-H1 sequence of SEQ ID NO:44, HVR-H2 sequence of SEQ ID NO:45, and HVR-H3 sequence of SEQ ID NO:46; and/or a light chain variable region (VLCD3) comprising HVR-L1 sequence of SEQ ID NO:47, HVR-L2 sequence of SEQ ID NO:48, and HVR-L3 sequence of SEQ ID NO:49. In some
embodiments, the first antigen binding domain comprises a heavy chain variable region (VHCD3) comprising the amino acid sequence of SEQ ID NO:50 and/or a light chain variable region (VLCD3) comprising the amino acid sequence of SEQ ID NO:51. In some
embodiments, the second (and third, if present) antigen binding domain comprises a heavy chain variable region (VHCEA) comprising HVR-H1 sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3 sequence of SEQ ID NO:40; and/or a light chain variable region (VLCEA) comprising HVR-L1 sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ ID NO:42, and HVR-L3 sequence of SEQ ID NO:43. In some
embodiment, the second (and third, if present) antigen binding domain comprises a heavy chain variable region (VHCEA) comprising the amino acid sequence of SEQ ID NO:34 and/or a light chain variable region (VLCEA) comprising the amino acid sequence of SEQ ID NO:35.
[0025] In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules. In particular such embodiments, the bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the first antigen binding domain is a crossover Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other) and the second (and third, if present) antigen binding domain is a conventional Fab molecule. In one embodiment, the first antigen binding domain is a crossover Fab molecule wherein the constant domains of the Fab heavy and light chain are exchanged. In one embodiment, the anti-CEA/anti-CD3 bispecific antibody comprises not more than one antigen binding domain that binds to CD3. In one embodiment, the first and the second antigen binding domain are fused to each other, optionally through a peptide linker. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules and (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain. In a particular embodiment, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding domain.
[0026] In some embodiments, the anti-CEA/anti-CD3 bispecific antibody further comprises an Fc domain composed of a first and a second subunit capable of stable association. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules and (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, and the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a particular embodiment, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
[0027] In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules and (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In a particular embodiment, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C- terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
[0028] In one embodiment the anti-CEA/anti-CD3 bispecific antibody comprises
(i) a first antigen binding domain that binds to CD3, comprising a heavy chain variable region (VHCD3) comprising HVR-H1 sequence of SEQ ID NO:44, HVR-H2 sequence of SEQ ID NO:45, and HVR-H3 sequence of SEQ ID NO:46; and a light chain variable region (VLCD3) comprising HVR-L1 sequence of SEQ ID NO:47, HVR-L2 sequence of SEQ ID NO:48, and HVR-L3 sequence of SEQ ID NO:49, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;
(ii) a second and a third antigen binding domain that bind to CEA, comprising a heavy chain variable region (VHCEA) comprising HVR-H1 sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3 sequence of SEQ ID NO:40; and a light chain variable region (VLCEA) comprising HVR-L1 sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ ID NO:42, and HVR-L3 sequence of SEQ ID NO:43, wherein the second and third antigen binding moiety are each a Fab molecule, particularly a conventional Fab molecule;
(iii) an Fc domain composed of a first and a second subunit capable of stable association, wherein the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
[0029] In some embodiments, the Fc domain comprised in the anti-CEA/anti-CD3 bispecific antibody is an IgG, specifically an IgGi or IgG4, Fc domain. In some embodiments, the Fc domain is a human Fc domain. In one embodiment, the Fc domain is a human IgGi Fc domain.
[0030] In some embodiments, the Fc domain comprised in the anti-CEA/anti-CD3 bispecific antibody comprises a modification promoting the association of the first and the second subunit of the Fc domain. In some such embodiments, in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
[0031] In some embodiments, the Fc domain comprised in the anti-CEA/anti-CD3 bispecific antibody exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain. In some embodiments, the Fc receptor is an Fey receptor and/or the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In some such embodiments, the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function. In one
embodiment, said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (EU numbering). In one embodiment, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function wherein said amino acid substitutions are L234A, L235A and P329G (EU numbering).
[0032] In one embodiment the anti-CEA/anti-CD3 bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 52, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 53, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 54, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55. In one embodiment, the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 52, a polypeptide comprising the sequence of SEQ ID NO: 53, a polypeptide comprising the sequence of SEQ ID NO: 54, and a polypeptide comprising the sequence of SEQ ID NO: 55. (CEA TCB)
[0033] In particular embodiments, the anti-CEA/anti-CD3 bispecific antibody is CEA TCB. In some embodiments, CEA TCB is administered at a dose of about 5 mg to about 400 mg every week (e.g., about 10 mg to about 60 mg every week, e.g., about 10 mg to about 40 mg every week, e.g. about 80 mg to about 200 mg every week, e.g. about 80 mg to about 400 mg every week, or e.g. about 160 mg to 400 mg every week). In some embodiments, CEA TCB is administered at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg about 160 mg, particular at least about 80 mg, every week.
[0034] In some embodiments of the methods, uses, compositions and kits described above and herein, the cancer is a CEA-positive cancer. In some embodiments, the cancer is colon cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, breast cancer, kidney cancer, esophageal cancer, prostate cancer, or other cancers described herein. In some embodiments, the individual has cancer or has been diagnosed with cancer. In some embodiments, the individual has locally advanced or metastatic cancer or has been diagnosed with locally advanced or metastatic cancer. In some embodiments, the cancer cells in the individual express PD-L1. In some embodiments, the expression of PD-L1 may be determined by an immunohistochemistry (IHC) assay.
[0035] In some embodiments of the methods, uses, compositions, and kits described above and herein, the treatment or administration of the human PD- 1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may result in a response in the individual. In some embodiments, the response is a complete response. In some embodiments, the response is a sustained response after cessation of the treatment. In some embodiments, the response is a complete response that is sustained after cessation of the treatment. In other
embodiments, the response is a partial response. In some embodiments, the response is a partial response that is sustained after cessation of the treatment.
[0036] In some embodiments of the methods, uses, compositions, and kits described above and herein, the anti-CEA/anti-CD3 bispecific antibody is administered before the PD-1 axis binding antagonist, simultaneous with the PD-1 axis binding antagonist, or after the PD-1 axis binding antagonist. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody is administered after the PD- 1 axis binding antagonist, particularly at least one hour after the PD-1 axis binding antagonist when they are both administered on the same day. In some embodiments, the treatment or administration of the human PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody comprises a dosing regimen comprising treatment cycles, wherein the individual is administered, on day 1 of each cycle, a human PD-1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 antibody at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg about 160 mg, particular at least about 80 mg, each cycle being repeated every 21 days. In some embodiments, the cycle is repeated until there is loss of clinical benefit and/or unacceptable toxicity. In some embodiments, cycles are repeated in which only the PD- 1 axis binding antagonist or only the anti-CEA/anti-CD3 antibody is administered.
[0037] In some embodiments, the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody are in the same composition. In some embodiments, the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody are in separate
compositions.
[0038] In some embodiments of the methods, uses, compositions, and kits described above and herein, the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibody is administered intravenously. In some embodiments of the methods, uses, compositions, and kits described above and herein, the treatment further comprises administering a chemotherapeutic agent for treating or delaying progression of cancer in an individual. In some embodiments, the individual has been treated with a chemotherapeutic agent before the combination treatment with the PD- 1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody. In some embodiments, the individual treated with the combination of the PD- 1 axis binding antagonist and/or the anti- CEA/anti-CD3 bispecific antibody is refractory to a chemotherapeutic agent treatment. In some embodiments, the individual treated with the combination of the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibody is intolerant to a
chemotherapeutic agent treatment. Some embodiments of the methods, uses, compositions, and kits described throughout the application, further comprise administering a
chemotherapeutic agent for treating or delaying progression of cancer.
[0039] In some embodiments of the methods, uses, compositions and kits described above and herein, CD8 T cells in the individual have enhanced priming, activation, proliferation and/or cytolytic activity relative to prior to the administration of the combination. In some embodiments, the number of CD8 T cells is elevated relative to prior to administration of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 antibody. In some embodiments, the CD8 T cell is an antigen-specific CD8 T cell. In some embodiments, Treg function is suppressed relative to prior to the administration of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 antibody. In some embodiments, T cell exhaustion is decreased relative to prior to the administration of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 antibody. [0040] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] Figure 1. CEA TCB-mediated lysis of MKN45 cells. Representative graphs of CEA TCB-mediated lysis of MKN-45 tumor cells assessed (A) 24 hours and (B) 48 hours after incubation of tumor cells with human PBMCs (E:T 10: 1). The EC50 values of tumor cell killing are: 615 pM (24 h), 362 pM (48 h). Target cell killing was assessed by quantification of LDH released into cell supernatants.
[0042] Figure 2. CEA TCB-mediated up-regulation of PD-1 on T cells, and of PD-Ll on surviving tumor cells after tumor cell lysis. Representative graphs of CEA TCB-mediated up- regulation of PD-1 receptor expression on CD8+ T cells (A), CD4+ T cells (B), and of PD-Ll ligand expression on tumor cells that resisted killing (C) analyzed 48 h post incubation of MKN-45 tumor cells with human PBMCs (E:T 10: 1). PD-1 and PD-Ll expression was analyzed by flow cytometry.
[0043] Figure 3. Tumor growth after treatment with CEA TCB (co-grafting, E:T 5: 1). Average tumor burden and standard error of mean (SEM, n = 12); tumor burden was measured by bioluminescence (total flux). CEA TCB was administered IV at doses of 0.5 and 2.5 mg/kg, starting one day (early treatment, panel A) or seven days (late treatment, panel B) after tumor/PBMCs co-grafting and SC injection. Control groups received phosphate-buffer saline (PBS, vehicle), MCSP TCB, or DP47 TCB (as untargeted controls, respectively). In all studies, TCBs were administered twice a week (2q7d). Arrows indicate the day of start of therapy. The curves were compared using non-linear regression analysis. (A) Start of therapy one day after co-grafting with human PBMCs. Curve comparison: vehicle vs. untargeted TCB p-value = 0.65, vehicle vs. CEA TCB (0.5 mg/kg) p-value < 0.0001, vehicle vs. CEA TCB (2.5 mg/kg) p-value < 0.0001, CEA TCB (2.5 mg/kg) vs. CEA TCB (0.5 mg/kg) p- value = 0.91. (B) Start of therapy seven days after co-grafting with human PBMCs. Curve comparison: vehicle vs. untargeted TCB p-value = 0.65, vehicle vs. CEA TCB (0.5 mg/kg) p- value < 0.0001, vehicle vs. CEA TCB (2.5 mg/kg) p-value < 0.0001, CEA TCB (2.5 mg/kg) vs. CEA TCB (0.5 mg/kg) p-value = 0.0008. [0044] Figure 4. Histological analysis of tumors after treatment with CEA TCB (co- grafting, E:T 5: 1). Hematoxylin and eosin (H&E, panels A and D) and anti-PD-Ll (panels B, C, E and F) staining of vehicle (panels A-C) and CEA TCB (panels D-F)-treated tumors (2.5 mg/kg, therapy given on Day 7) collected at termination in the co-grafting experiment of LS174T-fluc2 cells with hPBMCs (E:T 5: 1) showing strong induction of intra-tumor PD-L1 expression upon CEA TCB-treatment. Panel B and E correspond to the marked area of panel A and D, respectively. Panel C and F correspond to the marked area of panel B and E, respectively.
[0045] Figure 5. Tumor weight after treatment with CEA TCB. LS174T-fluc2 cells injected SC and grown until a volume of 100 mm was reached in NOG mice, followed by IP transfer of human PBMCs (10 x 106 cells). Three days after PBMCs transfer, mice received bi-weekly IV injections of CEA TCB (2.5 mg/kg), PBS (vehicle), or MCSP TCB (untargeted control, 2.5 mg/kg). Tumor mass was determined at study termination by weighing the explanted tumors. Graphs show average tumor weight and standard error of mean (SEM, 3 < n < 6 mice). ** p < 0.005 using unpaired, two-tailed t-test.
[0046] Figure 6. Flow cytometry analysis of tumor- infiltrating leukocytes after treatment with CEA TCB. Flow cytometry analysis of tumor tissues collected at termination in the human colon carcinoma xenograft model with IP transfer of human effector cells. The bar plot shows the number of intra-tumor human CD45+/CD3+ T-cells as assessed by flow cytometry in all treated groups. * p < 0.05 using unpaired, two-tailed t-test.
[0047] Figure 7. Immuno PD analysis after treatment with CEA TCB (IP transfer of human PBMCs). Flow cytometry analysis of blood (A-F) and tumor tissues (G-L) collected at Day 18 (corresponding to three days after the third CEA TCB administration) after tumor cell inoculation in the human colon carcinoma xenograft model (IP transfer of human effector cells). Dot plots show the expression of CD69 (B, E, H, K) and PD-1 (C, F, I, L) on CD3 T- cells in both compartments. Representative mice for each group are shown. A-C, G-I:
vehicle. D-F, J-L: CEA TCB
[0048] Figure 8. CEA TCB-induced tumor growth inhibition in fully humanized mice. Tumor growth inhibition of MKN45 in humanized mice. Tumor volumes at study termination (at day 32 after tumor cell injection) are shown. Each point represents an individual animal. CEA TCB was injected IV (2.5 mg/kg twice weekly), and tumor growth was measured by caliper.* p < 0.05 (unpaired t-test).
[0049] Figure 9. Increase of intra- tumoral human T-cell frequencies and T-cell activation by CEA TCB. Flow cytometric analysis of tumor samples collected at study termination (Day 32). Cells were stained for human antigens (huCD45, huCD3, huPD-1, huCD69, huKi-67, and huGZB). (A, B) Total human T-cell frequencies (A) and the ratio of CD8+ to CD4+ T- cells (B) in the tumor tissue. (C, D) The expression of activation markers in tumor is shown as percent of human CD8+ (C) or CD4+ (D) T-cells. Each point represents an individual animal. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (unpaired t-test).
[0050] Figure 10. Histological analyses of tumor tissues at study termination. Hematoxylin and eosin (H&E, panel A and D) and anti-PD-Ll (panel B, C, E and F) staining (Mab SP142) of vehicle (panel A-C) and CEA TCB (panel D-F) -treated tumors exhibiting strong induction of intra- tumoral PD-L1 expression upon CEA TCB treatment. All sections were
counterstained with hematoxylin (blue nuclei). Panel B and E correspond to the marked area of panel A and D, respectively. Panel C and F correspond to the marked area of panel B and E, respectively.
[0051] Figure 11. In vivo anti-tumor activity upon combination of CEA TCB with anti- human PD-L1 blocking antibody in MKN45 tumor model in fully humanized mice. (A) Average tumor burden and standard error of mean (SEM). Tumor burden was measured by digital caliper 3 times a week. Arrow indicates the day of start of therapy. At day 60: n=7 (vehicle), n=8 (CEA TCB); n=4 (a-PD-Ll); n=5 (combination CEA TCB with anti-PD-Ll). (B) Time-to-event statistical analysis. The event was defined as reaching 500 mm tumor volume. The pairwise log-rank test was used to compare the following treatment groups, as it can take the different drop-outs into account: vehicle vs. CEA TCB: p= 0.005; CEA TCB vs. anti-PD-Ll: p= 0.001; CEA TCB vs. combination of CEA TCB with anti-PD-Ll: p= 0.03.
[0052] Figure 12. Anti-tumor activity of CEA TCB in the MKN45 model in fully humanized NOG mice. For first line treatment, mice were administered either with vehicle, as control, or with CEA TCB (2.5 mg/kg, administered twice a week for 4 weeks, 7
administrations in total). At study day 34, mice in the CEA TCB-treated group were randomized into two sub-groups: one continued being administered with CEA TCB (2.5 mg/kg twice a week) and the other was administered with CEA TCB (2.5 mg/kg twice a week) in combination with anti-mouse PD-L1 antibody (10 mg/kg, administered once a week) for 5 weeks (11 administrations in total for CEA TCB and 6 administrations in total for anti-PD-Ll). ***p<0.0002 two-way ANOVA.
[0053] Figure 13. Anti-tumor activity of CEA TCB in the MC38-huCEA model in fully immunocompetent huCEA Tg mice. (A) For first line treatment, mice were administered either with vehicle, as control, with CEA TCB (2.5 mg/kg, administered twice a week for 4 weeks, 7 administrations in total), with anti-mouse PD-L1 antibody (10 mg/kg, administered once a week for 4 weeks, 4 administrations in total), and with a combination of CEA TCB and anti-mouse PD-L1, administered concomitantly with the respective dose and schedules used in the single therapeutic groups. Treatment was administered for 4 weeks. (B) Subcutaneous tumors were surgically removed, and after 5 weeks mice were rechallenged with fresh MC38-huCEA tumors cells injected sub-cutaneously on the opposite flank. As control, groups of naive huCEA Tg mice, either left un-manipulated or submitted to a surgical cut on the flank, were injected sub-cutaneously with MC38-huCEA on the opposite flank.
DETAILED DESCRIPTION
[0054] The inventors of this application demonstrated that a CEA T cell bispecific antibody and anti-PD-Ll immune therapy act synergistically in their anti-cancer properties and their combination could provide meaningful clinical benefit in patients with cancer. The data in the application show that the combination of a CEA T cell bispecific antibody (CEA TCB) with anti-PD-Ll immune therapy resulted in enhanced inhibition of tumor growth.
[0055] In one aspect, provided herein are methods, compositions and uses for treating or delaying progression of cancer in an individual comprising administering an effective amount of a human PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
[0056] In another aspect, provided herein are methods, compositions and uses for enhancing immune function in an individual having cancer comprising administering an effective amount of a human PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
I. Definitions
[0057] Before describing the invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0058] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.
[0059] As used herein, the terms "first", "second", "third" etc. with respect to antigen binding domains etc., are used for convenience of distinguishing when there is more than one of each type of domain. Use of these terms is not intended to confer a specific order or orientation unless explicitly so stated.
[0060] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
[0061] It is understood that aspects and embodiments of the invention described herein include "comprising " "consisting " and "consisting essentially of aspects and embodiments.
[0062] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partner, so as to remove T-cell dysfunction resulting from signaling on the PD-1 signaling axis - with a result being to restore or enhance T-cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-Ll binding antagonist and a PD-L2 binding antagonist. A "human" PD-1 axis binding antagonist refers to a PD- 1 axis binding antagonist which has the above- described effects on the human PD-1 signaling axis.
[0063] The term "PD-1 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- 1 with one or more of its binding partners, such as PD-Ll, PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-Ll and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins,
oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-Ll and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces the negative co- stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist is MDX-1106
(nivolumab) described herein. In another specific aspect, a PD-1 binding antagonist is MK- 3475 (pembrolizumab) described herein. In another specific aspect, a PD-1 binding antagonist is CT-011 (pidilizumab) described herein. In another specific aspect, a PD-1 binding antagonist is MEDI-0680 (AMP-514) described herein. In another specific aspect, a PD-1 binding antagonist is PDR001 described herein. In another specific aspect, a PD-1 binding antagonist is REGN2810 described herein. In another specific aspect, a PD-1 binding antagonist is BGB-108 described herein.
[0064] The term "PD-Ll binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- Ll with either one or more of its binding partners, such as PD-1, B7-1. In some
embodiments, a PD-Ll binding antagonist is a molecule that inhibits the binding of PD-Ll to its binding partners. In a specific aspect, the PD-Ll binding antagonist inhibits binding of PD-Ll to PD-1 and/or B7-1. In some embodiments, the PD-Ll binding antagonists include anti-PD-Ll antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-Ll with one or more of its binding partners, such as PD-1, B7-1. In one embodiment, a PD-Ll binding antagonist reduces the negative co- stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-Ll so as to render a dysfunctional T-cell less dysfunctional {e.g., enhancing effector responses to antigen recognition). In some
embodiments, a PD-Ll binding antagonist is an anti-PD-Ll antibody. In a specific aspect, an anti-PD-Ll antibody is YW243.55.S70 described herein. In another specific aspect, an anti- PD-Ll antibody is MDX-1105 described herein. In still another specific aspect, an anti-PD- Ll antibody is MPDL3280A (atezolizumab) described herein. In still another specific aspect, an anti-PD-Ll antibody is MDX-1105 described herein. In still another specific aspect, an anti-PD-Ll antibody is YW243.55.S70 described herein. In still another specific aspect, an anti-PD-Ll antibody is MEDI4736 (durvalumab) described herein. In still another specific aspect, an anti-PD-Ll antibody is MSB0010718C (avelumab) described herein.
[0065] The term "PD-Ll binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD- L2 with either one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins,
oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1. In one embodiment, a PD-L2 binding antagonist reduces the negative co- stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-L2 binding antagonist is an immunoadhesin.
[0066] The term "dysfunction" in the context of immune dysfunction, refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes the common elements of both exhaustion and/or anergy in which antigen recognition may occur, but the ensuing immune response is ineffective to control infection or tumor growth.
[0067] The term "dysfunctional", as used herein, also includes refractory or unresponsive to antigen recognition, specifically, impaired capacity to translate antigen recognition into down-stream T-cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing.
[0068] The term "anergy" refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T-cell receptor (e.g. increase in intracellular Ca2+ in the absence of ras-activation). T cell anergy can also result upon stimulation with antigen in the absence of co-stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of costimulation. The unresponsive state can often be overriden by the presence of interleukin-2. Anergic T-cells do not undergo clonal expansion and/or acquire effector functions.
[0069] The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Exhaustion can result from both extrinsic negative regulatory pathways (e.g.,
immunoregulatory cytokines) as well as cell intrinsic negative regulatory (co stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).
[0070] "Enhancing T-cell function" means to induce, cause or stimulate a T-cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T- cells. Examples of enhancing T-cell function include: increased secretion of γ-interferon from CD8+ T-cells, increased proliferation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) relative to such levels before the intervention. In one embodiment, the level of enhancement is as least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to one of ordinary skill in the art.
[0071] A "T cell dysfunctional disorder" is a disorder or condition of T-cells characterized by decreased responsiveness to antigenic stimulation. In a particular embodiment, a T-cell dysfunctional disorder is a disorder that is specifically associated with inappropriate increased signaling through PD-1. In another embodiment, a T-cell dysfunctional disorder is one in which T-cells are anergic or have decreased ability to secrete cytokines, proliferate, or execute cytolytic activity. In a specific aspect, the decreased responsiveness results in ineffective control of a pathogen or tumor expressing an immunogen. Examples of T cell dysfunctional disorders characterized by T-cell dysfunction include unresolved acute infection, chronic infection and tumor immunity.
[0072] "Tumor immunity" refers to the process in which tumors evade immune
recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated, and the tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.
[0073] "Immunogenicity" refers to the ability of a particular substance to provoke an immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the clearance of the tumor cells by the immune response. Examples of enhancing tumor immunogenicity include treatment with a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody.
[0074] "Sustained response" refers to the sustained effect on reducing tumor growth after cessation of a treatment. For example, the tumor size may remain to be the same or smaller as compared to the size at the beginning of the administration phase. In some embodiments, the sustained response has a duration at least the same as the treatment duration, at least 1.5X, 2. OX, 2.5X, or 3. OX length of the treatment duration.
[0075] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed. [0076] As used herein, the term "treatment" refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are mitigated or eliminated, including, but are not limited to, reducing the proliferation of (or destroying) cancerous cells, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging survival of individuals.
[0077] As used herein, "delaying progression of a disease" means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
[0078] An "effective amount" is at least the minimum amount required to effect a measurable improvement or prevention of a particular disorder. An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting {i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit {i.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or
pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
[0079] As used herein, "in conjunction with" refers to administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the individual.
[0080] A "disorder" is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
[0081] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell
proliferative disorder is a tumor.
[0082] "Tumor", as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive as referred to herein.
[0083] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non- small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, nodular melanomas, multiple myeloma and B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL;
intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade
immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and
Waldenstrom's Macro globulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), Meigs' syndrome, brain, as well as head and neck cancer, and associated metastases. In certain embodiments, cancers that are amenable to treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some
embodiments, the cancer is selected from: small cell lung cancer, glioblastoma,
neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Yet, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast carcinoma, including metastatic forms of those cancers. In some embodiments, the cancer is a CEA-positive cancer.
[0084] The term "cytotoxic agent" as used herein refers to any agent that is detrimental to cells (e.g., causes cell death, inhibits proliferation, or otherwise hinders a cellular function).
Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , 1131 , 1125 , Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
Exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid biosynthesis, cell cycle signalling inhibitors, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism. In one embodiment the cytotoxic agent is a taxane. In one embodiment the taxane is paclitaxel or docetaxel. In one embodiment the cytotoxic agent is a platinum agent. In one embodiment the cytotoxic agent is an antagonist of EGFR. In one embodiment the antagonist of EGFR is N-(3-ethynylphenyl)-6,7-bis(2- methoxyethoxy)quinazolin-4-amine (e.g., erlotinib). In one embodiment the cytotoxic agent is a RAF inhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or CRAF inhibitor. In one embodiment the RAF inhibitor is vemurafenib. In one embodiment the cytotoxic agent is a PI3K inhibitor.
[0085] " Chemotherapeutic agent" includes compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate , salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer/Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®,
GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, 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, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine;
acetogenins (especially bullatacin and bullatacinone); a camptothecin (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5a- reductases including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat dolastatin; aldesleukin, talc duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γΐΐ and calicheamicin coll (Angew Chem. Intl. Ed. Engl. 1994 33: 183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin,
methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE® (docetaxel, doxetaxel; Sanofi-Aventis); chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitoxantrone;
vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11 ; topoisomerase inhibitor RFS 2000; difluoromethylorni thine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.
[0086] Chemotherapeutic agent also includes (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®;
tamoxifen citrate), raloxifene, droloxifene, iodoxyfene , 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and AREVIIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin,
fluoxymesterone, all transretionic acid, fenretinide, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g. , ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.
[0087] Chemotherapeutic agent also includes antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idee), pertuzumab (OMNrfARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin
(MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab,
rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti- interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) which is a recombinant exclusively human- sequence, full-length IgGi λ antibody genetically modified to recognize interleukin-12 p40 protein.
[0088] Chemotherapeutic agent also includes "EGFR inhibitors," which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an "EGFR antagonist." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No. 4,943, 533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (US Patent No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in US Patent No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF- alpha for EGFR binding (EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as El. l, E2.4, E2.5, E6.2, E6.4, E2.l l, E6. 3 and E7.6. 3 and described in US 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al, J. Biol. Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g.,
