WO2020006511A1 - Trispecific antagonists - Google Patents

Trispecific antagonists Download PDF

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WO2020006511A1
WO2020006511A1 PCT/US2019/039982 US2019039982W WO2020006511A1 WO 2020006511 A1 WO2020006511 A1 WO 2020006511A1 US 2019039982 W US2019039982 W US 2019039982W WO 2020006511 A1 WO2020006511 A1 WO 2020006511A1
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amino acid
acid sequence
seq
antagonist
trispecific
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French (fr)
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Jackie Z. SHENG
Bo Liu
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Gensun Biopharma Inc
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Gensun Biopharma Inc
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Priority to JP2021522921A priority Critical patent/JP2021532170A/ja
Priority to CN201980056583.0A priority patent/CN112739717B/zh
Priority to EP19826335.2A priority patent/EP3814381A4/en
Publication of WO2020006511A1 publication Critical patent/WO2020006511A1/en
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
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    • 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
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    • 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
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    • 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
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    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
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    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/10—Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • the present application relates generally to cancer treatment and in particular, to bispecific and trispecific antagonists capable of modulating pathways associated with tumorigenesis, tumor immunity, and angiogenesis.
  • One aspect of the present application relates to a trispecific antagonist, comprising: an immunoglobulin scaffold comprising a CH1 domain, a CH2 domain and a CH3 domain; a first targeting domain comprising one or more variable domains selected from the group consisting of anti -PD- 1 variable domains, anti-PD-Ll variable domains, anti- TIGIT variable domains and anti-LAG-3 variable domains; a second targeting domain that binds specifically to VEGF and comprises one or more peptide domains derived from VEGFR; and a third targeting domain that comprises a peptide inhibitor of the
  • angiopoietin/Tie-2 signaling pathway wherein the second targeting domain is structurally linked to a carboxy -terminal of the CH3 domain and wherein the third targeting domain is inserted within the CH3 domain.
  • a trispecific antagonist comprising: an immunoglobulin scaffold comprising a CH1 domain, a CH2 domain and a CH3 domain; a first targeting domain comprising one or more variable domains selected from the group consisting of anti -PD- 1 variable domains, anti-PD-Ll variable domains, anti- TIGIT variable domains and anti-LAG-3 variable domains; a second targeting domain of anti -PD- 1 variable domains, anti-PD-Ll variable domains, anti-TIGIT variable domains and anti-LAG-3 variable domains; and a third targeting domain that comprises a peptide inhibitor of the angiopoietin/Tie-2 signaling pathway; wherein the second targeting domain is structurally linked to a carboxy -terminal of the CH3 domain and wherein the third targeting domain is inserted within the CH3 domain.
  • a trispecific antagonist comprising: an immunoglobulin scaffold comprising a CH1 domain, a CH2 domain and a CH3 domain; a first targeting domain comprising one or more variable domains selected from the group consisting of anti-VEGF variable domains; a second targeting domain comprising a TGF-b pathway inhibitor; and a third targeting domain that comprises a peptide inhibitor of the angiopoietin/Tie-2 signaling pathway; wherein the second targeting domain is structurally linked to a carboxy -terminal of the CH3 domain and wherein the third targeting domain is inserted within the CH3 domain.
  • a trispecific antagonist that comprises an immunoglobulin scaffold comprising a CH1 domain, a CH2 domain and a CH3 domain, a first targeting domain comprising one or more variable domains selected from the group consisting of anti-PD-l variable domains, anti-PD-Ll variable domains, anti-TIGIT variable domains and anti-LAG-3 variable domains; a second targeting domain comprising a component of VEGFR; and a third targeting domain comprising a TGF-b pathway inhibitor, wherein the second targeting domain is structurally linked to a carboxy -terminal of the CH3 domain, and wherein the third targeting domain is structurally linked to a carboxy -terminal of the second targeting domain.
  • a bispecific antagonist that comprises an immunoglobulin scaffold comprising a CH1 domain, a CH2 domain and a CH3 domain, a first targeting domain comprising one or more variable domains selected from the group consisting of anti-PD-l variable domains, anti-PD-Ll variable domains, anti-TIGIT variable domains and anti-LAG-3 variable domains; and a second targeting domain comprising a component of VEGFR, and wherein the third targeting domain is structurally linked to a carboxy -terminal of the second targeting domain.
  • Another aspect of the present application relates to a method for treating a cell proliferative disorder.
  • the method comprises the step of administering to a subject in need thereof an effective amount of the trispecific antagonists of the present application.
  • FIG. 1 shows HCVR and LCVR sequences of certain checkpoint antagonists, anti-PD-l, anti-PD-Ll, anti-TIGIT, anti-LAG-3, monoclonal antibodies (Mabs).
  • FIG. 2 shows HCVR and LCVR and other domain sequences for trebananib, bevacizumab, ranibizumab, Tie2, VEGF, and TGF-B antagonists.