EP659439A2, Merck Patent GmbH). EGFR antagonists include small molecules such as compounds described in US Patent Nos: 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, as well as the following PCT publications: W098/14451,
WO98/50038, WO99/09016, and WO99/24037. Particular small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSI Pharmaceuticals); PD 183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4- morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca);
BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(l-methyl-piperidin-4-yl)-pyrimido[5,4- d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(l- phenylethyl)amino]-lH-pyrrolo[2,3-d]pyrimidin-6-yl] -phenol); (R)-6-(4-hydroxyphenyl)-4- [(l-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3- bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4- fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3
fluorophenyl)methoxy] phenyl] -6 [5 [ [ [2methylsulf onyl)ethyl] amino] methyl] -2-furanyl] -4- quinazolinamine) .
[0089] Chemotherapeutic agents also include "tyrosine kinase inhibitors" including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan- HER inhibitors such as canertinib (CI- 1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non- HER targeted TK inhibitors such as imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib
(PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035,4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H- pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4- fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner- Lamber); antisense molecules (e.g. those that bind to HER-encoding nucleic acid); quinoxalines (US Patent No. 5,804,396); tryphostins (US Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI- 1033 (Pfizer); Affinitac (ISIS 3521 ; Isis/Lilly); imatinib mesylate (GLEEVEC®); PKI 166
(Novartis); GW2016 (Glaxo SmithKline); CI- 1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-lCl l (Imclone), rapamycin (sirolimus, RAPAMUNE®); or as described in any of the following patent publications: US Patent No. 5,804,396; WO 1999/09016 (American Cyanamid); WO
1998/43960 (American Cyanamid); WO 1997/38983 (Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).
[0090] Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.
[0091] Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone- 17-butyrate, hydrocortisone- 17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate,
prednicarbate, clobetasone- 17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate; immune selective anti-inflammatory peptides (ImSAIDs) such as phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG) (IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such as azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNFa) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra (Kineret), T cell costimulation blockers such as abatacept (Orencia), Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMRA®); Interleukin 13 (IL- 13) blockers such as lebrikizumab; Interferon alpha (IFN) blockers such as Rontalizumab; Beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as Anti-Mi prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTal/p2 blockers such as Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g. , At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); miscellaneous investigational agents such as thioplatin, PS-341,
phenylbutyrate, ET- 18- OCH3, or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone;
lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and
9-aminocamptothecin); podophyllotoxin; tegafur (UFTORAL®); bexarotene
(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate
(ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate
(SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone;
farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR™); and
pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.
[0092] Chemotherapeutic agents also include non-steroidal anti-inflammatory drugswith analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs can be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathies, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea, metastatic bone pain, headache and migraine, postoperative pain, mild-to-moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.
[0093] 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. In one embodiment, growth inhibitory agent is growth inhibitory antibody that prevents or reduces proliferation of a cell expressing an antigen to which the antibody binds. In another embodiment, the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase. Examples of 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 (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those 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. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. 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.
[0094] By "radiation therapy" is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one-time administration and typical dosages range from 10 to 200 units (Grays) per day. [0095] A "subject" or an "individual" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human. An individual or subject may be a patient.
[0096] The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies {e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
[0097] An "isolated' antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using, for example, Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
[0098] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
[0099] The term "constant domain" refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CHI , CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
[00100] The "variable region" or "variable domain" of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VR." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
[00101] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs 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, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
[00102] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.
[00103] The term IgG "isotype" or "subclass" as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
[00104] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B.
Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
[00105] The terms "full length antibody " "intact antibody" and "whole antibody" are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.
[00106] A "naked antibody" for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.
[00107] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
[00108] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
[0100] "Fv" is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
[0101] The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
[0102] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York, 1994), pp. 269-315.
[0103] The term "diabodies" refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).
[0104] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, 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. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, 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 target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
[0105] 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. For example, the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14 (3): 253-260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al, Nature, 352: 624-628 (1991); Marks et al, J. Mol Biol 222: 581-597 (1992); Sidhu et al, J. Mol Biol 338(2): 299-310 (2004); Lee et al, J. Mol Biol 340(5): 1073-1093 (2004); Fellouse, Proc. Natl Acad. Sci. USA 101(34): 12467- 12472 (2004); and Lee et al, J. Immunol Methods 284(1-2): 119-132 (2004), and
technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al, Nature 362: 255-258
(1993) ; Bruggemann et al, Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al, Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813
(1994) ; Fishwild et al, Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature
Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93
(1995) .
[0106] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
[0107] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one
embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non- human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody
performance. In general, a 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 will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et ah, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.
[0108] A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et ah, J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et ah, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
[0109] A "species-dependent antibody" is one which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody "binds specifically" to a human antigen (e.g., has a binding affinity (KD) value of no more than about 1x10" M, preferably no more than about 1x10 -"8 M and preferably no more than about 1x10 -"9 M) but has a binding affinity for a homologue of the antigen from a second nonhuman mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
[0110] The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446- 448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0111] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.
Loop Kabat AbM Chothia Contact
LI L24-L34 L24-L34 L26-L32 L30-L36
L2 L50-L56 L50-L56 L50-L52 L46-L55
L3 L89-L97 L89-L97 L91-L96 L89-L96
HI H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering)
HI H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering)
H2 H50-H65 H50-H58 H53-H55 H47-H58
H3 H95-H102 H95-H102 H96-H101 H93-H101
[0112] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (HI), 50-65 or 49-65 (H2) and 93- 102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.
[0113] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.
[0114] The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat " and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
[0115] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et ah, supra). The "EU index as in Kabat" refers to the residue numbering of the human IgGl EU antibody.
[0116] The expression "linear antibodies" refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CHl-VH-CHl) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
[0117] As use herein, the term "binds", "specifically binds to" or is "specific for" refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that binds to or specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of < ΙμΜ, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.
[0118] The term "bispecific" means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.
[0119] The term "antigen binding domain" refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). [0120] A "Fab molecule" refers to a protein consisting of the VH and CHI domain of the heavy chain (the "Fab heavy chain") and the VL and CL domain of the light chain (the "Fab light chain") of an immunoglobulin.
[0121] By a "crossover" Fab molecule (also termed "Crossfab") is meant a Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (VL-CH1, in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in N- to C- terminal direction). For clarity, in a crossover Fab molecule wherein the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CHI is referred to herein as the "heavy chain" of the
(crossover) Fab molecule. Conversely, in a crossover Fab molecule wherein the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.
[0122] In contrast thereto, by a "conventional" Fab molecule is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant domains (VH-CHl, in N- to C-terminal direction), and a light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction).
[0123] The term "Fc domain" or "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). This may be the case where the final two C- terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU
numbering). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). A "subunit" of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
[0124] By "fused" is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.
[0125] A "modification promoting heterodimerization" is a manipulation of the peptide backbone or the post-translational modifications of a polypeptide, e.g. an immunoglobulin heavy chain, that reduces or prevents the association of the polypeptide with an identical polypeptide to form a homodimer. A modification promoting heterodimerization as used herein particularly includes separate modifications made to each of two polypeptides desired to form a dimer, wherein the modifications are complementary to each other so as to promote association of the two polypeptides. For example, a modification promoting
heterodimerization may alter the structure or charge of one or both of the polypeptides desired to form a dimer so as to make their association sterically or electrostatically favorable, respectively. Heterodimerization occurs between two non-identical polypeptides, e.g. two immunoglobulin heavy chains wherein the variable regions are not the same. In some embodiments, the modification promoting heterodimerization comprises an amino acid mutation, specifically an amino acid substitution. In a particular embodiment, the
modification promoting heterodimerization comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two polypeptides desired to form a dimer.
[0126] An "activating Fc receptor" is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcyRIIIa (CD 16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).
[0127] The term "effector functions" when used in reference to antibodies refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.
II. PD-1 Axis Binding Antagonists
[0128] Provided herein is a method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody. Also provided herein is a method of enhancing immune function in an individual having cancer comprising
administering to the individual an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody. For example, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist. PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1", PDCD1, CD279 and SLEB2. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death 1 ligand 1", PDCD1LG1, CD274, B7-H, and PDL1. An exemplary human PD-L1 is shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1. PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2", PDCD1LG2, CD273, B7-DC, Btdc, and PDL2. An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot Accession No. Q9BQ51. In some embodiments, PD-1, PD- Ll, and PD-L2 are human PD-1, PD-L1 and PD-L2.
[0129] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect the PD-1 ligand binding partners are PD-L1 and/or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, PD- Ll binding partners are PD-1 and/or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners. In a specific aspect, a PD-L2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
[0130] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX 1106 (nivolumab), MK- 3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001,
REGN2810, and BGB-108. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1 binding antagonist is anti-PD-Ll antibody. In some embodiments, the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70,
MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). Antibody YW243.55.S70 is an anti-PD-Ll described in WO 2010/077634. MDX-1105, also known as BMS-936559, is an anti-PD-Ll antibody described in
WO2007/005874. MEDI4736, is an anti-PD-Ll monoclonal antibody described in
WO2011/066389 and US2013/034559. Nivolumab, also known as MDX-1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT, hBAT-1 or pidilizumab, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.
[0131] In some embodiments, the PD-1 axis binding antagonist is an anti-PD-Ll antibody. In some embodiments, the anti-PD-Ll antibody is capable of inhibiting binding between PD- Ll and PD-1 and/or between PD-L1 and B7-1. In some embodiments, the anti-PD-Ll antibody is a monoclonal antibody. In some embodiments, the anti-PD-Ll antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the anti-PD-Ll antibody is a humanized antibody. In some embodiments, the anti-PD-Ll antibody is a human antibody.
[0132] Examples of anti-PD-Ll antibodies useful for the methods, uses, compositions and kits of this invention, and methods for making thereof are described in PCT patent application WO 2010/077634, WO2007/005874, WO2011/066389, and US2013/034559, which are incorporated herein by reference. The anti-PD-Ll antibodies useful in this invention, including compositions containing such antibodies, may be used in combination with an anti-CEA/anti-CD3 bispecific antibody to treat cancer.
Anti-PDl antibodies
[0133] In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558 or nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). In a still further embodiment, provided is an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO: 1 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO:2. In a still further embodiment, provided is an isolated anti-PD-1 antibody comprising a heavy chain and/or a light chain sequence, wherein:
(a) the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence:
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDG
SKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT
VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE
FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP
QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:l), and
(b) the light chain sequences has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the light chain sequence:
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:2).
Anti-PD-Ll antibodies
[0134] Anti-PD-Ll antibodies described in WO 2010/077634 Al and US 8,217,149 may be used in the methods, uses, compositions and kits described herein. In some embodiments, the anti-PD-Ll antibody comprises a heavy chain variable region sequence of SEQ ID NO:3 and/or a light chain variable region sequence of SEQ ID NO:4. In a still further embodiment, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and/or a light chain sequence, wherein:
(a) the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSA (SEQ ID NO:3), and
(b) the light chain sequences has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:4).
[0135] In one embodiment, the anti-PD-Ll antibody comprises a heavy chain variable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3 sequence, wherein:
(a) the HVR-H1 sequence is GFTFSXiSWIH (SEQ ID NO:5)
(b) the HVR-H2 sequence is AWK2PYGGSX3YYADSVKG (SEQ ID NO:6)
(c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:7) further wherein: Xi is D or G; X2 is S or L; X3 is T or S. In one specific aspect, Xi is
D; X2 is S and X3 is T.
[0136] In another aspect, the polypeptide further comprises variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)- (HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In a further aspect, the framework sequences are VH subgroup III consensus framework. In a still further aspect, at least one of the framework sequences is the following:
HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8)
HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO:9)
HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 10) HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 11).
[0137] In a still further aspect, the heavy chain polypeptide is further combined with a variable region light chain comprising an HVR-L1, HVR-L2 and HVR-L3, wherein:
(a) the HVR-L1 sequence is R AS QX4X5X6TX7X8 A (SEQ ID NO: 12)
(b) the HVR-L2 sequence is SASX9LX10S, (SEQ ID NO: 13)
(c) the HVR-L3 sequence is QQXIIXI2XI3XI4PXI5T (SEQ ID NO: 14) wherein: X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; Xg is V or L; X9 is F or T; X10 is Y or A; Xn is Y, G, F, or S; Xi2 is L, Y, F or W; Xi3 is Y, N, A, T, G, F or I; X14 is H, V, P, T or I; X15 is A, W, R, P or T. In a still further aspect, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; Xg is F; X10 is Y; Xn is Y; Xi2 is L; X13 is Y; Xi4 is H; X15 is A.
[0138] In a still further aspect, the light chain further comprises variable region light chain framework sequences juxtaposed between the HVRs according to the formula: (LC-FR1)- (HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In a still further aspect, the framework sequences are derived from human consensus framework sequences. In a still further aspect, the framework sequences are VL kappa I consensus framework. In a still further aspect, at least one of the framework sequence is the following:
LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15)
LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 16)
LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17) LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 18).
[0139] In another embodiment, provided is an isolated anti-PD-Ll antibody or antigen binding fragment comprising a heavy chain and a light chain variable region sequence, wherein:
(a) the heavy chain comprises and HVR-H1, HVR-H2 and HVR-H3, wherein further:
(i) the HVR-H1 sequence is GFTFSXiSWIH; (SEQ ID
NO:5)
(ii) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID
NO:6)
(iii) the HVR-H3 sequence is RHWPGGFDY, and (SEQ ID
NO:7)
(b) the light chain comprises and HVR-L1, HVR-L2 and HVR-L3, wherein further:
(i) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 12)
(ii) the HVR-L2 sequence is SASX9LX10S; and (SEQ ID NO: 13)
(iii) the HVR-L3 sequence is QQXnXi2Xi3Xi4PXi5T; (SEQ ID NO: 14)
wherein: Xi is D or G; X2 is S or L; X3 is T or S; X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; Xg is V or L; X9 is F or T; X10 is Y or A; Xn is Y, G, F, or S; X12 is L, Y, F or W; X13 is Y, N, A, T, G, F or I; XM is H, V, P, T or I; Xi5 is A, W, R, P or T. In a specific aspect, Xi is D; X2 is S and X3 is T. In another aspect, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X10 is Y; Xn is Y; X12 is L; X13 is Y; X14 is H; X15 is A. In yet another aspect, Xi is D; X2 is S and X3 is T, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X10 is Y; Xn is Y; X12 is L; X13 is Y; X14 is H and X15 is A.
[0140] In a further aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC-FR1)-(HVR-H1)-(HC-FR2)- (HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FRl)-(HVR-Ll)- (LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In a still further aspect, the framework sequences are derived from human consensus framework sequences. In a still further aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10 and 11. In a still further aspect, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.
[0141] In a still further specific aspect, the antibody further comprises a human or murine constant region. In a still further aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In a still further specific aspect, the human constant region is IgGl. In a still further aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In a still further aspect, the murine constant region is IgG2A. In a still further specific aspect, the antibody has reduced or minimal effector function. In a still further specific aspect the minimal effector function results from an "effector- less Fc mutation" or aglycosylation. In still a further embodiment, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
[0142] In yet another embodiment, provided is an anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein:
(a) the heavy chain further comprises an HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO:20) and RHWPGGFDY (SEQ ID NO:21), respectively, or
(b) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3
sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO:22), SASFLYS (SEQ ID NO:23) and QQYLYHPAT (SEQ ID NO:24), respectively.
In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
[0143] In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC-FR1)-(HVR-H1)-(HC-FR2)- (HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FRl)-(HVR-Ll)- (LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In a still further aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10 and 11. In a still further aspect, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.
[0144] In a still further specific aspect, the antibody further comprises a human or murine constant region. In a still further aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In a still further specific aspect, the human constant region is IgGl. In a still further aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In a still further aspect, the murine constant region is IgG2A. In a still further specific aspect, the antibody has reduced or minimal effector function. In a still further specific aspect the minimal effector function results from an "effector- less Fc mutation" or aglycosylation. In still a further embodiment, the effector- less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
[0145] In another further embodiment, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein:
(a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSS (SEQ ID NO:25), and/or
(b) the light chain sequences has at least 85% sequence identity to the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:4).
In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC- FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In a further aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10 and
WGQGTLVTVSS (SEQ ID NO:27).
In a still further aspect, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18.
[0146] In a still further specific aspect, the antibody further comprises a human or murine constant region. In a still further aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In a still further specific aspect, the human constant region is IgGl. In a still further aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In a still further aspect, the murine constant region is IgG2A. In a still further specific aspect, the antibody has reduced or minimal effector function. In a still further specific aspect, the minimal effector function results from production in prokaryotic cells. In a still further specific aspect the minimal effector function results from an "effector-less Fc mutation" or aglycosylation. In still a further embodiment, the effector-less Fc mutation is an N297A or D265A/N297A
substitution in the constant region. [0147] In a further aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC-FR1)-(HVR-H1)-(HC-FR2)- (HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FRl)-(HVR-Ll)- (LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In a still further aspect, the framework sequences are derived from human consensus framework sequences. In a still further aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences is the following:
HC-FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:29)
HC-FR2 WVRQAPGKGLEWVA (SEQ ID NO:30)
HC-FR3 RFTIS ADTS KNT A YLQMNSLRAEDTA V Y YC AR (SEQ ID NO: 10) HC-FR4 WGQGTLVTVSS (SEQ ID NO:27).
[0148] In a still further aspect, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences is the following:
LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15)
LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 16)
LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17)
LC-FR4 FGQGTKVEIK (SEQ ID NO:28).
[0149] In a still further specific aspect, the antibody further comprises a human or murine constant region. In a still further aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In a still further specific aspect, the human constant region is IgGl. In a still further aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In a still further aspect, the murine constant region is IgG2A. In a still further specific aspect, the antibody has reduced or minimal effector function. In a still further specific aspect the minimal effector function results from an "effector- less Fc mutation" or aglycosylation. In still a further embodiment, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
[0150] In yet another embodiment, provided is an anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein: (c) the heavy chain further comprises an HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO:20) and RHWPGGFDY (SEQ ID NO:21), respectively, and/or
(d) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3
sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO:22), SASFLYS (SEQ ID NO:23) and QQYLYHPAT (SEQ ID NO:24), respectively.
In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
[0151] In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (HC-FR1)-(HVR-H1)-(HC-FR2)- (HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (LC-FRl)-(HVR-Ll)- (LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In a still further aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10 and WGQGTLVTVSSASTK (SEQ ID NO:31).
[0152] In a still further aspect, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17 and 18. In a still further specific aspect, the antibody further comprises a human or murine constant region. In a still further aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In a still further specific aspect, the human constant region is IgGl. In a still further aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In a still further aspect, the murine constant region is IgG2A. In a still further specific aspect, the antibody has reduced or minimal effector function. In a still further specific aspect the minimal effector function results from an "effector- less Fc mutation" or aglycosylation. In still a further embodiment, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
[0153] In a still further embodiment, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein:
(a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSS (SEQ ID NO:25), or
(b) the light chain sequences has at least 85% sequence identity to the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:4).
[0154] In some embodiments, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4 and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, one, two, three, four or five amino acid residues at the N-terminal of the heavy and/or light chain may be deleted, substituted or modified.
[0155] In a still further embodiment, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein:
(a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSSASTK (SEQ ID NO:26), or
(b) the light chain sequences has at least 85% sequence identity to the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:4).
[0156] In some embodiments, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain variable region sequence, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4 and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, one, two, three, four or five amino acid residues at the N-terminal of the heavy and/or light chain may be deleted, substituted or modified.
[0157] In a still further embodiment, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein:
(a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG
STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG
TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID
NO:32), and/or
(b) the light chain sequences has at least 85% sequence identity to the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:33) In a still further embodiment, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein:
(a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG
STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG
TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ
ID NO:56), and/or (b) the light chain sequences has at least 85% sequence identity to the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:33).
[0158] In some embodiments, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:33. In some embodiments, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:32 or 56. In some embodiments, provided is an isolated anti-PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:33 and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:32 or 56. In some embodiments, provided is an isolated anti- PD-Ll antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:33 and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:32.
[0159] In some embodiments, the isolated anti-PD-Ll antibody is aglycosylated.
Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X- serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites form an antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) is removed. The alteration may be made by substitution of an asparagine, serine or threonine residue within the glycosylation site another amino acid residue (e.g., glycine, alanine or a conservative substitution).
[0160] In any of the embodiments herein, the isolated anti-PD-Ll antibody can bind to a human PD-L1, for example a human PD-L1 as shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1, or a variant thereof.
[0161] In a still further embodiment, provided is an isolated nucleic acid encoding any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for expression of the nucleic acid encoding any of the previously described anti-PDLl antibodies. In a still further specific aspect, the vector is in a host cell suitable for expression of the nucleic acid. In a still further specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In a still further specific aspect, the eukaryotic cell is a mammalian cell, such as Chinese hamster ovary (CHO) cell.
[0162] The antibody or antigen binding fragment thereof, may be made using methods known in the art, for example, by a process comprising culturing a host cell containing nucleic acid encoding any of the previously described anti-PDLl antibodies or antigen- binding fragment in a form suitable for expression, under conditions suitable to produce such antibody or fragment, and recovering the antibody or fragment.
III. Anti-CEA/anti-CD3 bispecific Antibodies
[0163] Provided herein is a method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody. Also provided herein is a method of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody.
[0164] Provided herein are bispecific antibodies that bind to a human carcinoembryonic antigen (CEA). Alternative names for "CEA" include CEACAM5. The term "CEA" as used herein, refers to any native CEA from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses "full-length" and unprocessed CEA as well as any form of CEA that results from processing in the cell (e.g., mature protein). The term also encompasses naturally occurring variants and isoforms of CEA, e.g., splice variants or allelic variants. In one embodiment, CEA is human CEA. The amino acid sequence of human CEA is shown in UniProt (www.uniprot.org) accession no. P06731, or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004354.2.
[0165] In some embodiments, the antigen binding domain of a bispecific antibody that binds to CEA comprises a heavy chain variable region (VHCEA) comprising the amino acid sequence:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTK TGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMD YWGQGTTVTVSS (SEQ ID NO:34), and /or
a light chain variable region (VLCEA) comprising the amino acid sequence:
DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIK (SEQ ID NO:35).
[0166] In some embodiments, the antigen binding domain of a bispecific antibody that binds to CEA comprises a heavy chain variable region comprising an HVR-H1 sequence of EFGMN (SEQ ID NO:38), an HVR-H2 sequence of WINTKTGEATYVEEFKG (SEQ ID NO:39), and an HVR-H3 sequence of WDFAYYVEAMDY (SEQ ID NO:40) and a light chain variable region comprising an HVR-L1 sequence of KASAAVGTYVA (SEQ ID NO:41), an HVR-L2 sequence of SASYRKR (SEQ ID NO:42), and an HVR-L3 sequence of HQYYTYPLFT (SEQ ID NO:43).
[0167] In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds a human CD3 polypeptide, and a second antigen binding domain that binds CEA. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody further comprises a third antigen binding domain that binds CEA. In some embodiments, the second and the third antigen binding domain are identical (i.e. have the same amino acid sequences)
[0168] CD3 (cluster of differentiation 3) T-cell co-receptor is a protein complex and is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD35 chain, and two CD3e chains. These chains associate with the T-cell receptor (TCR) and the ζ-chain to generate an activation signal in T lymphocytes. The TCR, ζ-chain, and CD3 molecules together form the TCR complex. The term "CD3" as used herein, refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses "full-length" and unprocessed protein as well as any form of the protein or one or more of the CD3 chains (polypeptides) that result from processing in the cell (e.g., mature polypeptides). The term also encompasses naturally occurring variants and isoforms of CD3, e.g., splice variants or allelic variants. For example, descriptions of CD3y, CD35, and CD3e chains and sequences are provided at
www.uniprot.org/uniprot/P04234, www.uniprot.org/uniprot/P07766, and
www.uniprot.org/uniprot/P09693. In one embodiment, CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3s). The amino acid sequence of human CD3s is shown in UniProt (www.uniprot.org) accession no. P07766 (version 144), or NCBI
(www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. The amino acid sequence of cynomolgus [Macaca fascicularis] CD3s is shown in NCBI GenBank no. BAB71849.1.
[0169] In some embodiments, the bispecific antibody binds to a human CD3 epsilon (CD3e) polypeptide. In some embodiments, the bispecific antibody binds to a human CD3 epsilon polypeptide in native T-cell receptor (TCR) complex in association with other TCR subunits. In some embodiments, the bispecific antibody binds to a human CD3 gamma (CD3y) polypeptide. In some embodiments, the bispecific antibody binds a human CD3 gamma polypeptide in native T-cell receptor (TCR) complex in association with other TCR subunits.
[0170] In one aspect, assays are provided for identifying anti-CD3 antibodies thereof having biological activity. Biological activity may include, for example, binding to a CD3 polypeptide (e.g., CD3 on the surface of a T cell), or a peptide fragment thereof, either in vivo, in vitro, or ex vivo. In the case of a multispecific (e.g., bispecific) anti-CD3 antibody of the invention (e.g., a TCB antibody having an anti-CEA binding domain and a binding domain that recognizes a CD3 polypeptide), biological activity may also include, for example, effector cell activation (e.g., T cell (e.g., CD8+ and/or CD4+ T cell) activation), effector cell population expansion (i.e., an increase in T cell count), target cell population reduction (i.e., a decrease in the population of cells expressing CEA on their cell surfaces), and/or target cell killing. Antibodies having such biological activity in vivo and/or in vitro are provided. In certain embodiments, an antibody of the invention is tested for such biological activity.
[0171] In some embodiments, the antigen binding domain(s) of a bispecific antibody that binds to a CEA comprises a heavy chain variable region (VHCEA) comprising the amino acid sequence of SEQ ID NO:34, and a light chain variable region (VLCEA) comprising the amino acid sequence of SEQ ID NO:35. In some embodiments, the antigen binding domain(s) that binds to a CEA comprises a heavy chain variable region comprising an HVR-H1 sequence of EFGMN (SEQ ID NO:38), an HVR-H2 sequence of WINTKTGEATYVEEFKG (SEQ ID NO:39), and an HVR-H3 sequence of WDFAYYVEAMDY (SEQ ID NO:40) and comprises a light chain variable region comprising an HVR-L1 sequence of KASAAVGTYVA (SEQ ID NO:41), an HVR-L2 sequence of SASYRKR (SEQ ID NO:42), and an HVR-L3 sequence of HQYYTYPLFT (SEQ ID NO:43).
[0172] In some embodiments, the antigen binding domain of a bispecific antibody that binds to a CD3 comprises a heavy chain variable region (VHCD3) comprising the amino acid sequence of SEQ ID NO:50, and a light chain variable region (VLCD3) comprising the amino acid sequence of SEQ ID NO:51. In some embodiments, the antigen binding domain that binds to a CD3 comprises a heavy chain variable region comprising an HVR-H1 sequence of TYAMN (SEQ ID NO:44), an HVR-H2 sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO:45), and an HVR-H3 sequence of HGNFGNSYVSWFAY (SEQ ID NO:46) and comprises a light chain comprising an HVR-L1 sequence of GSSTGAVTTSNYAN (SEQ ID NO:47), an HVR-L2 sequence of GTNKRAP (SEQ ID NO:48), and an HVR-L3 sequence of ALWYSNLWV (SEQ ID NO:49).
[0173] In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules (e.g. conventional or crossover Fab molecules). In particular such embodiments, the bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the first antigen binding domain is a crossover Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other) and the second (and third, if present) antigen binding domain is a conventional Fab molecule. In one embodiment, the first antigen binding domain is a crossover Fab molecule wherein the constant domains of the Fab heavy and light chain are exchanged. In one embodiment, the anti-CEA/anti-CD3 bispecific antibody comprises not more than one antigen binding domain that binds to CD3, i.e. provides monovalent binding to CD3. In one embodiment, the first and the second antigen binding domain are fused to each other, optionally through a peptide linker. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3 and a second and optionally a third antigen binding domain that binds to CEA, wherein the antigen binding domains are Fab molecules and (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain. In a particular embodiment, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding domain. Optionally, the Fab light chain of the first antigen binding domain and the Fab light chain of the second antigen binding domain may additionally be fused to each other.
[0174] In some embodiments, the anti-CEA/anti-CD3 bispecific antibody further comprises an Fc domain composed of a first and a second subunit capable of stable association. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3, a second antigen binding domain that binds to CEA, and an Fc domain composed of a first and a second subunit capable of stable association, wherein the antigen binding domains are Fab molecules and wherein (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, and the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the first or second subunit of the Fc domain. In a particular embodiment, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody essentially consists of a first antigen binding domain that binds to CD3, a second antigen binding domain that binds to CEA, an Fc domain composed of a first and a second subunit capable of stable association, and optionally one or more peptide linkers, wherein the antigen binding domains are Fab molecules and wherein (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, and the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
[0175] In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3, a second and a third antigen binding domain that binds to CEA, and an Fc domain composed of a first and a second subunit capable of stable association, wherein the antigen binding domains are Fab molecules and wherein (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In a particular embodiment, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C- terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody essentially consists of a first antigen binding domain that binds to CD3, a second and a third antigen binding domain that binds to CEA, an Fc domain composed of a first and a second subunit capable of stable association, and optionally one or more peptide linkers, wherein the antigen binding domains are Fab molecules and wherein (i) the second antigen binding domain is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
[0176] In one embodiment the anti-CEA/anti-CD3 bispecific antibody comprises
(i) a first antigen binding domain that binds to CD3, comprising a heavy chain variable region (VHCD3) comprising HVR-H1 sequence of SEQ ID NO:44, HVR-H2 sequence of SEQ ID NO:45, and HVR-H3 sequence of SEQ ID NO:46; and a light chain variable region (VLCD3) comprising HVR-L1 sequence of SEQ ID NO:47, HVR-L2 sequence of SEQ ID NO:48, and HVR-L3 sequence of SEQ ID NO:49, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;
(ii) a second and a third antigen binding domain that bind to CEA, comprising a heavy chain variable region (VHCEA) comprising HVR-H1 sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3 sequence of SEQ ID NO:40; and a light chain variable region (VLCEA) comprising HVR-L1 sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ ID NO:42, and HVR-L3 sequence of SEQ ID NO:43, wherein the second and third antigen binding moiety are each a Fab molecule, particularly a conventional Fab molecule;
(iii) an Fc domain composed of a first and a second subunit capable of stable association, wherein the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
[0177] The Fab molecules may be fused to the Fc domain or to each other directly or through a peptide linker, comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable, non- immunogenic peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n or G4(SG4)n peptide linkers, "n" is generally an integer from 1 to 10, typically from 2 to 4. In one embodiment said peptide linker has a length of at least 5 amino acids, in one embodiment a length of 5 to 100, in a further embodiment of 10 to 50 amino acids. In one embodiment said peptide linker is (GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, n= 3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m= 0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in a further embodiment x=4 and n=2. In one embodiment said peptide linker is (G4S)2. A particularly suitable peptide linker for fusing the Fab light chains of the first and the second antigen binding domain to each other is (G4S)2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and the second antigen binding domain comprises the sequence (D)-(G4S)2. Another suitable such linker comprises the sequence (G4S)4. Additionally, linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where a Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.
[0178] In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises (i) a polypeptide wherein the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with a first Fc domain subunit (VH(i)-CHl(i)- VH(2)-CL(2)-CH2-CH3(-CH4)), (ii) a crossover Fab light chain polypeptide of the second Fab molecule, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL(2)-CH1(2)), (iii) the Fab light chain polypeptide of the first Fab molecule (VL(i)- CL(i)), (iv) a polypeptide wherein the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with a second Fc domain subunit (VH(3)-CH1(3)-CH2-CH3(- CH4)) and (v) the Fab light chain polypeptide of the third Fab molecule (VL(3)-CL(3)). In certain embodiments, the Fab light chain polypeptide of the first and the third Fab molecule are identical. In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond. In certain embodiments, the first and the third Fab molecule bind to CEA, and the second Fab molecule binds to CD3. [0179] In one embodiment the anti-CEA/anti-CD3 bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 52, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 53, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 54, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55. In one embodiment, the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 52, a polypeptide comprising the sequence of SEQ ID NO: 53, a polypeptide comprising the sequence of SEQ ID NO: 54, and a polypeptide comprising the sequence of SEQ ID NO: 55.
Fc domain
[0180] The anti-CEA/anti-CD3 bispecific antibody used in the present invention may comprise an Fc domain which consists of a pair of polypeptide chains comprising heavy chain domains of an antibody molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other.
[0181] In one embodiment, the Fc domain is an IgG Fc domain. In a particular embodiment the Fc domain is an IgGi Fc domain. In another embodiment the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (EU numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment the Fc domain is human. An exemplary sequence of a human IgGi Fc region is given in SEQ ID NO: 57.
Fc domain modifications promoting heterodimerization
[0182] The anti-CEA/anti-CD3 bispecific antibody used in the present invention may comprise different components (e.g. antigen binding domains) fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of such antibodies in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antibody a modification promoting the association of the desired polypeptides.
[0183] Accordingly, in particular embodiments the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain.
[0184] There exist several approaches for modifications in the CH3 domain of the Fc domain in order to enforce heterodimerization, which are well described e.g. in
WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901,
WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed).
[0185] In a specific embodiment said modification promoting the association of the first and the second subunit of the Fc domain is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other one of the two subunits of the Fc domain.
[0186] The knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
[0187] Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
[0188] Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).
[0189] Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
[0190] The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
[0191] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain
(the "knobs" subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the "hole" subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (EU numbering).
[0192] In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (EU numbering). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
[0193] In a particular embodiment, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (EU numbering).
[0194] In a particular embodiment the antigen binding domain that binds to CD3 in the anti-CEA/anti-CD3 bispecific antibody described herein is fused to the first subunit of the Fc domain (comprising the "knob" modification). Without wishing to be bound by theory, fusion of the CD3 binding domain to the knob-containing subunit of the Fc domain will (further) minimize the generation of bispecific antibodies comprising two CD3 binding domains (steric clash of two knob-containing polypeptides).
[0195] Other techniques of CH3-modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g. in
WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901,
WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545,
WO 2012/058768, WO 2013/157954, WO 2013/096291.
[0196] In one embodiment the heterodimerization approach described in EP 1870459 Al, is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain. One preferred embodiment are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (EU numbering).
[0197] In another embodiment the antibody comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (EU numbering).
[0198] In another embodiment the antibody comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to EU numbering).
[0199] In one embodiment the heterodimerization approach described in WO 2013/157953 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (EU numbering). In a further embodiment the first CH3 domain comprises further amino acid mutation L351K. In a further embodiment the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (EU numbering). [0200] In one embodiment the heterodimerization approach described in WO 2012/058768 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N, T411R, T411Q, T41 IK, T41 ID, T41 IE or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (EU numbering). In a further embodiment a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F. In a further embodiment a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (EU numbering).
[0201] In one embodiment the heterodimerization approach described in WO 2011/143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (EU numbering).
[0202] In one embodiment the heterodimerization approach described in WO 2011/090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A. In one embodiment a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (EU numbering).
[0203] In one embodiment the antibody or its Fc domain is of IgG2 subclass and the heterodimerization approach described in WO 2010/129304 is used alternatively.
[0204] In an alternative embodiment a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In one such embodiment a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, and more preferably D399K and E356K). In a further embodiment the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). In a further embodiment the first CH3 domain further or alternatively comprises amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to EU numbering).
[0205] In yet a further embodiment the heterodimerization approach described in WO 2007/147901 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (EU numbering).
[0206] In still another embodiment the heterodimerization approach described in WO 2007/110205 can be used alternatively.
[0207] In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (EU numbering).
Fc domain modifications reducing Fc receptor binding and/or effector function
[0208] The Fc domain confers to an antibody, such as a bispecific antibody, favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the antibody to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with other immuno stimulatory properties the antibody may have and the long half-life of the antibody, results in excessive activation of cytokine receptors and severe side effects upon systemic administration.
[0209] Accordingly, in particular embodiments, the Fc domain of the anti-CEA/anti-CD3 bispecific antibody exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain. In one such embodiment the Fc domain (or the molecule, e.g. antibody, comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc domain (or a corresponding molecule comprising a native IgGi Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgGi Fc domain domain (or a corresponding molecule comprising a native IgGi Fc domain). In one embodiment, the Fc domain (or the molecule, e.g. antibody, comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function. In a particular embodiment the Fc receptor is an Fey receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one embodiment the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC. In one embodiment the Fc domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the molecule, e.g. antibody, comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or the corresponding molecule comprising a native IgGi Fc domain) to FcRn.
[0210] In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5- fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment the molecule, e.g. antibody, comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a corresponding molecule comprising a non-engineered Fc domain. In a particular embodiment the Fc receptor is an Fey receptor. In some embodiments the Fc receptor is a human Fc receptor. In some embodiments the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
Preferably, binding to each of these receptors is reduced. In some embodiments binding affinity to a complement component, specifically binding affinity to Clq, is also reduced. In one embodiment binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the molecule, e.g. antibody, comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or a corresponding molecule comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or molecule (e.g. antibody) comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody- dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a corresponding molecule comprising a non-engineered Fc domain).
[0211] In one embodiment the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (EU numbering). In a more specific embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (EU numbering). In some embodiments the Fc domain comprises the amino acid substitutions L234A and L235A (EU numbering). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment the amino acid substitution is P329A or P329G, particularly P329G (EU numbering). In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (EU numbering). In a more specific embodiment the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 EU numbering). In more particular embodiments the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA"). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety. WO 2012/130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.
[0212] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgGi antibodies. Hence, in some embodiments the Fc domain is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment the IgG4 Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P (EU numbering). To further reduce its binding affinity to an Fc receptor and/or its effector function, in one embodiment the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (EU numbering). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (EU
numbering). In a particular embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (EU numbering). Such IgG4 Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
[0213] In a particular embodiment the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain, is a human IgGi Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (EU numbering). [0214] In certain embodiments N-glycosylation of the Fc domain has been eliminated. In one such embodiment the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) or glycine (N297G) (EU numbering).
[0215] In addition to the Fc domains described hereinabove and in PCT publication no. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (EU numbering). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
[0216] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.
[0217] Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression.
Alternatively, binding affinity of Fc domains or molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.
[0218] Effector function of an Fc domain, or a molecule (e.g. an antibody) comprising an Fc domain, can be measured by methods known in the art. A suitable assay for measuring ADCC is described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499- 1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc.
Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). [0219] In some embodiments, binding of the Fc domain to a complement component, specifically to Clq, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. Clq binding assays may be carried out to determine whether the Fc domain, or molecule (e.g. antibody) comprising the Fc domain, is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045- 1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).
[0220] In some embodiments, the bispecific antibody is a single-chain bispecific antibody comprising the first antigen binding domain and the second antigen binding domain. In some embodiments, the single-chain bispecific antibody comprises variable regions, as arranged from N-terminus to C-terminus, selected from the group consisting of (1) VHCEA-VLCEA- VHCD3-VLCD3, (2) VHCD3-VLCD3-VHCEA-VLCEA, (3) VHCD3-VLCD3-VLCEA-VHCEA, (4) VHCEA-VLCEA-VLCD3-VHCD3, (5) VLCEA-VHCEA-VHCD3-VLCD3, and (6) VLCD3- VHCD3-VHCEA-VLCEA.
IV. Antibody Preparation
[0221] Antibodies described herein are prepared using techniques available in the art for generating antibodies, exemplary methods of which are described in more detail in the following sections.
[0222] The antibody is directed against an antigen of interest (i.e., PD-L1 (such as a human PD-L1), CEA, or CD3 (such as a human CD3)). Preferably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disorder can result in a therapeutic benefit in that mammal.
[0223] In certain embodiments, an antibody provided herein has a dissociation constant (KD) of < ΙμΜ, < 150 nM, < 100 nM, < 50 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10"8 M or less, e.g. from 10"8 M to 10"13 M, e.g., from 10"9 M to 10"13 M).
[0224] In one embodiment, KD is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution binding affinity of Fabs for antigen is measured by equilibrating
Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100
125
pM or 26 pM [ I] -antigen are mixed with serial dilutions of a Fab of interest. The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20
(TWEEN-20®) in PBS. When the plates have dried, 150 μΐ/well of scintillant
(MICROSCINT-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.
[0225] According to another embodiment, KD is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE ®-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 iV-ethyl-iV'- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier' s instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (-0.2 μΜ) before injection at a flow rate of 5 μΐ/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μΐ/min. Association rates (kon) and dissociation rates (k0ff) are calculated using a simple one-to-one Langmuir binding model (BIACORE ® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio k0ff/kon See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M" 1 s" 1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer
(ThermoSpectronic) with a stirred cuvette.
(i) Antigen Preparation
[0226] Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for generating antibodies. For transmembrane molecules, such as receptors, fragments of these (e.g. the extracellular domain of a receptor) can be used as the immunogen. Alternatively, cells expressing the transmembrane molecule can be used as the immunogen. Such cells can be derived from a natural source (e.g. cancer cell lines) or may be cells which have been transformed by recombinant techniques to express the transmembrane molecule. Other antigens and forms thereof useful for preparing antibodies will be apparent to those in the art.
(ii) Certain Antibody-Based Methods
[0227] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example,
maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N- hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or RXN=C=NR, where R and R1 are different alkyl groups.
[0228] Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
[0229] Monoclonal antibodies can be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), and further described, e.g., in Hongo et al, Hybridoma, 14 (3): 253-260 (1995), Harlow et ah, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et ah, in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), and Ni, Xiandai
Mianyixue, 26(4):265-268 (2006) regarding human-human hybridomas. Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 regarding production of monoclonal human natural IgM antibodies from hybridoma cell lines. Human hybridoma technology (Trioma technology) is described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).
[0230] For various other hybridoma techniques, see, e.g., US 2006/258841; US
2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US
2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507. An exemplary protocol for producing monoclonal antibodies using the hybridoma method is described as follows. In one embodiment, a mouse or other appropriate host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of the invention or a fragment thereof, and an adjuvant, such as monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.). A
polypeptide of the invention (e.g., antigen) or a fragment thereof may be prepared using methods well known in the art, such as recombinant methods, some of which are further described herein. Serum from immunized animals is assayed for anti-antigen antibodies, and booster immunizations are optionally administered. Lymphocytes from animals producing anti-antigen antibodies are isolated. Alternatively, lymphocytes may be immunized in vitro.
[0231] Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. See, e.g., Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma cells may be used that fuse efficiently, support stable high-level production of antibody by the selected antibody- producing cells, and are sensitive to a medium such as HAT medium. Exemplary myeloma cells include, but are not limited to, murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[0232] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, e.g., a medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferably, serum-free hybridoma cell culture methods are used to reduce use of animal- derived serum such as fetal bovine serum, as described, for example, in Even et al. , Trends in Biotechnology, 24(3), 105-108 (2006).
[0233] Oligopeptides as tools for improving productivity of hybridoma cell cultures are described in Franek, Trends in Monoclonal Antibody Research, 111-122 (2005). Specifically, standard culture media are enriched with certain amino acids (alanine, serine, asparagine, proline), or with protein hydrolyzate fractions, and apoptosis may be significantly suppressed by synthetic oligopeptides, constituted of three to six amino acid residues. The peptides are present at millimolar or higher concentrations.
[0234] Culture medium in which hybridoma cells are growing may be assayed for production of monoclonal antibodies that bind to an antibody of the invention. The binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay (ELISA). The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis. See, e.g., Munson et al., Anal. Biochem., 107:220 (1980).
[0235] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. See, e.g., Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in an animal. Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. One procedure for isolation of proteins from hybridoma cells is described in US 2005/176122 and U.S. Pat. No. 6,919,436. The method includes using minimal salts, such as lyotropic salts, in the binding process and preferably also using small amounts of organic solvents in the elution process.
(iii) Library-Derived Antibodies
[0236] Antibodies useful in the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Additional methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).
[0237] In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol, 12: 433-455 (1994). Phage typically display antibody fragments, either as single- chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,
2007/0237764, 2007/0292936, and 2009/0002360.
[0238] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
(iv) Chimeric, Humanized and Human Antibodies [0239] In certain embodiments, an antibody provided herein is a chimeric antibody.
Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
[0240] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
[0241] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in
Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'lAcad. Sci. USA 86: 10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).
[0242] Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol, 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).
[0243] In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).
[0244] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous
immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
[0245] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol, 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005). [0246] Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
(v) Antibody Fragments
[0247] Antibody fragments may be generated by traditional means, such as enzymatic digestion, or by recombinant techniques. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9: 129-134.
[0248] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab') 2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab') 2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No.
5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibodies may be monospecific or bispecific.
(vi) Multispecific Antibodies
[0249] Multispecific antibodies have binding specificities for at least two different epitopes, where the epitopes are usually from different antigens. While such molecules normally will only bind two different epitopes (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies).
[0250] Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two
immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J, 10:3655-3659 (1991).
[0251] One approach known in the art for making bispecific antibodies is the "knobs-into- holes" or "protuberance-into-cavity" approach (see, e.g., US Pat. No. 5,731,168). In this approach, two immunoglobulin polypeptides {e.g., heavy chain polypeptides) each comprise an interface. An interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thereby allowing the two
immunoglobulin polypeptides to associate. These interfaces may be engineered such that a "knob" or "protuberance" (these terms may be used interchangeably herein) located in the interface of one immunoglobulin polypeptide corresponds with a "hole" or "cavity" (these terms may be used interchangeably herein) located in the interface of the other
immunoglobulin polypeptide. In some embodiments, the hole is of identical or similar size to the knob and suitably positioned such that when the two interfaces interact, the knob of one interface is positionable in the corresponding hole of the other interface. Without wishing to be bound to theory, this is thought to stabilize the heteromultimer and favor formation of the heteromultimer over other species, for example homomultimers. In some embodiments, this approach may be used to promote the heteromultimerization of two different immunoglobulin polypeptides, creating a bispecific antibody comprising two immunoglobulin polypeptides with binding specificities for different epitopes.
[0252] In some embodiments, a knob may be constructed by replacing a small amino acid side chain with a larger side chain. In some embodiments, a hole may be constructed by replacing a large amino acid side chain with a smaller side chain. Knobs or holes may exist in the original interface, or they may be introduced synthetically. For example, knobs or holes may be introduced synthetically by altering the nucleic acid sequence encoding the interface to replace at least one "original" amino acid residue with at least one "import" amino acid residue. Methods for altering nucleic acid sequences may include standard molecular biology techniques well known in the art. The side chain volumes of various amino acid residues are shown in the following table. In some embodiments, original residues have a small side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and import residues for forming a knob are naturally occurring amino acids and may include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, original residues have a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), and import residues for forming a hole are naturally occurring amino acids and may include alanine, serine, threonine, and valine.
Table 1. Properties of amino acid residues
Amino acid One-letter Massa Volume Accessible abbreviation (daltons) (A3) surface area0
(A2)
Alanine (Ala) A 71.08 88.6 115
Arginine (Arg) R 156.20 173.4 225
Asparagine (Asn) N 114.11 117.7 160
Aspartic Acid (Asp) D 115.09 111.1 150
Cysteine (Cys) C 103.14 108.5 135
Glutamine (Gin) Q 128.14 143.9 180
Glutamic Acid (Glu) E 129.12 138.4 190
Glycine (Gly) G 57.06 60.1 75
Histidine (His) H 137.15 153.2 195
Isoleucine (He) I 113.17 166.7 175
Leucine (Leu) L 113.17 166.7 170
Lysine (Lys) K 128.18 168.6 200
Methionine (Met) M 131.21 162.9 185
Phenylalanine (Phe) F 147.18 189.9 210
Proline (Pro) P 97.12 122.7 145
Serine (Ser) S 87.08 89.0 115 Amino acid One-letter Massa Volume Accessible abbreviation (daltons) (A3) surface area0
(A2)
Threonine (Thr) T 101.11 116.1 140
Tryptophan (Trp) W 186.21 227.8 255
Tyrosine (Tyr) Y 163.18 193.6 230
Valine (Val) V 99.14 140.0 155 aMolecular weight of amino acid minus that of water. Values from Handbook of Chemistry and Physics, 43rd ed. Cleveland, Chemical Rubber Publishing Co., 1961.
bValues from A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972.
cValues from C. Chothia, J. Mol. Biol. 105: 1-14, 1975. The accessible surface area is defined in Figures 6-20 of this reference.
[0253] In some embodiments, original residues for forming a knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining a three-dimensional structure may include X-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of an immunoglobulin constant domain. In these embodiments, the CH3/CH3 interface of human IgGi involves sixteen residues on each domain located on four anti-parallel β-strands. Without wishing to be bound to theory, mutated residues are preferably located on the two central anti-parallel β-strands to minimize the risk that knobs can be accommodated by the surrounding solvent, rather than the compensatory holes in the partner CH3 domain. In some embodiments, the mutations forming corresponding knobs and holes in two immunoglobulin polypeptides correspond to one or more pairs provided in the following table.
Table 2. Exemplary sets of corresponding knob-and hole-forming mutations
CH3 of first immunoglobulin CH3 of second immunoglobulin
T366Y Y407T
T366W Y407A
F405A T394W
Y407T T366Y
T366Y:F405A T394W:Y407T
T366W:F405W T394S:Y407A
F405W:Y407A T366W:T394S
F405W T394S Mutations are denoted by the original residue, followed by the position using the EU numbering system, and then the import residue (all residues are given in single-letter amino acid code). Multiple mutations are separated by a colon.
[0254] In some embodiments, an immunoglobulin polypeptide comprises a CH3 domain comprising one or more amino acid substitutions listed in Table 2 above. In some embodiments, a bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising one or more amino acid substitutions listed in the left column of Table 2, and a second immunoglobulin polypeptide comprising a CH3 domain comprising one or more corresponding amino acid substitutions listed in the right column of Table 2.
[0255] Following mutation of the DNA as discussed above, polynucleotides encoding modified immunoglobulin polypeptides with one or more corresponding knob- or hole- forming mutations may be expressed and purified using standard recombinant techniques and cell systems known in the art. See, e.g., U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; U.S. Pub. No. 2013/0089553; and Spiess et al., Nature Biotechnology 31: 753-758, 2013. Modified immunoglobulin polypeptides may be produced using prokaryotic host cells, such as E. coli, or eukaryotic host cells, such as CHO cells. Corresponding knob- and hole-bearing immunoglobulin polypeptides may be expressed in host cells in co-culture and purified together as a heteromultimer, or they may be expressed in single cultures, separately purified, and assembled in vitro. In some embodiments, two strains of bacterial host cells (one expressing an immunoglobulin polypeptide with a knob, and the other expressing an immunoglobulin polypeptide with a hole) are co-cultured using standard bacterial culturing techniques known in the art. In some embodiments, the two strains may be mixed in a specific ratio, e.g., so as to achieve equal expression levels in culture. In some embodiments, the two strains may be mixed in a 50:50, 60:40, or 70:30 ratio. After polypeptide expression, the cells may be lysed together, and protein may be extracted. Standard techniques known in the art that allow for measuring the abundance of homo-multimeric vs. hetero-multimeric species may include size exclusion chromatography. In some embodiments, each modified immunoglobulin polypeptide is expressed separately using standard recombinant techniques, and they may be assembled together in vitro.
Assembly may be achieved, for example, by purifying each modified immunoglobulin polypeptide, mixing and incubating them together in equal mass, reducing disulfides (e.g., by treating with dithiothreitol), concentrating, and reoxidizing the polypeptides. Formed bispecific antibodies may be purified using standard techniques including cation-exchange chromatography and measured using standard techniques including size exclusion chromatography. For a more detailed description of these methods, see Spiess et al., Nat Biotechnol 31:753-8, 2013. In some embodiments, modified immunoglobulin polypeptides may be expressed separately in CHO cells and assembled in vitro using the methods described above.
[0256] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is typical to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
[0257] In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
[0258] According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. One interface comprises at least a part of the CH 3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end- products such as homodimers.
[0259] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
[0260] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytic ally cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
[0261] Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
[0262] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol, 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444- 6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368 (1994).
[0263] Another technique for making bispecific antibody fragments is the "bispecific T cell engager" or BiTE® approach (see, e.g., WO2004/ 106381, WO2005/061547,
WO2007/042261, and WO2008/119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain includes two single chain Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of a length sufficient to allow intramolecular association between the two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different cell types, such that cells of two different cell types are brought into close proximity or tethered when each scFv is engaged with its cognate epitope. One particular embodiment of this approach includes a scFv recognizing a cell-surface antigen expressed by an immune cell, e.g., a CD3 polypeptide on a T cell, linked to another scFv that recognizes a cell-surface antigen expressed by a target cell, such as a malignant or tumor cell.
[0264] As it is a single polypeptide, the bispecific T cell engager may be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., a CHO cell line. However, specific purification techniques (see, e.g., EP1691833) may be necessary to separate monomeric bispecific T cell engagers from other multimeric species, which may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing secreted polypeptides is first subjected to a metal affinity chromatography, and polypeptides are eluted with a gradient of imidazole
concentrations. This eluate is further purified using anion exchange chromatography, and polypeptides are eluted using with a gradient of sodium chloride concentrations. Finally, this eluate is subjected to size exclusion chromatography to separate monomers from multimeric species.
[0265] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60 (1991).
(vii) Single-Domain Antibodies
[0266] A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 Bl). In one embodiment, a single-domain antibody consists of all or a portion of the heavy chain variable domain of an antibody.
(viii) Antibody Variants
[0267] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made.
(ix) Substitution, Insertion, and Deletion Variants
[0268] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Table 3. Exemplary Substitutions.
Figure imgf000092_0001
[0269] Amino acids may be grouped according to common side-chain properties:
a. hydrophobic: Norleucine, Met, Ala, Val, Leu, He;
b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
c. acidic: Asp, Glu;
d. basic: His, Lys, Arg;
e. residues that influence chain orientation: Gly, Pro;
f. aromatic: Trp, Tyr, Phe.
[0270] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. [0271] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
[0272] Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
[0273] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
[0274] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
[0275] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
(x) Glycosylation variants
[0276] In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
[0277] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The
oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
[0278] In one embodiment, antibody variants are provided comprising an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. Specifically, antibodies are contemplated herein that have reduced fucose relative to the amount of fucose on the same antibody produced in a wild-type CHO cell. That is, they are characterized by having a lower amount of fucose than they would otherwise have if produced by native CHO cells (e.g., a CHO cell that produce a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene). In certain embodiments, the antibody is one wherein less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon comprise fucose. For example, the amount of fucose in such an antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. In certain embodiments, the antibody is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the antibody is completely without fucose, or has no fucose or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about + 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6- fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al.
Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).
[0279] Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by
GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean- Mairet et al.); US Patent No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5): 851-861 (2006). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0280] In certain embodiments, the antibody variants comprising an Fc region described herein are capable of binding to an FcyRIII. In certain embodiments, the antibody variants comprising an Fc region described herein have ADCC activity in the presence of human effector cells or have increased ADCC activity in the presence of human effector cells compared to the otherwise same antibody comprising a human wild-type IgGlFc region,
(xi) Fc region variants
[0281] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
[0282] In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.
166: 1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'lAcad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101: 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759- 1769 (2006)).
[0283] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
[0284] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)
[0285] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). In an exemplary embodiment, the antibody comprising the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.
[0286] In some embodiments, alterations are made in the Fc region that result in altered {i.e., either improved or diminished) Clq binding and/or Complement Dependent
Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[0287] Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.)). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
(xii) Antibody Derivatives
[0288] The antibodies used in the invention can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. In certain embodiments, the moieties suitable for derivatization of the antibody are water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1,3- dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
(xiii) Vectors, Host Cells, and Recombinant Methods
[0289] Antibodies may also be produced using recombinant methods. For recombinant production of an anti-antigen antibody, nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
(a) Signal Sequence Component
[0290] An antibody may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process a native antibody signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, a factor leader (including Saccharomyces and Kluyveromyces a-factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available.
(b) Origin of Replication
[0291] Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria, the 2μ, plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the 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.
(c) Selection Gene Component
[0292] Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. [0293] 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.
[0294] Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up antibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
[0295] For example, cells transformed with the DHFR gene are identified by culturing the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other co- transformed nucleic acid. A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFR activity (e.g., ATCC CRL-9096) may be used.
[0296] Alternatively, cells transformed with the GS gene are identified by culturing the transformants in a culture medium containing L-methionine sulfoximine (Msx), an inhibitor of GS. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acid. The GS selection/amplification system may be used in combination with the DHFR selection/amplification system described above.
[0297] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody of interest, wild-type DHFR gene, and another selectable marker such as aminoglycoside 3'- phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.
[0298] A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et ah, Nature, 282:39 (1979)). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly, Lew2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leul gene.
[0299] In addition, vectors derived from the 1.6 μιη circular plasmid pKDl can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8: 135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
(d) Promoter Component
[0300] Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding an antibody. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding an antibody.
[0301] Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
[0302] Examples of suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
[0303] Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde- 3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.
[0304] Antibody transcription from vectors in mammalian host cells can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
[0305] 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 Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et ah, Nature 297:598- 601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.
(e) Enhancer Element Component
[0306] Transcription of a DNA encoding an antibody of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a- fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100- 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
(f) Transcription Termination Component
[0307] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will 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 antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See W094/11026 and the expression vector disclosed therein.
(g) Selection and Transformation of Host Cells [0308] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 4 IP disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
[0309] Full length antibody, antibody fusion proteins, and antibody fragments can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) that by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half-life in circulation. Production in E. coli is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.
5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523 (Simmons et al.), which describes translation initiation region (TIR) and signal sequences for optimizing expression and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli. After expression, the antibody may be isolated from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g., in CHO cells.
[0310] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K.
thermotolerans, Άηά Κ. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. For a review discussing the use of yeasts and filamentous fungi for the production of therapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22: 1409-1414 (2004).
[0311] Certain fungi and yeast strains may be selected in which glycosylation pathways have been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describing humanization of the glycosylation pathway in Pichia pastoris); and Gerngross et al., supra.
[0312] Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa calif ornica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the invention, particularly for transfection of Spodoptera frugiperda cells.
[0313] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed {Leninaceae), alfalfa (M. truncatula), and tobacco can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing
PLANTIBODIES™ technology for producing antibodies in transgenic plants).
[0314] Vertebrate cells may be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals NY. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR" CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268.
[0315] 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.
(h) Culturing the Host Cells
[0316] The host cells used to produce an antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et ah, Meth. Enz. 58:44 (1979), Barnes et ah, Anal. Biochem.
102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ 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.
(xiv) Purification of Antibody
[0317] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, 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. Carter et ah, Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. 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.
[0318] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being among one of the typically preferred purification steps. 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 γΐ, γ2, or γ4 heavy chains (Lindmark et ah, J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et ah, 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. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin
SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
[0319] In general, various methodologies for preparing antibodies for use in research, testing, and clinical are well-established in the art, consistent with the above-described methodologies and/or as deemed appropriate by one skilled in the art for a particular antibody of interest.
C. Selecting Biologically Active Antibodies
[0320] Antibodies produced as described above may be subjected to one or more
"biological activity" assays to select an antibody with beneficial properties from a therapeutic perspective or selecting formulations and conditions that retain biological activity of the antibody. The antibody may be tested for its ability to bind the antigen against which it was raised. For example, methods known in the art (such as ELISA, Western Blot, etc.) may be used. [0321] For example, for an anti-PD-Ll antibody, the antigen binding properties of the antibody can be evaluated in an assay that detects the ability to bind to PD-L1. In some embodiments, the binding of the antibody may be determined by saturation binding; ELISA; and/or competition assays (e.g. RIA's), for example. Also, the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic. Such assays are known in the art and depend on the target antigen and intended use for the antibody. For example, the biological effects of PD-L1 blockade by the antibody can be assessed in CD8+T cells, a lymphocytic choriomeningitis virus (LCMV) mouse model and/or a syngeneic tumor model e.g., as described in US Patent 8,217,149.
[0322] To screen for antibodies which bind to a particular epitope on the antigen of interest (e.g., those which block binding of the anti-PD-Ll antibody of the example to PD-L1), a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed.
Alternatively, epitope mapping, e.g. as described in Champe et al., J. Biol. Chem. 270: 1388- 1394 (1995), can be performed to determine whether the antibody binds an epitope of interest.
D. Pharmaceutical Compositions and Formulations
[0323] Also provided herein are pharmaceutical compositions and formulations comprising a PD-1 axis binding antagonist and/or an antibody described herein (such as an anti-PD-Ll antibody, or a bispecific antibody that binds CEA and CD3) and a pharmaceutically acceptable carrier.
[0324] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (e.g. a PD-1 axis binding antagonist and/or an anti- CEA/anti-CD3 bispecific antibody) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20
(HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
[0325] Exemplary lyophilized antibody formulations are described in US Patent No.
6,267,958. Aqueous antibody formulations include those described in US Patent No.
6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.
[0326] The composition and formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
[0327] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in
macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[0328] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. IV. Methods of Treatment
[0329] Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody. In some embodiments, the treatment results in a response in the individual after treatment. In some embodiments, the response is a partial response. In some embodiments, the response is a complete response. In some embodiments, the treatment results in a sustained response (e.g., a sustained partial response or complete response) in the individual after cessation of the treatment. The methods described herein may find use in treating conditions where enhanced
immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer. Also provided herein are methods of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of a PD-1 axis binding antagonist (e.g., MPDL3280A) and an anti-CEA/anti-CD3 bispecific antibody (e.g. CEA TCB).
[0330] In some instances, the methods provided herein include administration of an effective amount of a PD- 1 axis binding antagonist selected from the group consisting of a PD-1 binding antagonist, a PD-Ll binding antagonist, and a PD-L2 binding antagonist. In some instances, the PD-Ll binding antagonist is an antibody, such as an antibody that is capable of inhibiting PD-Ll binding to PD-1 and B7.1, but does not disrupt binding of PD-1 to PD-L2. In some instances, the PD-Ll binding antagonist antibody is MPDL3280A, which may be administered at a dose of about 800 mg to about 1500 mg every three weeks (e.g., about 1000 mg to about 1300 mg every three weeks, e.g., about 1100 mg to about 1200 mg every three weeks). In some embodiments, MPDL3280A is administered at a dose of about 1200 mg every three weeks.
[0331] As a general proposition, the therapeutically effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-Ll antibody, e.g., MPDL3280A) may be administered to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight whether by one or more administrations. In some embodiments, for example, the antagonist (e.g., anti-PD-Ll antibody, e.g., MPDL3280A) is administered in a dose of about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example. In some embodiments, the antagonist (e.g., anti-PD- Ll antibody, e.g., MPDL3280A) is administered at 15 mg/kg. However, other dosage regimens may be useful. In one embodiment, a PD-1 axis binding antagonist (e.g., anti-PD- Ll antibody, e.g., MPDL3280A) is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg. In some embodiments, a PD-1 axis binding antagonist (e.g., anti-PD-Ll antibody, e.g., MPDL3280A) is administered at a dose of about 1150 mg to about 1250 mg every three weeks. In some embodiments, a PD-1 axis binding antagonist (e.g., anti-PD-Ll antibody, e.g., MPDL3280A) is administered at a dose of about 1200 mg every three weeks. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The dose of the antibody administered in a combination treatment may be reduced as compared to a single treatment. In some embodiments, for example, the method for treating or delaying progression of cancer in an individual comprises a dosing regimen comprising treatment cycles, wherein the individual is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist (e.g., anti-PD-Ll antibody, e.g.,
MPDL3280A) at a dose of about 1200 mg, wherein each cycle is 21 days (i.e., each cycle is repeated every 21 days). The progress of this therapy is easily monitored by conventional techniques.
[0332] In some instances, the methods provided herein include administration of an effective amount of an anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB). In some instances, the anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) is administered to the individual at a dose of about 5 mg to about 400 mg every week (e.g., about 10 mg to about 60 mg every week, e.g., about 10 mg to about 40 mg every week, e.g. about 80 mg to about 200 mg every week, e.g. about 80 mg to about 400 mg every week, or e.g. about 160 mg to 400 mg every week). In some embodiments, the anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) is administered at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg or about 160 mg, every week. As a general proposition, the therapeutically effective amount of an anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) administered to a human will be in the range of about 5 to about 400 mg (e.g., about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150 mg, about 160 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg), whether by one or more administrations. For example, in some embodiments, about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg or about 160 mg, of CEA TCB is administered. In some embodiments, CEA TCB is administered at 5 mg, 10 mg, 20 mg, 40 mg, 80 mg or 160 mg, once a week. In particular embodiments, at least about 80 mg of CEA TCB is administered. In some embodiments, CEA TCB is administered at 80 mg once a week. In some embodiments, CEA TCB is administered at 80-200 mg, 80-400 mg or 160-400 mg once a week. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) may be administered weekly, every 2 weeks, every 3 weeks, every 4 weeks, on days 1, 8 and 15 of each 21-day cycle, or on days 1, 8, and 15 of each 28-day cycle.
[0333] In some instances, the PD-1 axis binding antagonist (e.g., an anti-PD-Ll antibody, e.g., MPDL3280A) and the anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) are administered in a single dosing regimen. The administration of these agents may be concurrent or separate within the context of the dosing regimen. For example, in some instances, the methods provided herein include a dosing regimen comprising treatment cycles, wherein the individual is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecific antibody at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg or about 160 mg, each cycle being repeated every 21 days. In one embodiment, the individual is administered, on days 1 of each cycle, a human PD- 1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecific antibody at a dose of about 80 mg, each cycle being repeated every 21 days. In a particular embodiment, the individual is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecific antibody at a dose of at least about 80 mg, each cycle being repeated every 21 days. In still a further embodiment, the individual is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecific antibody at a dose of about 80 mg to about 200 mg. about 80 mg to about 400 mg, or about 160 mg to about 400 mg, each cycle being repeated every 21 days.
[0334] In some embodiments, the individual is a human. In some embodiments, the individual is suffering from locally advanced or metastatic breast cancer. In some embodiments, the individual has CEA positive cancer. In some embodiments, CEA positive cancer is colon cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, breast cancer, kidney cancer, esophageal cancer, or prostate cancer. In a particular embodiment, the cancer is colorectal carcinoma. In some
embodiments, the breast cancer is a breast carcinoma or a breast adenocarcinoma. In some embodiments, the breast carcinoma is an invasive ductal carcinoma. In some embodiments, the lung cancer is a lung adenocarcinoma. In some embodiments, the colon cancer is a colorectal adenocarcinoma. In some embodiments, the cancer cells in the individual express PD-L1. In some embodiments, the cancer cells in the individual express CEA protein at a level that is detectable (e.g., detectable using methods known in the art).
[0335] In some embodiments, the individual has been treated with a cancer therapy before the combination treatment with a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody. In some embodiments, the individual has cancer that is resistant to one or more cancer therapies. In some embodiments, resistance to cancer therapy includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment. In some embodiments, resistance to a cancer therapy includes progression of the cancer during treatment with the anti-cancer therapy. In some embodiments, resistance to a cancer therapy includes cancer that does not response to treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, the cancer is at early stage or at late stage.
[0336] In some embodiments, the combination therapy of the invention comprises administration of a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody. The PD- 1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may be administered in any suitable manner known in the art. For example, The PD- 1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the PD-1 axis binding antagonist is in a separate composition as the anti-CEA/anti-CD3 bispecific antibody. In some embodiments, the PD-1 axis binding antagonist is in the same composition as the anti-CEA/anti-CD3 bispecific antibody.
[0337] The PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally,
intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally,
intraventricularly, or intranasally. In some embodiments, the anti-CEA/anti-CD3 bispecific antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody may be administered for prevention or treatment of disease. The appropriate dosage of the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibody may be determined based on the type of disease to be treated, the type of the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
[0338] In some embodiments, the methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a
mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some
embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting PI3K/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent. The additional therapy may be one or more of the chemotherapeutic agents described herein.
Other Combination Therapies
[0339] Also provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual a human PD-1 axis binding antagonist (e.g., anti-PD-Ll antibody, e.g., MPDL3280A) and an anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) in conjunction with another anti-cancer agent or cancer therapy.
[0340] In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a chemotherapy or
chemotherapeutic agent. In some embodiments, a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with a radiation therapy or radiotherapeutic agent. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a targeted therapy or targeted therapeutic agent. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an immunotherapy or immunotherapeutic agent, for example a monoclonal antibody.
[0341] Without wishing to be bound to theory, it is thought that enhancing T cell stimulation, by promoting an activating co- stimulatory molecule or by inhibiting a negative co-stimulatory molecule, may promote tumor cell death thereby treating or delaying progression of cancer. In some embodiments, a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against an activating co- stimulatory molecule. In some embodiments, an activating co-stimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist directed against an activating co- stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against an inhibitory co-stimulatory molecule. In some embodiments, an inhibitory co- stimulatory molecule may include CTLA-4 (also known as CD 152), PD-1, TIM-3, BTLA, VISTA, LAG- 3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some embodiments, the antagonist directed against an inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, PD-1, TEVI-3, BTLA, VISTA, LAG- 3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.
[0342] In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against CTLA-4 (also known as CD152), e.g., a blocking antibody. In some embodiments, a PD-1 axis binding antagonist may be administered in conjunction with ipilimumab (also known as MDX-010, MDX-101, or YERVOY®). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against B7-H3 (also known as CD276), e.g., a blocking antibody. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with MGA271. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against a TGF beta, e.g., metelimumab (also known as CAT- 192), fresolimumab (also known as GC1008), or LY2157299.
[0343] In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment comprising adoptive transfer of a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment comprising adoptive transfer of a T cell comprising a dominant- negative TGF beta receptor, e.g, a dominant- negative TGF beta type II receptor. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment comprising a HERCREEM protocol (see, e.g., ClinicalTrials.gov Identifier NCT00889954).
[0344] In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or ILA), e.g., an activating antibody. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with urelumab (also known as BMS-663513). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against CD40, e.g., an activating antibody. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with CP-870893. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against OX40 (also known as CD134), e.g., an activating antibody. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an anti-OX40 antibody (e.g., AgonOX). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agonist directed against CD27, e.g., an activating antibody. In some embodiments, a PD-1 axis binding antagonist may be administered in conjunction with CDX-1127. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antagonist directed against indoleamine-2,3- dioxygenase (IDO). In some embodiments, with the IDO antagonist is 1-methyl-D- tryptophan (also known as 1-D-MT). [0345] In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE). In some embodiments, a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with and anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599). In some
embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYLA®, Genentech). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with DMUC5754A. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), e.g., an antibody directed against EDNBR conjugated with MMAE.
[0346] In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an angiogenesis inhibitor. In some embodiments, a PD-1 axis binding antagonist may be administered in conjunction with an antibody directed against a VEGF, e.g., VEGF-A. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with bevacizumab (also known as AVASTIN®, Genentech). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody directed against angiopoietin 2 (also known as Ang2). In some embodiments, a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with MED 13617.
[0347] In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antineoplastic agent. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an agent targeting CSF-IR (also known as M-CSFR or CD115). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody may be administered in conjunction with anti-CSF-lR (also known as IMC-CS4). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody may be administered in conjunction with an interferon, for example interferon alpha or interferon gamma. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with Roferon-A (also known as recombinant Interferon alpha-2a). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or Leukine®). In some embodiments, a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with IL-2 (also known as aldesleukin or Proleukin®). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with IL-12. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody targeting CD20. In some embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or Gazyva®) or rituximab. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an antibody targeting GITR. In some embodiments, the antibody targeting GITR is TRX518.
[0348] In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide cocktail, a hybrid peptide, or a peptide- pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104: 14-21, 2013). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an adjuvant. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment comprising a TLR agonist, e.g., Poly-ICLC (also known as Hiltonol®), LPS, MPL, or CpG ODN. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with tumor necrosis factor (TNF) alpha. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with IL-1. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with HMGB1. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an IL-10 antagonist. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an IL-4 antagonist. In some embodiments, a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with an IL-13 antagonist. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an HVEM antagonist. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an ICOS agonist, e.g., by administration of ICOS-L, or an agonistic antibody directed against ICOS. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment targeting CX3CL1. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment targeting CXCL9. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment targeting CXCL10. In some embodiments, a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with a treatment targeting CCL5. In some embodiments, a PD-1 axis binding antagonist may be administered in conjunction with an LFA-1 or ICAMl agonist. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a Selectin agonist.
[0349] In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a targeted therapy. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of B-Raf. In some embodiments, a PD- 1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with vemurafenib (also known as Zelboraf®). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with dabrafenib (also known as Tafinlar®). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with erlotinib (also known as Tarceva®). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of a MEK, such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with cobimetinib (also known as GDC-0973 or XL-518). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with trametinib (also known as Mekinist®). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of K-Ras. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of c-Met. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with onartuzumab (also known as MetMAb). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of Alk. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with AF802 (also known as CH5424802 or alectinib). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of a phosphatidylinositol 3- kinase (PI3K). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody may be administered in conjunction with BKM120. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with perifosine (also known as KRX-0401). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of an Akt. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with MK2206. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with GSK690693. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with GDC-0941. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with an inhibitor of mTOR. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with sirolimus (also known as rapamycin). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with temsirolimus (also known as CCI-779 or Torisel®). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with everolimus (also known as RAD001). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with ridaforolimus (also known as AP-23573, MK-8669, or deforolimus). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with OSI-027. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with AZD8055. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with INK128. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with a dual PBK/mTOR inhibitor. In some embodiments, a PD-1 axis binding antagonist and an anti- CEA/anti-CD3 bispecific antibody may be administered in conjunction with XL765. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with GDC-0980. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with BEZ235 (also known as NVP-BEZ235). In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with BGT226. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with
GSK2126458. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody may be administered in conjunction with PF-04691502. In some embodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody may be administered in conjunction with PF-05212384 (also known as PKI-587).
V. Articles of Manufacture or Kits
[0350] In another embodiment of the invention, an article of manufacture or a kit is provided comprising a PD-1 axis binding antagonist and/or an anti-CEA/anti-CD3 bispecific antibody. In some embodiments, the article of manufacture or kit further comprises package insert comprising instructions for suing the PD- 1 axis binding antagonist in conjunction with an anti-CEA/anti-CD3 bispecific antibody to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer. Any of the PD- 1 axis binding antagonist and/or an anti-CEA/anti-CD3 bispecific antibodies described herein may be included in the article of manufacture or kits.
[0351] In some embodiments, the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecific antibody are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of
manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
[0352] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
EXAMPLES
[0353] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
Example 1: CEA TCB-mediated tumor lysis leads to up-regulation of PD-1 on human T cells and of PD-L1 on surviving tumor cells in vitro
[0354] CEA TCB is a novel T cell bispecific antibody targeting the carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells. It is currently being investigated as a single agent in a Phase I study in patients with advanced and /or metastatic CEA expressing tumors.
[0355] The lysis of CEA-expressing MKN-45 target cells mediated by CEA TCB was assessed after 24 h and 48 h of incubation with human PBMCs (E:T 10: 1, LDH release) (Figure 1, A 24 h, B 48 h). [0356] PBMCs were isolated from a buffy coat obtained from the "Blutspende Zurich". Blood was diluted 3: 1 with PBS. About 30 ml of the blood/PBS mixture was layered on 15 ml of Histopaque and centrifuged for 30 min at 450 g without brake at room temperature (RT). Lymphocytes were collected with a 10 ml pipette into 50 ml tubes containing PBS and filled up to 50 ml with PBS and centrifuged 10 min at 350 g. The supernatant was discarded, the pellet re-suspended in 50 ml PBS and centrifuged for 10 min at 300 g. The washing step was repeated once. Cells were re-suspended in RPMI containing 10% FCS and 1% GlutaMax and stored at 37°C, 5% C02 in the incubator until the initiation of the assay.
[0357] Adherent MKN-45 target cells were harvested with Trypsin/EDTA one day before the assay, washed and plated at density of 1.4 mio cells/well using flat-bottom 24- well plates. Cells were left to adhere overnight in a humidified incubator. On the day of the assay, plates were centrifuged at 350 g for 5 min and the medium was aspirated. 550 μΐ per well of assay medium (RPMI1640, 2% FCS, 1% GlutaMax) were added.
[0358] CEA TCB or untargeted TCB were added at indicated concentrations (range of 6.4 pM - 100 nM, in triplicates). PBMCs were added to target cells at the final EffectonTarget ratio E:T 10: 1, final volume per well was 1.1 ml.
[0359] Target cell killing was assessed after 24 h and 48 h of incubation by quantification of LDH (lactate dehydrogenase) released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (= 100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (= 0%) refers to target cells co-incubated with effector cells without bispecific antibody. The EC50 values were calculated using GraphPadPrism5.
[0360] Following killing, the surface expression of PD-1 receptor (on CD4+ or CD8+ T cells) and of PD-L1 (on tumor cells that survived killing) was assessed by flow cytometry.
[0361] Upon removal of 50 μΐ of the supernatant for the second LDH read-out (48h time point), the assay plates were centrifuged for 5 min at 350 g and the supernatants were discarded. Cell pellets were re-suspended in 100 μΐ PBS and transferred into wells of a 96- round-bottom well plate. The wells were washed with 80 μΐ PBS and this washing step was pooled with the already transferred supernatants from the steps before. The adherent MKN45 cells were detached with 80 μΐ Cell Dissociation Buffer (Gibco) and transferred to the FACS plate as well. Plates were centrifuged for 4 min at 400 g. Cell pellets were washed once again with 150 μΐ of FACS-buffer. After centrifugation (400 g for 4 min), the supernatant was removed and cells were re-suspended by careful vortexing. The cell suspension per well was split in two and the assay plates were centrifuged again. 25 μΐ of the antibody mix (antibodies directed against CD4, CD8, PD-1 (Biolegend, #329920) and PD-L1 (Biolegend #329708)) were added per well. To remove unbound antibody, the cells were washed twice with 150 μΐ FACS buffer per well. After centrifugation (400 g for 4 min), the supernatant was removed and cells were re-suspended in 120 μΐ FACS buffer.
[0362] Figure 2 displays a dose-dependent up-regulation of PD-1 receptor on CD8+ (A) and on CD4+ (B) T cells as well as of PD-L1 on tumor cells that resisted to killing (C) and were harvested after 48 h of incubation.
Example 2: CEA TCB-mediated tumor lysis leads to up-regulation of PD-1 on human T cells and of PD-L1 on surviving tumor cells in vivo
[0363] Nonclinical in vivo pharmacology studies were conducted using a human colon carcinoma xenograft tumor model (LS174T-fluc2) in immunodeficient NOG mice. Two different experimental settings were used: 1) tumor cells were co-grafted with human PBMCs (subcutaneous [SC]) at different E:T ratios, and 2) tumor cells were injected SC followed by intraperitoneal (IP) transfer of PBMCs once tumors were established (reached a palpable mass).
Xenograft Experiment with Co-Grafting of Effector Cells
[0364] Results of CEA TCB in vivo efficacy obtained upon co-grafting of LS174T-fluc2 cells with human PBMCs at an effectontarget ratio (E:T) 5: 1 are displayed in Figure 3.
[0365] When administered one day after tumor/PBMCs co-grafting, both doses of CEA TCB (0.5 and 2.5 mg/kg) induced tumor regression (Figure 3A). When administered seven days after the co-grafting, only the higher dose (2.5 mg/kg) induced tumor regression, whereas the lower dose (0.5 mg/kg) was unable to induce tumor regression but led to tumor growth control and tumor stasis (Figure 3B).
[0366] The histological analysis of tumors collected at termination of the same experiment confirmed that both early (Day 1) treatment with 0.5 mg/kg CEA TCB and delayed (Day 7) treatment with 2.5 mg/kg CEA TCB led to complete elimination of CEA-expressing tumor cells and resulted in prominent T-cell infiltration into tumors (not shown). Whereas high- CEA expression on tumor cells along with variable T-cell infiltration was detected in control tumors (vehicle and untargeted MCSP TCB), CEA TCB treatment (0.5 and 2.5 mg/kg) led to a complete elimination of CEA-expressing tumor cells along with formation of
necrotic/fibrotic areas filled with infiltrating T- cells (not shown). The staining of the same tumors with anti-PD-Ll antibody demonstrated a strong induction of intra- tumoral PD-L1 expression upon CEA TCB-treatment as compared to vehicle control (Figure 4). Xenograft Experiment with IP Transfer of Effector Cells
[0367] The in vivo efficacy of CEA TCB was also evaluated in xenograft models, in which tumor cells were injected SC followed by IP transfer of PBMCs once tumors reached a palpable mass. CEA TCB (2.5 mg/kg IV bi-weekly) led to significant tumor regression (reduction of tumor weight, Figure 5) and to an increase in T-cell infiltration into tumors (flow cytometry analysis (Figure 6) and histology (not shown)), with T-cells having activated phenotype (as shown by flow cytometry analysis indicating upregulation of CD25 and CD69 activation markers, not shown). This was in contrast to the vehicle (PBS) and MCSP TCB (untargeted control), where only a low number of T-cells infiltrated tumors and showed a resting phenotype.
[0368] Additional experiments studying PD- 1 and CD69 biomarkers showed that, compared to vehicle, CEA TCB induced up-regulation of the early activation marker CD69 and of PD-1 (Figure 7L, arrow) on tumor infiltrating T-cells. In contrast, peripheral blood T- cells showed a resting phenotype in both groups (Figure 7 A-F).
[0369] The staining of the same tumors with anti-PD-Ll antibody showed a strong induction of intra-tumor PD-Ll expression upon CEA TCB-treatment as compared to vehicle control.
Xenograft Experiment in Fully Humanized Mice
[0370] The antitumor activity of CEA TCB was assessed in vivo in a humanized mouse model bearing the CEA-expressing MKN45 cancer cell line subcutaneously.
[0371] CEA TCB was injected IV (2.5 mg/kg, twice/week) on Day 11 after tumor cell injection, and tumor growth was measured twice weekly by caliper. Figure 8 shows individual tumor volumes at study termination; a statistically significant (p < 0.05) difference in tumor volume was found.
[0372] In addition to tumor volume measurements, tumors, blood, spleen, and lymph nodes were collected at study termination (corresponding to 32 days after tumor cell injection) and analyzed by flow cytometry. CEA TCB treatment resulted in a significant increase of human T-cell frequencies in the tumor and changed the ratio of CD8+/CD4+ T-cells in favor of CD8+ T-cells (Figure 9 A and B). At the same time, both CD4+ and CD8+ T-cells exhibited an activated phenotype in the CEA TCB-treated tumors shown by up-regulation of CD69 and PD-1 expression on intratumoral CD4+ and CD8+ T-cells (Figure 9 C and D). CD8+ T-cells revealed an up-regulation of the intracellular Granzyme B (GZB) expression as well as a higher proliferation rate (Ki-67 marker). Analyses of spleen and blood samples of CEA TCB- treated animals did not show any difference in terms of T-cell frequencies, T-cell ratio, or activation status as compared to vehicle.
[0373] Thus, the immunological changes triggered by CEA TCB treatment were tumor- specific indicating that the cross-linking and activation of human T-cells occurs exclusively in CEA expressing tumors and not in other areas that are negative for CEA like blood and spleen.
[0374] Histological analysis of tumors collected at study termination confirmed the results of flow cytometry (not shown). Whereas human CEA was expressed in tumors of both vehicle and CEA TCB-treated groups, the size of tumor was significantly reduced by CEA TCB treatment. Moreover, vehicle tumors showed low infiltration of human immune cells that were mostly confined to stromal areas and the tumor border, whereas the CEA TCB- treated groups displayed a strong immune cell infiltration through the whole tumor area and not only in the periphery. In addition, human T-cells displayed increased expression of GZB within tumors. The staining of the same tumors with anti-PD-Ll antibody demonstrated a strong induction of intra-tumoral PD-Ll expression upon CEA TCB treatment compared with the vehicle control (Figure 10).
Example 3: Assessment of in vivo anti-tumor activity upon combination of CEA TCB with anti-PD-Ll blocking antibody in gastric carcinoma mouse model
[0375] The anti-tumor efficacy of CEA TCB in combination with anti-human PD-Ll blocking antibody was assessed in fully humanized mice bearing the gastric carcinoma tumor cell line (MKN45).
[0376] An anti-mouse PD-Ll surrogate antibody based on the YW243.55.S70 PD-Ll antibody described in WO 2010/077634 (sequence shown in Figure 11) was generated for use in vivo tumor models. This antibody contained a DAPG mutation to abolish FcyR interaction. The variable region of YW243.55.S70 was attached to a murine IgGl constant domain with DAPG Fc mutations.
[0377] The polypeptide sequences of YW243.55.S70 PD-Ll muIgGl are as follows: YW243.55.S70 PD-Ll muIgGl DAPG HC (SEQ ID NO:36):
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGV HTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDAPEVQFSWFVDDVEVHTAQT QPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQ VYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSY FVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
YW243.55.S70 PD-L1 muIgGl LC (SEQ ID NO:37):
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRADAAPTV SIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDST YSMSSTLTLTKDEYERHNSYTCEATHKTSTSPrVKSFNRNEC
[0378] In summary, 1 xlO6 MKN45 tumor cells were injected sub-cutaneously in fully humanized NOG mice. 7 days after tumor cell injection, mice were randomized in four groups: the first group received phosphate-buffer saline (PBS, vehicle) as control, the second group received CEA TCB (at the dose of 2.5 mg/kg, administered twice a week for 7 weeks, 15 administrations in total), the third group received anti-PD-Ll antibody (at the dose of 10 mg/kg, administered once a week for 7 weeks, 8 administrations in total), and the fourth group received a combination of CEA TCB and anti-PD-Ll antibody, administered concomitantly with the respective dose and schedules used in the single therapeutic groups for 7 weeks (8 administrations of anti-PD-Ll antibody 10 mg/kg given once a week and 15 administrations of CEA TCB 2.5 mg/kg given twice a week).
[0379] The results of this study show a significant increase in anti-tumor activity upon combination of CEA TCB with anti-PD-Ll blocking antibody as compared to CEA TCB single agent (Figure 11). Treatment with anti-PD-Ll antibody as single agent did not show any anti-tumor activity. No overt test item-related toxicities were observed following treatment with the combination.
[0380] A second study was designed to assess whether, after a first line treatment with CEA TCB monotherapy, the combination of CEA TCB with a-PD-Ll could exert superior anti-tumor activity than continuing treatment with CEA TCB as monotherapy.
[0381] For this study, 1 xlO6 MKN45 tumor cells were injected sub-cutaneously in fully humanized NOG mice. 10 days after tumor cell injection, mice received either phosphate- buffer saline (PBS, vehicle) as control, or CEA TCB (at the dose of 2.5 mg/kg, administered twice a week for 4 weeks, 7 administrations in total). At study day 34, CEA TCB- treated mice were randomized into two sub-groups: one continued receiving CEA TCB administration (2.5 mg/kg, twice a week), the other received CEA TCB (2.5 mg/kg, twice a week) in combination with anti-PD-Ll antibody (10 mg/kg, once a week) for 5 weeks (11 administrations in total for CEA TCB, and 6 administrations in total for anti-PD-Ll antibody).
[0382] The results clearly show that CEA TCB + anti-PD-Ll combination therapy as second line treatment mediates significant superior anti-tumor activity than CEA TCB monotherapy (Figure 12).
Example 4: Assessment of in vivo anti-tumor activity upon combination of CEA TCB with anti-PD-Ll blocking antibody in colorectal carcinoma mouse model
[0383] The anti-tumor efficacy of CEA TCB in combination with anti- PD-L1 blocking antibody was also assessed in fully immunocompetent mice bearing the colorectal carcinoma tumor cell line MC38 engineered in-house to express human CEA on the cell surface (MC38- huCEA).
[0384] In summary, 0.5 xlO6 MC38-huCEA tumor cells were injected sub-cutaneously in fully immunocompetent C57BL/6 transgenic for human CEA (huCEA Tg mice). 17 days after tumor cell injection, mice were randomized in four groups for first line treatment: the first group received phosphate-buffer saline (PBS, vehicle) as control, the second group received CEA TCB (at the dose of 2.5 mg/kg, administered twice a week for 4 weeks, 7 administrations in total), the third group received an anti-mouse PD-L1 antibody (at the dose of 10 mg/kg, administered once a week for 4 weeks, 4 administrations in total), and the fourth group received a combination of CEA TCB and anti- PD-L1 antibody, administered concomitantly with the respective dose and schedules used in the single therapeutic groups for 4 weeks (4 administrations of anti-PD-Ll 10 mg/kg given once a week and 7
administrations of CEA TCB 2.5 mg/kg given twice a week).
[0385] The results show a significant increase in anti-tumor activity upon combination of CEA TCB with anti-PD-Ll blocking antibody as compared to CEA TCB and anti-PD-Ll single agents (Figure 13). Moreover, at study day 42, 4 out of 9 mice from the combination treated group were tumor free, while no tumor-free mice were observed upon CEA TCB treatment, and only 1 out of 8 tumor-free mice were observed upon anti-PD-Ll therapy.
[0386] No overt test item-related toxicities were observed following treatment with the combination.
[0387] At study day 42, sub-cutaneous tumors were surgically removed from all groups and mice were left untreated for 5 weeks. After this resting period, first line- treated mice plus two groups of naive huCEA Tg mice (one left untouched, and the other previously submitted to a surgical cut on the flank to mimic surgical intervention) were injected sub- cutaneously with 0.5 xlO6 MC38-huCEA tumor cell lines on the opposite flank. No therapy injection was applied to any of the groups, while tumor growth was monitored. The results show that in all first line treated mice, tumor protection upon tumor rechallenge was observed, with significant tumor control as compared to naive mice. The group who had received the combination of CEA TCB with anti-PD-Ll as first line treatment, showed 3 out of 8 tumor-free mice at study day 37 upon tumor rechallenge. These data indicate that in the MC38-huCEA model, the combination of CEA TCB with anti-PD-Ll antibody not only improved first line treatment response, but also improved anti-tumor protection upon subsequent tumor challenge.
Example 5: An open-label, multi-center, dose escalation and expansion Phase IB study to evaluate the safety, pharmacokinetics and therapeutic activity of CEA TCB in combination with atezolizumab in patients with locally advanced and/or metastatic CEA-positive solid tumors
[0388] Data discussed above demonstrate that CEA TCB and atezolizumab act
synergistically in their anti-cancer properties and collectively their combination could provide meaningful clinical benefit in patients with cancer.
[0389] An open-label, multi-center, dose escalation and expansion Phase lb clinical study of CEA TCB in combination with atezolizumab is conducted. Each treatment cycle is 21 days in duration and consists of IV infusions of CEA TCB given weekly (QW) (+ 1 day) in combination with atezolizumab given every 3 weeks (Q3W) (+ 2 days). Patients receive atezolizumab (1200 mg fixed dose) IV on Day 1 of each cycle, followed by CEA TCB at least one hour later, given IV on Day 1, Day 8 and Day 15 of each cycle.
[0390] For CEA TCB, the starting dose is 5 mg administered on a QW schedule, to be administered at least one hour after the administration of 1200 mg of atezolizumab when they are both administered on the same day (Day 1 of each cycle).
[0391] Patients are treated until loss of clinical benefit, unacceptable toxicities, or withdrawal of consent. In case one of the treatments is permanently discontinued, treatment with the other drug alone may be continued.
[0392] Key eligibility criteria include confirmed locally advanced and/or metastatic solid tumor in patients who have progressed on a standard therapy, are intolerant to standard therapy, and/or are non-amenable to standard therapy; Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) 0-1; and locally confirmed CEA expression in tumor tissue ( > 50% of tumor cells staining with at least moderate to high intensity of CEA expression for all solid tumors with exception of NSCLC > 20% of tumor cells staining with at least moderate to high intensity of CEA expression) or centrally confirmed CEA expression.
[0393] Primary objectives of this study are safety and tolerability, with secondary objectives of PK and clinical activity (objective overall response rate (ORR), disease control rate (DCR; defined as response rate [RR] + stable disease rate [SDR]), progression-free survival (PFS) and overall survival (OS) data according to Response Evaluation Criteria in Solid Tumors (RECIST), Version 1.1 criteria and modified RECIST criteria).
[0394] Tumor response is evaluated according to RECIST vl .1 and modified RECIST criteria using unidimensional measurement such as computed tomography (CT) scan or magnetic resonance imaging.
Results
[0395] As of 15 November 2016, 30 patients have been treated at the following dose cohort levels: 5 mg of CEA TCB QW+ 1200 atezolizumab Q3W: 4 patients
10 mg of CEA TCB QW+ 1200 atezolizumab Q3W: 4 patients
20 mg of CEA TCB QW+ 1200 atezolizumab Q3W: 4 patients
40 mg of CEA TCB QW+ 1200 atezolizumab Q3W: 6 patients
80 mg of CEA TCB QW+ 1200 atezolizumab Q3W: 5 patients
160 mg of CEA TCB QW+ 1200 atezolizumab Q3W: 7 patients
[0396] The combination treatment was at current dose levels (up to 160 mg CEA TCB QW + 1200 mg atezolizumab Q3W) safe and well tolerated. No patients were discontinued due to a treatment related safety event.
[0397] All patients in this study had an FDG PET scan tumor assessment at week 4 and CT scan tumor assessment at week 8 and then every 8 weeks.
[0398] At 20 mg CEA TCB, 2/4 patients have experienced prolonged stable disease according to RECIST criteria. One patient with Pancreas adenocarcinoma has been in the study for 32 weeks, the patient experienced stable metabolic response assessed by FDG PET at week 4. The second patient, a gastro-esophageal junction adenocarcinoma with MSI instability has been in the study for 30 weeks. This patient experienced a partial metabolic response at week 4 FDG PET assessment.
[0399] At 40 mg CEA TCB, 1/6 patients experienced prolonged stable disease according to RECIST criteria and it's been on study for 20 weeks. This patient, a colorectal carcinoma patient, was previously treated in the monotherapy trial (BP29541) and received 31 cycles of treatment.
[0400] At 80 mg CEA TCB, 5/5 patients experienced tumor volume reduction at week 8 CT scan and metabolic partial response at week 4 FDG PET scan. In this cohort 4/5 patients were colorectal carcinoma patients and 1 patient with ampulla Vater carcinoma.
[0401] In summary, the clinical data generated so far in this trial showed promising relevant clinical antitumor activity in 2 /4 patients treated at 20 mg CEA TCB + 1200 mg atezolizumab, 1/6 patients treated at 40 mg CEA TCB + 1200 mg atezolizumab, and 5/5 patients treated at 80 mg CEA TCB + 1200 mg atezolizumab.
[0402] The data discussed above regarding the 80 mg CEA TCB cohort in a population that do not respond to anti PD-1 or anti PD-L1 treatment, supports that CEA TCB and
atezolizumab act synergistically in their anti-cancer properties and collectively their combination could provide meaningful clinical benefit in patients with cancer.

Claims

1. A method for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of a human PD-1 axis binding antagonist and an anti-CEA/anti-CD3 antibody.
2. The method of claim 1, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-Ll binding antagonist and a PD-L2 binding antagonist.
3. The method of claim 1 or 2, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.
4. The method of claim 3, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners.
5. The method of claim 3 or 4, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-Ll.
6. The method of any one of claims 3-5, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2.
7. The method of any one of claims 3-6, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-Ll and PD-L2.
8. The method of any one of claims 3-7, wherein the PD-1 binding antagonist is an antibody.
9. The method of any one claims 3-8, wherein the PD-1 binding antagonist is selected from the group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108.
10. The method of claim 2, wherein the PD-1 axis binding antagonist is a PD-Ll binding antagonist.
11. The method of claim 10, wherein the PD-Ll binding antagonist inhibits the binding of PD-Ll to PD-1.
12. The method of claim 10 or 11, wherein the PD-Ll binding antagonist inhibits the binding of PD-Ll to B7-1.
13. The method of any one of claims 10-12, wherein the PD-Ll binding antagonist inhibits the binding of PD-Ll to both PD-1 and B7-1.
14. The method of any one of claims 10-13, wherein the PD-Ll binding antagonist is an antibody.
15. The method of any one of claims 10-14, wherein the PD-Ll binding antagonist is selected from the group consisting of: MPDL3280A (atezolizumab), YW243.55.S70, MDX- 1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab).
16. The method of claim 14, wherein the antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO: 19, HVR-H2 sequence of SEQ ID NO:20, and HVR-H3 sequence of SEQ ID NO:21; and a light chain comprising HVR-Ll sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ ID NO:24.
17. The method of claim 14 or 16, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 or 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4.
18. The method of claim 2, wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.
19. The method of claim 18, wherein the PD-L2 binding antagonist is an antibody.
20. The method of claim 18, wherein the PD-L2 binding antagonist is an immunoadhesin.
21. The method of any one of claims 1-19, wherein the PD-1 axis binding antagonist is an antibody and comprises an aglycosylation site mutation.
22. The method of claim 21, wherein the aglycosylation site mutation is a substitution mutation.
23. The method of claim 22, wherein the substitution mutation is at amino acid residue N297, L234, L235, and/or D265 (EU numbering).
24. The method of claim 21 or 22, wherein the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, and D265A.
25. The method of any one of claims 21-23, wherein the substitution mutation is a D265A mutation and an N297G mutation.
26. The method of any one of claims 1-25, wherein the anti-CEA/anti-CD3 bispecific antibody comprises a first antigen binding domain that binds to CD3, and a second antigen binding domain that binds to CEA.
27. The method of claim 26, wherein the first antigen binding domain comprises a heavy chain variable region (VHCD3) and a light chain variable region (VLCD3), and the second antigen binding domain comprises a heavy chain variable region (VHCEA) and a light chain variable region (VLCEA).
28. The method of claim 26 or 27, wherein the first antigen binding domain comprises a heavy chain variable region (VHCD3) comprising HVR-H1 sequence of SEQ ID NO:44, HVR-H2 sequence of SEQ ID NO:45, and HVR-H3 sequence of SEQ ID NO:46; and/or a light chain variable region (VLCD3) comprising HVR-Ll sequence of SEQ ID NO:47, HVR- L2 sequence of SEQ ID NO:48, and HVR-L3 sequence of SEQ ID NO:49.
29. The method of any one of claims 26-28, wherein the first antigen binding domain comprises a heavy chain variable region (VHCD3) comprising the amino acid sequence of SEQ ID NO:50 and/or a light chain variable region (VLCD3) comprising the amino acid sequence of SEQ ID NO:51.
30. The method of any one of claims 26-29, wherein the second antigen binding domain comprises a heavy chain variable region (VHCEA) comprising HVR-Hl sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3 sequence of SEQ ID NO:40; and/or a light chain variable region (VLCEA) comprising HVR-L1 sequence of SEQ ID NO:41 , HVR-L2 sequence of SEQ ID NO:42, and HVR-L3 sequence of SEQ ID NO:43.
31. The method of any one of claims 26-30, wherein the second antigen binding domain comprises a heavy chain variable region (VHCEA) comprising the amino acid sequence of SEQ ID NO:34 and/or a light chain variable region (VLCEA) comprising the amino acid sequence of SEQ ID NO:35.
32. The method of any one of claims 26-31 , wherein the anti-CEA/anti-CD3 bispecific antibody comprises a third antigen binding domain that binds to CEA.
33. The method of claim 32, wherein the third antigen binding domain comprises a heavy chain variable region (VHCEA) comprising HVR-Hl sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3 sequence of SEQ ID NO:40; and/or a light chain variable region (VLCEA) comprising HVR-L1 sequence of SEQ ID NO:41 , HVR-L2 sequence of SEQ ID NO:42, and HVR-L3 sequence of SEQ ID NO:43.
34. The method of claim 32 or 33, wherein the third antigen binding domain comprises a heavy chain variable region (VHCEA) comprising the amino acid sequence of SEQ ID NO:34 and/or a light chain variable region (VLCEA) comprising the amino acid sequence of SEQ ID NO:35.
35. The method of any one of claims 26-34, wherein the antigen binding domains are Fab molecules.
36. The method of any one of claims 26-35, wherein the first antigen binding domain is a crossover Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged, and the second and third, if present, antigen binding domain is a conventional Fab molecule.
37. The method of claims 35 or 36, wherein (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, or (ii) the first antigen binding domain is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain.
38. The method of any one of claims 26-37, wherein the anti-CEA/anti-CD3 bispecific antibody further comprises an Fc domain composed of a first and a second subunit capable of stable association.
39. The method of claim 38, wherein (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the second antigen binding domain, and the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.
40. The method of claims 38 or 39, wherein (i) the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.
41. The method of any one of claims 38-40, wherein the Fc domain is an IgG, specifically an IgGi or IgG4, Fc domain.
42. The method of any one of claims 38-41, wherein the Fc domain is a human Fc domain.
43. The method of any one of claims 38-42, wherein the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain.
44. The method of claim 43, wherein in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
45. The method of any one of claims 38-44, wherein the Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain.
46. The method of any one of claims 38-45, wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.
47. The method of claim 46, wherein said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (EU numbering).
48. The method of any one of claims 38-47, wherein each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (EU numbering).
49. The method of any one of claims 1-48, wherein the cancer is a CEA-positive cancer.
50. The method of any one of claims 1-49, wherein the cancer is colon cancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, breast cancer, kidney cancer, esophageal cancer, or prostate cancer.
51. The method of any one of claims 1-50, wherein the individual has cancer or has been diagnosed with cancer.
52. The method of any one of claims 1-51, wherein cancer cells in the individual express PD-L1.
53. The method of any one of claims 1-52, wherein the human PD-1 axis binding antagonist is administered at a dose of about 1200 mg every three weeks.
54. The method of any one of claims 1-52, wherein the anti-CEA/anti-CD3 bispecific antibody is administered at a dose of about 5 mg to about 100 mg every week.
55. The method of any one of claims 1-53, comprising treatment cycles wherein the individual is administered, on day 1 of each cycle, a human PD-1 axis binding antagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 antibody at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg or about 160 mg, particularly at least about 80 mg, each cycle being repeated every 21 days.
56. The method of any one of claims 1-55, wherein the individual is refractory to a chemotherapeutic agent treatment.
57. The method of any one of claims 1-55, wherein the individual is intolerant to a chemotherapeutic agent treatment.
58. The method of any one of claims 1-57, wherein the treatment results in a response in the individual.
59. The method of any one of claims 1-58, wherein the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibody are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
60. The method of any one of claims 1-59, further comprising administering a
chemotherapeutic agent for treating or delaying progression of cancer.
61. A method of enhancing immune function in an individual having cancer comprising administering an effective amount of a PD- 1 axis binding antagonist and an anti-CEA/anti- CD3 bispecific antibody.
62. The method of claim 61, wherein CD8 T cells in the individual have enhanced priming, activation, proliferation and/or cytolytic activity relative to prior to the
administration of the PD-1 axis binding antagonist and the anti-HER2 antibody.
63. The method of claim 61 or 62, wherein the number of CD8 T cells is elevated relative to prior to administration of the combination.
64. The method of claim 62 or 63, wherein the CD8 T cell is an antigen- specific CD8 T cell.
65. The method of any one of claims 61-64, wherein Treg function is suppressed relative to prior to the administration of the combination.
66. The method of any one of claims 61-65, wherein T cell exhaustion is decreased relative to prior to the administration of the combination.
67. Use of a human PD-1 axis binding antagonist in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the human PD- 1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier.
68. Use of an anti-CEA/anti-CD3 bispecific antibody in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.
69. A composition comprising a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises administration of said composition in
combination with a second composition, wherein the second composition comprises an anti- CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier.
70. A composition comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises administration of said composition in
combination with a second composition, wherein the second composition comprises a human PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier.
71. A kit comprising a medicament comprising a PD- 1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the medicament in combination with a composition comprising an anti- CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.
72. A kit comprising a first medicament comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier, and a second medicament comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier.
73. The kit of claim 72, wherein the kit further comprises a package insert comprising instructions for administration of the first medicament and the second medicament for treating or delaying progression of cancer in an individual.
74. A kit comprising a medicament comprising an anti-CEA/anti-CD3 bispecific antibody and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the medicament in combination with a composition comprising a PD-1 axis binding antagonist and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.
PCT/EP2017/050168 2016-01-08 2017-01-05 Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies WO2017118675A1 (en)

Priority Applications (13)

Application Number Priority Date Filing Date Title
BR112018011029A BR112018011029A2 (en) 2016-01-08 2017-01-05 methods for treating or delaying cancer progression and improving immune function in a cancer individual, uses of a binding antagonist and a bispecific antibody, compositions and kits
ES17700491T ES2837428T3 (en) 2016-01-08 2017-01-05 CEA-Positive Cancer Treatment Procedures Using PD-1 Axis Binding Antagonists and Anti-CEA / Anti-CD3 Bispecific Antibodies
KR1020187019336A KR20180097615A (en) 2016-01-08 2017-01-05 Methods for the treatment of CEA-positive cancers using PD-1 axis-binding antagonists and anti-CEA / anti-CD3 bispecific antibodies
CN201780004427.0A CN108368179B (en) 2016-01-08 2017-01-05 Methods of treating CEA positive cancers using PD-1 axis binding antagonists and anti-CEA/anti-CD 3 bispecific antibodies
PL17700491T PL3400246T3 (en) 2016-01-08 2017-01-05 Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies
EP17700491.8A EP3400246B1 (en) 2016-01-08 2017-01-05 Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies
IL259588A IL259588B2 (en) 2016-01-08 2017-01-05 Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies
CA3006529A CA3006529A1 (en) 2016-01-08 2017-01-05 Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies
MX2018008347A MX2018008347A (en) 2016-01-08 2017-01-05 Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies.
AU2017205089A AU2017205089B2 (en) 2016-01-08 2017-01-05 Methods of treating CEA-positive cancers using PD-1 axis binding antagonists and anti-CEA/anti-CD3 bispecific antibodies
JP2018535339A JP6949030B2 (en) 2016-01-08 2017-01-05 Treatment of CEA-Positive Cancer Using PD-1 Axial Binding Antagonists and Anti-CEA / Anti-CD3 Bispecific Antibodies
EP20201657.2A EP3862365A1 (en) 2016-01-08 2017-01-05 Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies
HK19101508.2A HK1259020A1 (en) 2016-01-08 2019-01-29 Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP16150506 2016-01-08
EP16150506.0 2016-01-08

Publications (1)

Publication Number Publication Date
WO2017118675A1 true WO2017118675A1 (en) 2017-07-13

Family

ID=55168132

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/050168 WO2017118675A1 (en) 2016-01-08 2017-01-05 Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies

Country Status (15)

Country Link
US (2) US10596257B2 (en)
EP (2) EP3862365A1 (en)
JP (2) JP6949030B2 (en)
KR (1) KR20180097615A (en)
CN (1) CN108368179B (en)
AR (1) AR107303A1 (en)
AU (1) AU2017205089B2 (en)
BR (1) BR112018011029A2 (en)
CA (1) CA3006529A1 (en)
ES (1) ES2837428T3 (en)
HK (1) HK1259020A1 (en)
IL (1) IL259588B2 (en)
MX (1) MX2018008347A (en)
PL (1) PL3400246T3 (en)
WO (1) WO2017118675A1 (en)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018099539A1 (en) * 2016-11-29 2018-06-07 Horst Lindhofer Combination of t-cell redirecting multifunctional antibodies with immune checkpoint modulators and uses thereof
WO2019020745A1 (en) * 2017-07-28 2019-01-31 F. Hoffmann-La Roche Ag Bispecific antibody formulation
WO2019096136A1 (en) * 2017-11-14 2019-05-23 拜西欧斯（北京）生物技术有限公司 Anti-pd-1 antibody, preparation method therefor and use thereof
WO2019115659A1 (en) * 2017-12-14 2019-06-20 F. Hoffmann-La Roche Ag Use of a cea cd3 bispecific antibody and a pd-1 axis binding antagonist in a dosage regime to treat cancer
WO2019234576A1 (en) 2018-06-03 2019-12-12 Lamkap Bio Beta Ltd. Bispecific antibodies against ceacam5 and cd47
CN111315781A (en) * 2017-11-01 2020-06-19 豪夫迈·罗氏有限公司 Combination therapy with a targeted OX40 agonist
WO2021053587A1 (en) 2019-09-18 2021-03-25 Klaus Strein Bispecific antibodies against ceacam5 and cd3
KR20210064264A (en) * 2018-09-27 2021-06-02 고에끼 자이단 호우징 짓껭 도부쯔 쥬오 겡뀨쇼 immunocompromised mouse
EP3831849A1 (en) 2019-12-02 2021-06-09 LamKap Bio beta AG Bispecific antibodies against ceacam5 and cd47
WO2021121215A1 (en) * 2019-12-20 2021-06-24 Shandong Boan Biotechnology Co., Ltd. Optimized anti-cd3 arm in the generation of t-cell bispecific antibodies for immunotherapy
EP3861854A4 (en) * 2018-09-27 2022-01-26 Central Institute For Experimental Animals Immunodeficient mouse
WO2022090439A1 (en) * 2020-10-30 2022-05-05 F. Hoffmann-La Roche Ag TREATMENT OF CANCER USING A CEA CD3 BISPECIFIC ANTIBODY AND A TGFβ SIGNALING INHIBITOR
WO2022130348A1 (en) 2020-12-18 2022-06-23 Lamkap Bio Beta Ag Bispecific antibodies against ceacam5 and cd47
US11459394B2 (en) 2017-02-24 2022-10-04 Macrogenics, Inc. Bispecific binding molecules that are capable of binding CD137 and tumor antigens, and uses thereof
RU2798246C2 (en) * 2018-06-03 2023-06-20 Ламкап Био Бета Лтд. Bispecific antibodies against ceacam5 and cd47
US11685781B2 (en) 2018-02-15 2023-06-27 Macrogenics, Inc. Variant CD3-binding domains and their use in combination therapies for the treatment of disease
WO2023242351A1 (en) 2022-06-16 2023-12-21 Lamkap Bio Beta Ag Combination therapy of bispecific antibodies against ceacam5 and cd47 and bispecific antibodies against ceacam5 and cd3

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2018008347A (en) * 2016-01-08 2018-12-06 Hoffmann La Roche Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies.
AU2017339856A1 (en) * 2016-10-06 2019-05-23 Merck Patent Gmbh Dosing regimen of avelumab for the treatment of cancer
US20190376069A1 (en) * 2017-02-07 2019-12-12 The Regents Of The University Of California Use of microbial consortia in the production of multi-protein complexes
WO2018181425A1 (en) 2017-03-29 2018-10-04 塩野義製薬株式会社 Medicinal composition for treating cancer
CN110573528B (en) * 2017-03-29 2023-06-09 豪夫迈·罗氏有限公司 Bispecific antigen binding molecules to costimulatory TNF receptors
EP3409322A1 (en) * 2017-06-01 2018-12-05 F. Hoffmann-La Roche AG Treatment method
US11208485B2 (en) * 2018-10-11 2021-12-28 Inhibrx, Inc. PD-1 single domain antibodies and therapeutic compositions thereof
CN114778815B (en) * 2022-03-29 2023-06-09 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Marker panel for predicting prognosis of gastric cancer

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014131711A1 (en) * 2013-02-26 2014-09-04 Roche Glycart Ag Bispecific t cell activating antigen binding molecules
WO2015112534A2 (en) * 2014-01-21 2015-07-30 Medimmune, Llc Compositions and methods for modulating and redirecting immune responses

Family Cites Families (315)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CU22545A1 (en) 1994-11-18 1999-03-31 Centro Inmunologia Molecular OBTAINING A CHEMICAL AND HUMANIZED ANTIBODY AGAINST THE RECEPTOR OF THE EPIDERMAL GROWTH FACTOR FOR DIAGNOSTIC AND THERAPEUTIC USE
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
FR2413974A1 (en) 1978-01-06 1979-08-03 David Bernard DRYER FOR SCREEN-PRINTED SHEETS
US4419446A (en) 1980-12-31 1983-12-06 The United States Of America As Represented By The Department Of Health And Human Services Recombinant DNA process utilizing a papilloma virus DNA as a vector
NZ201705A (en) 1981-08-31 1986-03-14 Genentech Inc Recombinant dna method for production of hepatitis b surface antigen in yeast
US4601978A (en) 1982-11-24 1986-07-22 The Regents Of The University Of California Mammalian metallothionein promoter system
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
DD266710A3 (en) 1983-06-06 1989-04-12 Ve Forschungszentrum Biotechnologie Process for the biotechnical production of alkaline phosphatase
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US4943533A (en) 1984-03-01 1990-07-24 The Regents Of The University Of California Hybrid cell lines that produce monoclonal antibodies to epidermal growth factor receptor
US4965199A (en) 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
GB8516415D0 (en) 1985-06-28 1985-07-31 Celltech Ltd Culture of animal cells
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US6548640B1 (en) 1986-03-27 2003-04-15 Btg International Limited Altered antibodies
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
IL85035A0 (en) 1987-01-08 1988-06-30 Int Genetic Eng Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same
EP0307434B2 (en) 1987-03-18 1998-07-29 Scotgen Biopharmaceuticals, Inc. Altered antibodies
DE68925971T2 (en) 1988-09-23 1996-09-05 Cetus Oncology Corp CELL GROWING MEDIUM FOR INCREASED CELL GROWTH, FOR INCREASING LONGEVITY AND EXPRESSION OF PRODUCTS
GB8823869D0 (en) 1988-10-12 1988-11-16 Medical Res Council Production of antibodies
KR0184860B1 (en) 1988-11-11 1999-04-01 메디칼 리써어치 카운실 Single domain ligands receptors comprising said ligands methods for their production and use of said ligands
FR2646437B1 (en) 1989-04-28 1991-08-30 Transgene Sa NOVEL DNA SEQUENCES, THEIR APPLICATION AS A SEQUENCE ENCODING A SIGNAL PEPTIDE FOR THE SECRETION OF MATURE PROTEINS BY RECOMBINANT YEASTS, EXPRESSION CASSETTES, PROCESSED YEASTS AND PROCESS FOR PREPARING THE SAME
DE3920358A1 (en) 1989-06-22 1991-01-17 Behringwerke Ag BISPECIFIC AND OLIGO-SPECIFIC, MONO- AND OLIGOVALENT ANTI-BODY CONSTRUCTS, THEIR PRODUCTION AND USE
DE69029036T2 (en) 1989-06-29 1997-05-22 Medarex Inc SPECIFIC REAGENTS FOR AIDS THERAPY
US5081235A (en) 1989-07-26 1992-01-14 City Of Hope Chimeric anti-cea antibody
AU6290090A (en) 1989-08-29 1991-04-08 University Of Southampton Bi-or trispecific (fab)3 or (fab)4 conjugates
CA2066428C (en) 1989-09-08 2000-11-28 Bert Vogelstein Structural alterations of the egf receptor gene in human gliomas
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
DE69120146T2 (en) 1990-01-12 1996-12-12 Cell Genesys Inc GENERATION OF XENOGENIC ANTIBODIES
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
ES2246502T3 (en) 1990-08-29 2006-02-16 Genpharm International, Inc. TRANSGENIC NON-HUMAN ANIMALS ABLE TO PRODUCE HETEROLOGICAL ANTIBODIES.
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
ES2113940T3 (en) 1990-12-03 1998-05-16 Genentech Inc ENRICHMENT METHOD FOR PROTEIN VARIANTS WITH ALTERED UNION PROPERTIES.
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
EP0586505A1 (en) 1991-05-14 1994-03-16 Repligen Corporation Heteroconjugate antibodies for treatment of hiv infection
ES2206447T3 (en) 1991-06-14 2004-05-16 Genentech, Inc. HUMANIZED ANTIBODY FOR HEREGULINE.
GB9114948D0 (en) 1991-07-11 1991-08-28 Pfizer Ltd Process for preparing sertraline intermediates
US7018809B1 (en) 1991-09-19 2006-03-28 Genentech, Inc. Expression of functional antibody fragments
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
AU661533B2 (en) 1992-01-20 1995-07-27 Astrazeneca Ab Quinazoline derivatives
AU675929B2 (en) 1992-02-06 1997-02-27 Curis, Inc. Biosynthetic binding protein for cancer marker
ATE149570T1 (en) 1992-08-17 1997-03-15 Genentech Inc BISPECIFIC IMMUNOADHESINS
CA2149329C (en) 1992-11-13 2008-07-15 Darrell R. Anderson Therapeutic application of chimeric and radiolabeled antibodies to human b lymphocyte restricted differentiation antigen for treatment of b cell lymphoma
JPH08511420A (en) 1993-06-16 1996-12-03 セルテック・セラピューテイクス・リミテッド Body
GB9314893D0 (en) 1993-07-19 1993-09-01 Zeneca Ltd Quinazoline derivatives
ATE207366T1 (en) 1993-12-24 2001-11-15 Merck Patent Gmbh IMMUNOCONJUGATES
IL112248A0 (en) 1994-01-25 1995-03-30 Warner Lambert Co Tricyclic heteroaromatic compounds and pharmaceutical compositions containing them
IL112249A (en) 1994-01-25 2001-11-25 Warner Lambert Co Pharmaceutical compositions containing di and tricyclic pyrimidine derivatives for inhibiting tyrosine kinases of the epidermal growth factor receptor family and some new such compounds
US5654307A (en) 1994-01-25 1997-08-05 Warner-Lambert Company Bicyclic compounds capable of inhibiting tyrosine kinases of the epidermal growth factor receptor family
US5798100A (en) 1994-07-06 1998-08-25 Immunomedics, Inc. Multi-stage cascade boosting vaccine
BR9508409A (en) 1994-07-21 1997-12-23 Akzo Nobel Nv Storage stable transportable peroxide composition and use of an organic peroxide formulation
US5804396A (en) 1994-10-12 1998-09-08 Sugen, Inc. Assay for agents active in proliferative disorders
US5789199A (en) 1994-11-03 1998-08-04 Genentech, Inc. Process for bacterial production of polypeptides
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5840523A (en) 1995-03-01 1998-11-24 Genetech, Inc. Methods and compositions for secretion of heterologous polypeptides
EP2295415A1 (en) 1995-03-30 2011-03-16 OSI Pharmaceuticals, Inc. Quinazoline derivatives
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
ES2304786T3 (en) 1995-04-27 2008-10-16 Amgen Fremont Inc. ANTI-IL-8 HUMAN ANTIBODIES, DERIVED FROM IMMUNIZED XENORATONES.
GB9508565D0 (en) 1995-04-27 1995-06-14 Zeneca Ltd Quiazoline derivative
GB9508538D0 (en) 1995-04-27 1995-06-14 Zeneca Ltd Quinazoline derivatives
EP0823941A4 (en) 1995-04-28 2001-09-19 Abgenix Inc Human antibodies derived from immunized xenomice
US5747498A (en) 1996-05-28 1998-05-05 Pfizer Inc. Alkynyl and azido-substituted 4-anilinoquinazolines
CA2222231A1 (en) 1995-06-07 1996-12-19 Imclone Systems Incorporated Antibody and antibody fragments for inhibiting the growth of tumors
SI9620103A (en) 1995-07-06 1998-10-31 Novartis Ag Pyrrolopyrimidines and processes for the preparation thereof
US6267958B1 (en) 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
US7727761B2 (en) 1995-08-01 2010-06-01 Vegenics Limited Vascular endothelial growth factor C (VEGF-C) protein and gene, mutants thereof, and uses thereof
US5760041A (en) 1996-02-05 1998-06-02 American Cyanamid Company 4-aminoquinazoline EGFR Inhibitors
GB9603095D0 (en) 1996-02-14 1996-04-10 Zeneca Ltd Quinazoline derivatives
GB9603256D0 (en) 1996-02-16 1996-04-17 Wellcome Found Antibodies
PL190489B1 (en) 1996-04-12 2005-12-30 Warner Lambert Co Irreversible inhibitors of tyrosine kinases
ES2186908T3 (en) 1996-07-13 2003-05-16 Glaxo Group Ltd HETEROCICICLES CONDENSED COMPOUNDS AS INHIBITORS OF PPROTEINA-TIROSINA-QUINASAS.
AU729039C (en) 1996-08-30 2001-08-02 Upfront Chromatography A/S Isolation of immunoglobulins
ID18494A (en) 1996-10-02 1998-04-16 Novartis Ag PIRAZOLA DISTRIBUTION IN THE SEQUENCE AND THE PROCESS OF MAKING IT
DK1500329T3 (en) 1996-12-03 2012-07-09 Amgen Fremont Inc Human antibodies that specifically bind TNF-alpha
FR2757863B1 (en) 1996-12-27 1999-03-26 Inovat Sarl NOVEL BIOLOGICALLY ACTIVE SUBSTANCES, PROCESS FOR OBTAINING SAME AND COMPOSITIONS CONTAINING SAME
US20080318254A9 (en) 1997-03-10 2008-12-25 The Regents Of The University Of California PSCA antibodies and hybridomas producing them
UA73073C2 (en) 1997-04-03 2005-06-15 Уайт Холдінгз Корпорейшн Substituted 3-cyan chinolines
US6002008A (en) 1997-04-03 1999-12-14 American Cyanamid Company Substituted 3-cyano quinolines
DE69830901T2 (en) 1997-05-02 2006-05-24 Genentech Inc., San Francisco A method for producing multispecific antibodies having heteromultimeric and common components
US20020173629A1 (en) 1997-05-05 2002-11-21 Aya Jakobovits Human monoclonal antibodies to epidermal growth factor receptor
US6235883B1 (en) 1997-05-05 2001-05-22 Abgenix, Inc. Human monoclonal antibodies to epidermal growth factor receptor
WO1998050038A1 (en) 1997-05-06 1998-11-12 American Cyanamid Company Use of quinazoline compounds for the treatment of polycystic kidney disease
US6171586B1 (en) 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
DE69830315T2 (en) 1997-06-24 2006-02-02 Genentech Inc., San Francisco GALACTOSYLATED GLYCOPROTEIN CONTAINING COMPOSITIONS AND METHOD FOR THE PRODUCTION THEREOF
ZA986729B (en) 1997-07-29 1999-02-02 Warner Lambert Co Irreversible inhibitors of tyrosine kinases
ZA986732B (en) 1997-07-29 1999-02-02 Warner Lambert Co Irreversible inhibitiors of tyrosine kinases
TW436485B (en) 1997-08-01 2001-05-28 American Cyanamid Co Substituted quinazoline derivatives
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
ATE419009T1 (en) 1997-10-31 2009-01-15 Genentech Inc METHODS AND COMPOSITIONS CONSISTING OF GLYCOPROTEIN GLYCOFORMS
KR20010031813A (en) 1997-11-06 2001-04-16 윌리암 에이취 캘넌, 에곤 이 버그 Use of quinazoline derivatives as tyrosine kinase inhibitors for treating colonic polyps
US6610833B1 (en) 1997-11-24 2003-08-26 The Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
ATE531812T1 (en) 1997-12-05 2011-11-15 Scripps Research Inst HUMANIZATION OF RODENT ANTIBODIES
EP1049787B1 (en) 1998-01-23 2004-11-24 Vlaams Interuniversitair Instituut voor Biotechnologie Multipurpose antibody derivatives
ATE375365T1 (en) 1998-04-02 2007-10-15 Genentech Inc ANTIBODIES VARIANTS AND FRAGMENTS THEREOF
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
AU3657899A (en) 1998-04-20 1999-11-08 James E. Bailey Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
ES2188254T3 (en) 1998-11-19 2003-06-16 Warner Lambert Co N- (4- (3-CHLORO-4-FLUORO-PHENYLAMINE) -7- (3-MORFOLIN-4-IL-PROPOXI) -QUIN AZOLIN-6-IL) -ACRILAMADA, AN IRREVERSIBLE THYROSINE KINASE INHIBITOR.
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
HUP0104865A3 (en) 1999-01-15 2004-07-28 Genentech Inc Polypeptide variants with altered effector function
DE60039596D1 (en) 1999-03-30 2008-09-04 Japan Tobacco Inc PROCESS FOR THE PREPARATION OF MONOCLONAL ANTIBODIES
EP2275540B1 (en) 1999-04-09 2016-03-23 Kyowa Hakko Kirin Co., Ltd. Method for controlling the activity of immunologically functional molecule
AU782626B2 (en) 1999-10-04 2005-08-18 Medicago Inc. Method for regulating transcription of foreign genes
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
EP1229125A4 (en) 1999-10-19 2005-06-01 Kyowa Hakko Kogyo Kk Process for producing polypeptide
AU784983B2 (en) 1999-12-15 2006-08-17 Genentech Inc. Shotgun scanning, a combinatorial method for mapping functional protein epitopes
ES2528794T3 (en) 2000-04-11 2015-02-12 Genentech, Inc. Multivalent antibodies and uses thereof
US6773883B2 (en) 2000-07-31 2004-08-10 The Brigham & Women's Hospital, Inc. Prognostic classification of endometrial cancer
CA2424602C (en) 2000-10-06 2012-09-18 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-producing cell
US6946292B2 (en) 2000-10-06 2005-09-20 Kyowa Hakko Kogyo Co., Ltd. Cells producing antibody compositions with increased antibody dependent cytotoxic activity
US7064191B2 (en) 2000-10-06 2006-06-20 Kyowa Hakko Kogyo Co., Ltd. Process for purifying antibody
US6596541B2 (en) 2000-10-31 2003-07-22 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
JP3523245B1 (en) 2000-11-30 2004-04-26 メダレックス，インコーポレーテッド Transgenic chromosome-introduced rodents for the production of human antibodies
WO2002092017A2 (en) 2001-05-16 2002-11-21 Albert Einstein College Of Medicine Of Yeshiva University Human antipneumococcal antibodies from non-human animals
NZ592087A (en) 2001-08-03 2012-11-30 Roche Glycart Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
JP4359503B2 (en) 2001-08-23 2009-11-04 ゲンマブ エー／エス Human antibody specific for interleukin-15 (IL-15)
ES2326964T3 (en) 2001-10-25 2009-10-22 Genentech, Inc. GLICOPROTEIN COMPOSITIONS.
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US7317091B2 (en) 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
AU2003236018A1 (en) 2002-04-09 2003-10-20 Kyowa Hakko Kirin Co., Ltd. METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcGamma RECEPTOR IIIa
US7691568B2 (en) 2002-04-09 2010-04-06 Kyowa Hakko Kirin Co., Ltd Antibody composition-containing medicament
ATE503829T1 (en) 2002-04-09 2011-04-15 Kyowa Hakko Kirin Co Ltd CELL WITH REDUCED OR DELETED ACTIVITY OF A PROTEIN INVOLVED IN GDP-FUCOSE TRANSPORT
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
AU2003236019A1 (en) 2002-04-09 2003-10-20 Kyowa Hakko Kirin Co., Ltd. Drug containing antibody composition appropriate for patient suffering from Fc Gamma RIIIa polymorphism
CA2481837A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
EP1513879B1 (en) 2002-06-03 2018-08-22 Genentech, Inc. Synthetic antibody phage libraries
US7361740B2 (en) 2002-10-15 2008-04-22 Pdl Biopharma, Inc. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
DE60332957D1 (en) 2002-12-16 2010-07-22 Genentech Inc IMMUNOGLOBULIN VARIANTS AND ITS USES
AU2004205631A1 (en) 2003-01-16 2004-08-05 Genentech, Inc. Synthetic antibody phage libraries
CA2513308A1 (en) 2003-01-17 2004-08-05 Josef Michl Pancreatic cancer associated antigen, antibody thereto, and diagnostic and treatment methods
US20060104968A1 (en) 2003-03-05 2006-05-18 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases
US7871607B2 (en) 2003-03-05 2011-01-18 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
AU2004270103B2 (en) 2003-05-21 2012-02-23 E. R. Squibb & Sons, L.L.C. Human monoclonal antibodies against Bacillusanthracis protective antigen
US7635472B2 (en) 2003-05-31 2009-12-22 Micromet Ag Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders
US20080241884A1 (en) 2003-10-08 2008-10-02 Kenya Shitara Fused Protein Composition
AU2004280065A1 (en) 2003-10-09 2005-04-21 Kyowa Hakko Kirin Co., Ltd. Process for producing antibody composition by using RNA inhibiting the function of alpha1,6-fucosyltransferase
EA036531B1 (en) 2003-11-05 2020-11-19 Роше Гликарт Аг Type ii anti-cd20 humanized antibody (variants), pharmaceutical composition comprising these antibody variants, and use thereof
DK1691833T3 (en) 2003-11-28 2010-05-03 Micromet Ag Compositions comprising polypeptides
JPWO2005053742A1 (en) 2003-12-04 2007-06-28 協和醗酵工業株式会社 Medicament containing antibody composition
US7235641B2 (en) 2003-12-22 2007-06-26 Micromet Ag Bispecific antibodies
WO2005086875A2 (en) 2004-03-11 2005-09-22 City Of Hope A humanized anti-cea t84.66 antibody and uses thereof
MXPA06011199A (en) 2004-03-31 2007-04-16 Genentech Inc Humanized anti-tgf-beta antibodies.
US7785903B2 (en) 2004-04-09 2010-08-31 Genentech, Inc. Variable domain library and uses
CA2885854C (en) 2004-04-13 2017-02-21 F. Hoffmann-La Roche Ag Anti-p-selectin antibodies
TWI380996B (en) 2004-09-17 2013-01-01 Hoffmann La Roche Anti-ox40l antibodies
JO3000B1 (en) 2004-10-20 2016-09-05 Genentech Inc Antibody Formulations.
AR051836A1 (en) 2004-11-30 2007-02-14 Centocor Inc RECEIVER ANTAGONISTS 3 SIMIL TOLL METHODS AND USES
NZ556286A (en) 2005-02-07 2010-11-26 Glycart Biotechnology Ag Antigen binding molecules that bind EGFR, vectors encoding same, and uses thereof
US10011858B2 (en) 2005-03-31 2018-07-03 Chugai Seiyaku Kabushiki Kaisha Methods for producing polypeptides by regulating polypeptide association
WO2006114115A1 (en) 2005-04-26 2006-11-02 Trion Pharma Gmbh Combination of antibodies and glucocorticoids for treating cancer
EP1888649A2 (en) 2005-05-09 2008-02-20 GlycArt Biotechnology AG Antigen binding molecules having modified fc regions and altered binding to fc receptors
LT2439273T (en) 2005-05-09 2019-05-10 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
CN105330741B (en) 2005-07-01 2023-01-31 E.R.施贵宝&圣斯有限责任公司 Human monoclonal antibodies to programmed death ligand 1 (PD-L1)
US7612181B2 (en) 2005-08-19 2009-11-03 Abbott Laboratories Dual variable domain immunoglobulin and uses thereof
RU2515108C2 (en) 2005-08-19 2014-05-10 Эббви Инк Immunoglobulin with double variable domains and its applications
AU2006301492B2 (en) 2005-10-11 2011-06-09 Amgen Research (Munich) Gmbh Compositions comprising cross-species-specific antibodies and uses thereof
ES2577292T3 (en) 2005-11-07 2016-07-14 Genentech, Inc. Binding polypeptides with diversified VH / VL hypervariable sequences and consensus
US20070237764A1 (en) 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
SG153825A1 (en) 2005-12-16 2009-07-29 Ibc Pharmaceuticals Inc Multivalent immunoglobulin-based bioactive assemblies
DK1976880T3 (en) * 2005-12-21 2016-09-26 Amgen Res (Munich) Gmbh Pharmaceutical compositions with resistance to soluble cea
JP5463036B2 (en) 2005-12-21 2014-04-09 アムゲン リサーチ （ミュンヘン） ゲーエムベーハー Pharmaceutical antibody composition having resistance to soluble CEA
AU2007229698B9 (en) 2006-03-24 2012-11-08 Merck Patent Gmbh Engineered heterodimeric protein domains
CA2651567A1 (en) 2006-05-09 2007-11-22 Genentech, Inc. Binding polypeptides with optimized scaffolds
JP2009539413A (en) 2006-06-12 2009-11-19 トゥルビオン・ファーマシューティカルズ・インコーポレーテッド Single-chain multivalent binding protein with effector function
JP2009541275A (en) 2006-06-22 2009-11-26 ノボ・ノルデイスク・エー／エス Production of bispecific antibodies
SI2502938T1 (en) 2006-10-27 2015-05-29 Genentech, Inc. Antibodies and immunoconjugates and uses therefor
US20080226635A1 (en) 2006-12-22 2008-09-18 Hans Koll Antibodies against insulin-like growth factor I receptor and uses thereof
MX2009010611A (en) 2007-04-03 2010-03-26 Micromet Ag Cross-species-specific bispecific binders.
LT2520590T (en) 2007-04-03 2018-09-10 Amgen Research (Munich) Gmbh Cross-species-specific binding domain
CN109456410B (en) 2007-04-03 2022-01-28 安进研发（慕尼黑）股份有限公司 Cross-species specific CD 3-epsilon binding domains
CN100592373C (en) 2007-05-25 2010-02-24 群康科技(深圳)有限公司 Liquid crystal panel drive device and its drive method
JP5490714B2 (en) 2007-11-28 2014-05-14 メディミューン，エルエルシー Protein preparation
US20090162359A1 (en) 2007-12-21 2009-06-25 Christian Klein Bivalent, bispecific antibodies
US9266967B2 (en) 2007-12-21 2016-02-23 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
US8227577B2 (en) 2007-12-21 2012-07-24 Hoffman-La Roche Inc. Bivalent, bispecific antibodies
US8242247B2 (en) 2007-12-21 2012-08-14 Hoffmann-La Roche Inc. Bivalent, bispecific antibodies
PT2235064E (en) 2008-01-07 2016-03-01 Amgen Inc Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
BRPI0907718A2 (en) 2008-02-11 2017-06-13 Curetech Ltd method for treating a tumor, method for improving tolerability to at least one chemotherapeutic agent, method for increasing survival of an individual having a tumor, method for reducing or preventing tumor recurrence, use of a humanized monoclonal antibody or fragment and antibody thereof humanized monoclonal or fragment thereof
EP2262837A4 (en) 2008-03-12 2011-04-06 Merck Sharp & Dohme Pd-1 binding proteins
US20110159023A1 (en) 2008-08-25 2011-06-30 Solomon Langermann Pd-1 antagonists and methods for treating infectious disease
ES2592216T3 (en) 2008-09-26 2016-11-28 Dana-Farber Cancer Institute, Inc. Human anti-PD-1, PD-L1 and PD-L2 antibodies and their uses
SI2376535T1 (en) 2008-12-09 2017-07-31 F. Hoffmann-La Roche Ag Anti-pd-l1 antibodies and their use to enhance t-cell function
CA2756244A1 (en) 2009-04-02 2010-10-07 Roche Glycart Ag Multispecific antibodies comprising full length antibodies and single chain fab fragments
EP2417156B1 (en) 2009-04-07 2015-02-11 Roche Glycart AG Trivalent, bispecific antibodies
CN102459346B (en) 2009-04-27 2016-10-26 昂考梅德药品有限公司 The method manufacturing heteromultimers molecule
WO2010136172A1 (en) 2009-05-27 2010-12-02 F. Hoffmann-La Roche Ag Tri- or tetraspecific antibodies
EP2261258A1 (en) 2009-06-08 2010-12-15 Schuler, Gerold MCSP-aligned scFv antibody fragments and use of same
US9676845B2 (en) 2009-06-16 2017-06-13 Hoffmann-La Roche, Inc. Bispecific antigen binding proteins
US8703132B2 (en) 2009-06-18 2014-04-22 Hoffmann-La Roche, Inc. Bispecific, tetravalent antigen binding proteins
BR112012003983A2 (en) 2009-08-31 2021-09-14 Roche Glycart Ag ABM BINDING MOLECULE ABM ANTIGEN BINDING MOLECULES ISOLATED POLYPEPTIDE VARIANT HUMANIZED ANTIGEN BINDING MOLECULE ISOLATED POLYNUCLEOTIDE ANTIBODY VECTOR COMPOSITION HOST CELL ABM PRODUCTION METHOD TUMOR CELL LYSIS INDUCTION METHOD, DIAGNOSTIC DIAGNOSIS METHOD OF TUMOR DIAGNOSIS METHOD CANCER METHOD OF INCREASE TIME OF SURVIVAL IN PATIENTS THAT HAVE A CANCER INDUCTION METHOD IN REGRESSION PATIENTS OF A ENGINE USE ABM AND INVENTION
US9493578B2 (en) 2009-09-02 2016-11-15 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens
JP2013512251A (en) 2009-11-24 2013-04-11 アンプリミューン、インコーポレーテッド Simultaneous inhibition of PD-L1 / PD-L2
NZ599405A (en) 2009-11-24 2014-09-26 Medimmune Ltd Targeted binding agents against b7-h1
SI2519543T1 (en) 2009-12-29 2016-08-31 Emergent Product Development Seattle, Llc Heterodimer binding proteins and uses thereof
TW201138821A (en) 2010-03-26 2011-11-16 Roche Glycart Ag Bispecific antibodies
US9527926B2 (en) 2010-05-14 2016-12-27 Rinat Neuroscience Corp. Heterodimeric proteins and methods for producing and purifying them
AU2011325833C1 (en) 2010-11-05 2017-07-13 Zymeworks Bc Inc. Stable heterodimeric antibody design with mutations in the Fc domain
AR084053A1 (en) 2010-11-30 2013-04-17 Chugai Pharmaceutical Co Ltd THERAPEUTIC AGENT THAT INDUCES CYTOTOXICITY
PE20140303A1 (en) 2011-02-10 2014-03-22 Roche Glycart Ag MUTANT INTERLEUQUIN-2 POLYPEPTIDES
NZ614125A (en) 2011-03-02 2015-07-31 Roche Glycart Ag Cea antibodies
PT2691417T (en) 2011-03-29 2018-10-31 Roche Glycart Ag Antibody fc variants
EA201892619A1 (en) 2011-04-29 2019-04-30 Роше Гликарт Аг IMMUNOCONJUGATES CONTAINING INTERLEUKIN-2 MUTANT POLYPETIPS
PE20141454A1 (en) 2011-05-06 2014-10-23 Us Gov Health & Human Serv RECOMBINANT IMMUNOTOXIN TARGETING MESOTHELIN
CN103842383B (en) 2011-05-16 2017-11-03 健能隆医药技术（上海）有限公司 Polyspecific FAB fusion proteins and its application method
SG195072A1 (en) 2011-05-21 2013-12-30 Macrogenics Inc Cd3-binding molecules capable of binding to human and non-human cd3
WO2013012414A1 (en) 2011-07-18 2013-01-24 Medimmune, Llc Dosing regimens for treatment of cea-expressing cancers
TW201840336A (en) 2011-08-01 2018-11-16 美商建南德克公司 Methods of treating cancer using pd-1 axis binding antagonists and mek inhibitors
SI2748202T1 (en) 2011-08-23 2018-10-30 Roche Glycart Ag Bispecific antigen binding molecules
KR101870555B1 (en) 2011-08-23 2018-06-22 로슈 글리카트 아게 Bispecific antibodies specific for t-cell activating antigens and a tumor antigen and methods of use
CN103748114B (en) 2011-08-23 2017-07-21 罗切格利卡特公司 T cell activation bispecific antigen binding molecules
RU2617970C2 (en) 2011-08-23 2017-04-28 Рош Гликарт Аг ANTIBODIES WITHOUT Fc-FRAGMENT INCLUDING TWO FAB-FRAGMENT AND METHODS OF APPLICATION
US9309306B2 (en) 2011-08-23 2016-04-12 Roche Glycart Ag Anti-MCSP antibodies
US20130078250A1 (en) 2011-08-23 2013-03-28 Oliver Ast Bispecific t cell activating antigen binding molecules
EP2578230A1 (en) 2011-10-04 2013-04-10 Trion Pharma Gmbh Removal of Tumor Cells from Intraoperative Autologous Blood Salvage
US10851178B2 (en) 2011-10-10 2020-12-01 Xencor, Inc. Heterodimeric human IgG1 polypeptides with isoelectric point modifications
CA2859667C (en) 2011-12-20 2022-05-24 Medimmune, Llc Modified polypeptides for bispecific antibody scaffolds
MX360109B (en) 2012-04-20 2018-10-23 Merus Nv Methods and means for the production of ig-like molecules.
EP3556776A1 (en) 2012-05-31 2019-10-23 F. Hoffmann-La Roche AG Methods of treating cancer using pd-1 axis binding antagonists and vegf antagonists
US20150203591A1 (en) 2012-08-02 2015-07-23 Regeneron Pharmaceuticals, Inc. Mutivalent antigen-binding proteins
SG10201800535XA (en) 2012-08-07 2018-02-27 Roche Glycart Ag Composition comprising two antibodies engineered to have reduced and increased effector function
US9315567B2 (en) 2012-08-14 2016-04-19 Ibc Pharmaceuticals, Inc. T-cell redirecting bispecific antibodies for treatment of disease
US10131712B2 (en) 2012-08-14 2018-11-20 Ibc Pharmaceuticals, Inc. Combination therapy with T-cell redirecting bispecific antibodies and checkpoint inhibitors
JOP20200236A1 (en) 2012-09-21 2017-06-16 Regeneron Pharma Anti-cd3 antibodies, bispecific antigen-binding molecules that bind cd3 and cd20, and uses thereof
WO2014056783A1 (en) 2012-10-08 2014-04-17 Roche Glycart Ag Fc-free antibodies comprising two fab-fragments and methods of use
ES2824024T3 (en) 2012-10-10 2021-05-11 Sangamo Therapeutics Inc T cell modifying compounds and uses thereof
WO2014079886A1 (en) 2012-11-20 2014-05-30 Sanofi Anti-ceacam5 antibodies and uses thereof
UY35148A (en) 2012-11-21 2014-05-30 Amgen Inc HETERODIMERIC IMMUNOGLOBULINS
US9605084B2 (en) 2013-03-15 2017-03-28 Xencor, Inc. Heterodimeric proteins
AU2014205086B2 (en) 2013-01-14 2019-04-18 Xencor, Inc. Novel heterodimeric proteins
US10131710B2 (en) 2013-01-14 2018-11-20 Xencor, Inc. Optimized antibody variable regions
US10487155B2 (en) 2013-01-14 2019-11-26 Xencor, Inc. Heterodimeric proteins
US9701759B2 (en) 2013-01-14 2017-07-11 Xencor, Inc. Heterodimeric proteins
US10968276B2 (en) 2013-03-12 2021-04-06 Xencor, Inc. Optimized anti-CD3 variable regions
JP6636803B2 (en) 2013-02-05 2020-01-29 エンクマフ エスアーエールエル Method for selection of antibodies to BCMA
JO3529B1 (en) 2013-02-08 2020-07-05 Amgen Res Munich Gmbh Anti-leukocyte adhesion for the mitigation of potential adverse events caused by CD3-specific binding domains
MY192312A (en) 2013-02-26 2022-08-17 Roche Glycart Ag Bispecific t cell activating antigen binding molecules
RU2015140915A (en) 2013-02-26 2017-04-03 Роше Гликарт Аг BSPECIFIC ANTI-BINDING MOLECULES ACTIVATING T-CELLS
WO2014153164A1 (en) 2013-03-14 2014-09-25 The California Institute For Biomedical Research Targeting agent antibody conjugates and uses thereof
US10858417B2 (en) 2013-03-15 2020-12-08 Xencor, Inc. Heterodimeric proteins
SG11201506499YA (en) 2013-03-15 2015-09-29 Glaxosmithkline Ip No 2 Ltd Low concentration antibody formulations
EP2972373B1 (en) 2013-03-15 2019-10-09 F.Hoffmann-La Roche Ag Biomarkers and methods of treating pd-1 and pd-l1 related conditions
AR095363A1 (en) 2013-03-15 2015-10-14 Genentech Inc BIOMARKERS AND METHODS FOR THE TREATMENT OF CONDITIONS RELATED TO PD-1 AND PD-L1
US20140302037A1 (en) 2013-03-15 2014-10-09 Amgen Inc. BISPECIFIC-Fc MOLECULES
WO2014163684A1 (en) 2013-04-03 2014-10-09 Ibc Pharmaceuticals, Inc. Combination therapy for inducing immune response to disease
EP2789630A1 (en) 2013-04-09 2014-10-15 EngMab AG Bispecific antibodies against CD3e and ROR1
SG11201509361TA (en) 2013-05-28 2015-12-30 Numab Ag Novel antibodies
KR20160030936A (en) 2013-07-16 2016-03-21 제넨테크, 인크. Methods of treating cancer using pd-1 axis binding antagonists and tigit inhibitors
AR097584A1 (en) 2013-09-12 2016-03-23 Hoffmann La Roche ANTIBODY COMBINATION THERAPY AGAINST HUMAN CSF-1R AND ANTIBODIES AGAINST HUMAN PD-L1
SG11201602283UA (en) 2013-09-27 2016-04-28 Genentech Inc Anti-pdl1 antibody formulations
EP3083692B1 (en) 2013-12-17 2020-02-19 F.Hoffmann-La Roche Ag Methods of treating her2-positive cancers using pd-1 axis binding antagonists and anti-her2 antibodies
WO2015095410A1 (en) 2013-12-17 2015-06-25 Genentech, Inc. Methods of treating cancer using pd-1 axis binding antagonists and an anti-cd20 antibody
CA2934028A1 (en) 2013-12-17 2015-06-25 Genentech, Inc. Combination therapy comprising ox40 binding agonists and pd-1 axis binding antagonists
DK3083689T3 (en) 2013-12-17 2020-08-03 Genentech Inc Anti-CD3 antibodies and methods of use
EP3092251B1 (en) 2014-01-06 2021-01-20 F. Hoffmann-La Roche AG Monovalent blood brain barrier shuttle modules
UA117289C2 (en) 2014-04-02 2018-07-10 Ф. Хоффманн-Ля Рош Аг Multispecific antibodies
EP3149042B1 (en) 2014-05-29 2019-08-28 Spring Bioscience Corporation Pd-l1 antibodies and uses thereof
JP6652557B2 (en) 2014-05-30 2020-02-26 ヴェンタナ メディカル システムズ， インク． Multiplexed assay for improved scoring of tumor tissue stained for PD-L1
AU2015289672A1 (en) 2014-07-15 2017-03-02 Genentech, Inc. Compositions for treating cancer using PD-1 axis binding antagonists and MEK inhibitors
EP2982692A1 (en) 2014-08-04 2016-02-10 EngMab AG Bispecific antibodies against CD3epsilon and BCMA
PE20170263A1 (en) 2014-08-04 2017-03-30 Hoffmann La Roche T-CELL ACTIVATING ANTIGEN-BINDING BI-SPECIFIC MOLECULES
WO2016019969A1 (en) 2014-08-08 2016-02-11 Ludwig-Maximilians-Universität München Subcutaneously administered bispecific antibodies for use in the treatment of cancer
RU2017108173A (en) 2014-08-14 2018-09-17 Ф.Хоффманн-Ля Рош Аг COMBINED THERAPY BASED ON ANTIBODIES ACTIVATING HUMAN CD40 AND ANTIBODIES TO HUMAN PD-L1
WO2016030350A1 (en) 2014-08-29 2016-03-03 F. Hoffmann-La Roche Ag Combination therapy of tumor-targeted il-2 variant immunocytokines and antibodies against human pd-l1
WO2016036678A1 (en) 2014-09-02 2016-03-10 Medimmune, Llc Formulations of bispecific antibodies
WO2016044189A1 (en) 2014-09-15 2016-03-24 Genentech, Inc. Methods of treating cancer using pd-1 axis binding antagonist and il-17 binding antagonists
MA40764A (en) 2014-09-26 2017-08-01 Chugai Pharmaceutical Co Ltd THERAPEUTIC AGENT INDUCING CYTOTOXICITY
ES2850325T3 (en) 2014-10-09 2021-08-27 Engmab Sarl Bispecific antibodies against CD3epsilon and ROR1
AU2015329966A1 (en) 2014-10-09 2017-04-27 Engmab Sàrl Bispecific antibodies against CD3epsilon and ROR1 for use in the treatment of ovarian cancer
EP3224275B1 (en) 2014-11-14 2020-03-04 F.Hoffmann-La Roche Ag Antigen binding molecules comprising a tnf family ligand trimer
WO2016081384A1 (en) 2014-11-17 2016-05-26 Genentech, Inc. Combination therapy comprising ox40 binding agonists and pd-1 axis binding antagonists
MX2017006571A (en) 2014-11-20 2017-09-29 Hoffmann La Roche T cell activating bispecific antigen binding molecules agiant folr1 and cd3.
PL3221357T3 (en) 2014-11-20 2020-11-02 F. Hoffmann-La Roche Ag Common light chains and methods of use
EP3023437A1 (en) 2014-11-20 2016-05-25 EngMab AG Bispecific antibodies against CD3epsilon and BCMA
EP3029068A1 (en) 2014-12-03 2016-06-08 EngMab AG Bispecific antibodies against CD3epsilon and BCMA for use in the treatment of diseases
EP3227337A1 (en) 2014-12-05 2017-10-11 F. Hoffmann-La Roche AG Methods and compositions for treating cancer using pd-1 axis antagonists and hpk1 antagonists
EP3240908A2 (en) 2014-12-30 2017-11-08 F. Hoffmann-La Roche AG Methods and compositions for prognosis and treatment of cancers
JP2018515450A (en) 2015-04-23 2018-06-14 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト Combination therapy of antibodies that bind to angiopoietin 2 with antibodies that bind to programmed death ligand 1
JP2018520642A (en) 2015-05-01 2018-08-02 ジェネンテック， インコーポレイテッド Mask anti-CD3 antibody and method of use thereof
ES2835866T3 (en) 2015-05-12 2021-06-23 Hoffmann La Roche Therapeutic and diagnostic procedures for cancer
KR20180012753A (en) 2015-05-29 2018-02-06 제넨테크, 인크. Treatment and Diagnosis Methods for Cancer
WO2016196381A1 (en) 2015-05-29 2016-12-08 Genentech, Inc. Pd-l1 promoter methylation in cancer
TWI731861B (en) 2015-06-16 2021-07-01 美商建南德克公司 HUMANIZED AND AFFINITY MATURED ANTIBODIES TO FcRH5 AND METHODS OF USE
MX2017016353A (en) 2015-06-17 2018-05-02 Genentech Inc Methods of treating locally advanced or metastatic breast cancers using pd-1 axis binding antagonists and taxanes.
SI3331910T1 (en) 2015-08-03 2020-07-31 Engmab Sarl Monoclonal antibodies against human b cell maturation antigen (bcma)
WO2017055388A2 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Bispecific t cell activating antigen binding molecules
JP6937746B2 (en) 2015-10-02 2021-09-22 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト Bispecific anti-CD19 × CD3T cell-activating antigen-binding molecule
WO2017055318A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Cd33xcd3 bispecific t cell activating antigen binding molecules
CN107849137B (en) 2015-10-02 2021-11-26 豪夫迈·罗氏有限公司 Bispecific anti-CEAXCD 3T cell activating antigen binding molecules
US20170096485A1 (en) 2015-10-02 2017-04-06 Hoffmann-La Roche Inc. Bispecific t cell activating antigen binding molecules
AU2016329251B2 (en) 2015-10-02 2023-02-02 F. Hoffmann-La Roche Ag Anti-PD1 antibodies and methods of use
PL3377107T3 (en) 2015-11-19 2020-12-14 F. Hoffmann-La Roche Ag Methods of treating cancer using b-raf inhibitors and immune checkpoint inhibitors
JP7325186B2 (en) 2015-12-09 2023-08-14 エフ・ホフマン－ラ・ロシュ・アクチェンゲゼルシャフト Type II anti-CD20 antibody for reducing the formation of anti-drug antibodies
MX2018008347A (en) * 2016-01-08 2018-12-06 Hoffmann La Roche Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies.
CN109196121B (en) 2016-02-29 2022-01-04 基因泰克公司 Methods for treatment and diagnosis of cancer
ES2850428T3 (en) 2016-04-15 2021-08-30 Hoffmann La Roche Cancer monitoring and treatment procedures
JP7250674B2 (en) 2016-08-08 2023-04-03 エフ・ホフマン－ラ・ロシュ・アクチェンゲゼルシャフト CANCER TREATMENT AND DIAGNOSTIC METHOD
WO2018031865A1 (en) 2016-08-12 2018-02-15 Genentech, Inc. Combination therapy with a mek inhibitor, a pd-1 axis inhibitor, and a vegf inhibitor
EP3516396A1 (en) 2016-09-26 2019-07-31 H. Hoffnabb-La Roche Ag Predicting response to pd-1 axis inhibitors
AU2017339517B2 (en) 2016-10-06 2024-03-14 Foundation Medicine, Inc. Therapeutic and diagnostic methods for cancer
EP3551663A1 (en) 2016-12-12 2019-10-16 H. Hoffnabb-La Roche Ag Methods of treating cancer using anti-pd-l1 antibodies and antiandrogens
MX2019008348A (en) 2017-01-18 2019-10-21 Genentech Inc Idiotypic antibodies against anti-pd-l1 antibodies and uses thereof.
TW201837467A (en) 2017-03-01 2018-10-16 美商建南德克公司 Diagnostic and therapeutic methods for cancer
AU2018250875A1 (en) 2017-04-13 2019-10-03 F. Hoffmann-La Roche Ag An interleukin-2 immunoconjugate, a CD40 agonist, and optionally a PD-1 axis binding antagonist for use in methods of treating cancer
MX2019012192A (en) 2017-04-14 2020-01-21 Genentech Inc Diagnostic and therapeutic methods for cancer.
JP2020527351A (en) 2017-07-21 2020-09-10 ジェネンテック， インコーポレイテッド Cancer treatment and diagnosis
JP2020530003A (en) 2017-08-07 2020-10-15 アムジェン インコーポレイテッド Treatment of three-type negative breast cancer or colorectal cancer with liver metastasis with anti-PD-L1 antibody and oncolytic virus
MX2020006125A (en) * 2017-12-14 2020-08-24 Hoffmann La Roche Use of a cea cd3 bispecific antibody and a pd-1 axis binding antagonist in a dosage regime to treat cancer.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014131711A1 (en) * 2013-02-26 2014-09-04 Roche Glycart Ag Bispecific t cell activating antigen binding molecules
WO2015112534A2 (en) * 2014-01-21 2015-07-30 Medimmune, Llc Compositions and methods for modulating and redirecting immune responses

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HOFFMANN-LA ROCHE: "A Study of the Safety, Pharmacokinetics, and Therapeutic Activity of RO6958688 in Combination With Atezolizumab in Participants With Locally Advanced and/or Metastatic Carcinoembryonic Antigen (CEA)-Positive Solid Tumors - Full Text View - ClinicalTrials.gov", CLINICAL TRIALS, 6 January 2016 (2016-01-06), XP055355741, Retrieved from the Internet <URL:https://clinicaltrials.gov/ct2/show/NCT02650713?term=nct02650713&rank=1> [retrieved on 20170316] *
HOFFMANN-LAROCHE: "A Study of RO6958688 in Patients With Locally Advanced and/or Metastatic Solid Tumors - Full Text View - ClinicalTrials.gov", CLINICAL TRIALS, 12 December 2014 (2014-12-12), XP055355740, Retrieved from the Internet <URL:https://clinicaltrials.gov/ct2/show/NCT02324257?term=nct02324257&rank=1> [retrieved on 20170316] *
MARINA BACAC ET AL: "Abstract 1494: Combination of CEA TCB, a novel T-cell bispecific antibody for the treatment of solid tumors, with PD-L1 checkpoint blockade", CANCER RESEARCH, 15 July 2016 (2016-07-15), pages 1494, XP055355787, DOI: 10.1158/1538-7445.AM2016-1494 *
MARINA BACAC ET AL: "Abstract 2481: CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors | Cancer Research", CANCER RESEARCH, 1 August 2015 (2015-08-01), pages 2481, XP055355796, DOI: 10.1158/1538-7445.AM2015-2481 *
TAKUYA OSADA ET AL: "CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1", CANCER IMMUNOLOGY, IMMUNOTHERAPY, vol. 64, no. 6, 1 June 2015 (2015-06-01), Berlin/Heidelberg, pages 677 - 688, XP055236106, ISSN: 0340-7004, DOI: 10.1007/s00262-015-1671-y *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018099978A1 (en) * 2016-11-29 2018-06-07 Horst Lindhofer Combination of t-cell redirecting multifunctional antibodies with immune checkpoint modulators and uses thereof
WO2018099539A1 (en) * 2016-11-29 2018-06-07 Horst Lindhofer Combination of t-cell redirecting multifunctional antibodies with immune checkpoint modulators and uses thereof
US11942149B2 (en) 2017-02-24 2024-03-26 Macrogenics, Inc. Bispecific binding molecules that are capable of binding CD137 and tumor antigens, and uses thereof
US11459394B2 (en) 2017-02-24 2022-10-04 Macrogenics, Inc. Bispecific binding molecules that are capable of binding CD137 and tumor antigens, and uses thereof
WO2019020745A1 (en) * 2017-07-28 2019-01-31 F. Hoffmann-La Roche Ag Bispecific antibody formulation
US20200392237A1 (en) * 2017-11-01 2020-12-17 Hoffmann-La Roche Inc. Combination therapy with targeted OX40 agonists
CN111315781A (en) * 2017-11-01 2020-06-19 豪夫迈·罗氏有限公司 Combination therapy with a targeted OX40 agonist
WO2019096136A1 (en) * 2017-11-14 2019-05-23 拜西欧斯（北京）生物技术有限公司 Anti-pd-1 antibody, preparation method therefor and use thereof
CN111212660A (en) * 2017-12-14 2020-05-29 豪夫迈·罗氏有限公司 Use of CEA CD3 bispecific antibodies and PD-1 axis binding antagonists in dosage regimens for the treatment of cancer
WO2019115659A1 (en) * 2017-12-14 2019-06-20 F. Hoffmann-La Roche Ag Use of a cea cd3 bispecific antibody and a pd-1 axis binding antagonist in a dosage regime to treat cancer
JP2021506817A (en) * 2017-12-14 2021-02-22 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト Use of CEA CD3 bispecific antibody and PD-1 axis binding antagonist in dosing regimens to treat cancer
US11685781B2 (en) 2018-02-15 2023-06-27 Macrogenics, Inc. Variant CD3-binding domains and their use in combination therapies for the treatment of disease
US11555071B2 (en) 2018-06-03 2023-01-17 Lamkap Bio Beta Ltd. Bispecific antibodies against CEACAM5 and CD47
RU2798246C2 (en) * 2018-06-03 2023-06-20 Ламкап Био Бета Лтд. Bispecific antibodies against ceacam5 and cd47
WO2019234576A1 (en) 2018-06-03 2019-12-12 Lamkap Bio Beta Ltd. Bispecific antibodies against ceacam5 and cd47
KR20210064264A (en) * 2018-09-27 2021-06-02 고에끼 자이단 호우징 짓껭 도부쯔 쥬오 겡뀨쇼 immunocompromised mouse
KR102581970B1 (en) 2018-09-27 2023-09-22 고에끼 자이단 호우징 짓껭 도부쯔 쥬오 겡뀨쇼 immunocompromised mouse
EP3861854A4 (en) * 2018-09-27 2022-01-26 Central Institute For Experimental Animals Immunodeficient mouse
WO2021053587A1 (en) 2019-09-18 2021-03-25 Klaus Strein Bispecific antibodies against ceacam5 and cd3
WO2021110647A1 (en) 2019-12-02 2021-06-10 Lamkap Bio Beta Ag Bispecific antibodies against ceacam5 and cd47
EP3831849A1 (en) 2019-12-02 2021-06-09 LamKap Bio beta AG Bispecific antibodies against ceacam5 and cd47
WO2021121215A1 (en) * 2019-12-20 2021-06-24 Shandong Boan Biotechnology Co., Ltd. Optimized anti-cd3 arm in the generation of t-cell bispecific antibodies for immunotherapy
WO2022090439A1 (en) * 2020-10-30 2022-05-05 F. Hoffmann-La Roche Ag TREATMENT OF CANCER USING A CEA CD3 BISPECIFIC ANTIBODY AND A TGFβ SIGNALING INHIBITOR
WO2022130348A1 (en) 2020-12-18 2022-06-23 Lamkap Bio Beta Ag Bispecific antibodies against ceacam5 and cd47
US11753481B2 (en) 2020-12-18 2023-09-12 Lamkap Bio Beta Ltd Bispecific antibodies against CEACAM5 and CD47
WO2023242351A1 (en) 2022-06-16 2023-12-21 Lamkap Bio Beta Ag Combination therapy of bispecific antibodies against ceacam5 and cd47 and bispecific antibodies against ceacam5 and cd3

Also Published As

Publication number Publication date
EP3400246B1 (en) 2020-10-21
US20180000931A1 (en) 2018-01-04
MX2018008347A (en) 2018-12-06
IL259588B1 (en) 2023-05-01
HK1259020A1 (en) 2019-11-22
CN108368179B (en) 2022-08-23
AU2017205089A1 (en) 2018-06-21
EP3862365A1 (en) 2021-08-11
IL259588B2 (en) 2023-09-01
BR112018011029A2 (en) 2018-11-21
JP2019502712A (en) 2019-01-31
US20200376119A1 (en) 2020-12-03
CN108368179A (en) 2018-08-03
KR20180097615A (en) 2018-08-31
EP3400246A1 (en) 2018-11-14
JP6949030B2 (en) 2021-10-13
AU2017205089B2 (en) 2023-10-05
PL3400246T3 (en) 2021-03-08
IL259588A (en) 2018-07-31
JP2021193096A (en) 2021-12-23
CA3006529A1 (en) 2017-07-13
ES2837428T3 (en) 2021-06-30
AR107303A1 (en) 2018-04-18
US10596257B2 (en) 2020-03-24

Similar Documents

Publication Publication Date Title
US20200376119A1 (en) Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies
US20240076404A1 (en) Methods of treating cancers using pd-1 axis binding antagonists and taxanes
US11154616B2 (en) Methods of treating locally advanced or metastatic breast cancers using PD-1 axis binding antagonists and taxanes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17700491

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 259588

Country of ref document: IL

ENP Entry into the national phase

Ref document number: 3006529

Country of ref document: CA

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112018011029

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 2017205089

Country of ref document: AU

Date of ref document: 20170105

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: MX/A/2018/008347

Country of ref document: MX

ENP Entry into the national phase

Ref document number: 20187019336

Country of ref document: KR

Kind code of ref document: A

Ref document number: 2018535339

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 112018011029

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20180530