  • FIGS. 3A-3H show eight trispecific antitumor antagonists, TS-ZPT-l. l, TS- ZPT-1.2, TS-ZPT-1.3, TS-ZPT-2, TS-ZPT-3, TS-ZPT-4, TS-ZPT-5 and TS-ZPT-6, respectively, with IgGl or IgG4 backbones comprising: (1) anti-PD-l variable region (VH1, VL1) domains or other checkpoint antibody variable domains; (2) an aflibercept fusion protein domain: (i) at the amino-terminal end of one or both IgG arms (FIGS. 3A-3C, 3F);
  • each antagonist (FIGS. 3D, 3E, 3H); or (iii) between the carboxy -terminal end of the CH3 domain and a trebananib peptide (or other biological peptide) (FIG. 3G) (3) a trebananib peptide (or other biological peptide): (i) fused to the carboxy -terminal end of each CH3 region (FIGS. 3A-3D); (ii) inserted within each of the two CH3 regions (FIG. 3E); (iii) fused to the carboxy -terminal end of each CH1 region (FIG.
  • the aflibercept fusion protein domain comprises vascular endothelial growth factor (VEGF)-binding portions from the extracellular domains of human VEGF receptors 1 and 2, while the trebananib peptide targets and binds to Angl and Ang2, thereby preventing the interaction of Angl and/or Ang2 with their cognate Tie2 receptors.
  • VEGF vascular endothelial growth factor
  • FIGS. 4A-4C show three different trispecific antitumor antagonists, TS-ZPT- 7, TS-ZPT-8 and TS-ZPT-9, respectively, comprising (1) an aflibercept fusion protein domain at each carboxy -terminal end; (2) anti-PD-l or other checkpoint antagonist antibody variable domain (VH1, VL1); and (3) a trebananib peptide (or other biological peptide): (i) inserted within each of the two CH3 regions (FIG. 4A); (ii) inserted between the carboxy - terminal end of each CH2 region and an aflibercept fusion protein domain at the carboxy - terminal end of each polypeptide chain in the antagonist (FIG. 4B); or (iii) fused to the amino terminal end of each IgGl arm (FIG. 4C).
  • VH1, VL1 checkpoint antagonist antibody variable domain
  • FIGS. 5A-5G show seven different trispecific antitumor antagonists, TS-LPT- 1, TS-LPT-2, TS-LPT-3, TS-LPT-4, TS-LPT-5, TS-M3, and TS-M4, respectively, comprising: (1) VH2 and VL2 regions corresponding to ranibizumab (Lucentis),
  • bevacizumab Avastin or other anti -VEGF variable domains; (2) VH1 and VL1 regions corresponding to anti-PD-l or other checkpoint antagonist antibody variable domains; and (3) a trebananib peptide (or other biological peptide): (i) inserted within each of the two CH3 regions (FIG. 5A); (ii) fused to the carboxy-terminal end of each CL1 region (FIG. 5B) (iii) fused to the carboxy-terminal end of the CH1 region in each of the two polypeptide chains (FIG. 5C); (iv) fused to the carboxy-terminal end of the CL region in each of the two polypeptide chains (FIG.
  • ranibizumab variable domains are derived from bevacizumab and both are known to block binding of human VEGF-A to VEGFR 1 and 2.
  • TS-LPT-l Exemplary sequences of TS-LPT-l, TS-LPT-2, TS-LPT-3, TS-LPT-4, TS-LPT-5 TS-M3, and TS-M4 are shown in SEQ ID NOS:247-269.
  • FIGS. 6A and 6B show non-reducing SDS-polyacrylamide gel
  • FIGS. 7A and 7B show IC50 values (nM) calculated from a cell-based binding assay reflecting the ability of the trispecific antitumor antagonists depicted in FIGS. 3A-3H to block the interaction between human PD-l and human PD-L1.
  • FIGS. 8A and 8B show IC50 values (nM) calculated from a binding assay reflecting the ability of the trispecific antitumor antagonists depicted in FIGS. 3D, 3E and 3G to block the interaction between VEGF and VEGFR-2.
  • FIG. 9 shows IC50 values (nM) calculated from a binding assay reflecting the ability of the trispecific antitumor antagonists depicted in FIGS. 3B, 3D, 3G, 3H and 5A to block the interaction between Ang2 and Tie2, compared to a bevacizumab-trebananib fusion control molecule.
  • FIG. 10 shows the results of a size exclusion chromatography analysis (SEC) of purified trispecific antitumor antagonists TS-ZPT-2(2Pl7), TS-ZPT-3L(2Pl7) and TS- ZPT-5(2Pl7) and anti-PD-l monospecific antibody 2P17, produced by transient transfection of HEK293 cells.
  • SEC size exclusion chromatography analysis
  • FIG. 11 shows the results of a size exclusion chromatography analysis (SEC) of purified trispecific antitumor antagonists TS-ZPT-2(2Pl7), TS-ZPT-3L(2Pl7) and TS- ZPT-5(2Pl7) expressed by CHO stable pools. Two transfection pools were assessed for TS- ZPT-3L(2Pl7). The percentage of high molecular weight (HWM%) species, low molecular weight (LMW%) species and dimerized molecule (Dimer%) are shown.
  • HWM high molecular weight
  • LMW low molecular weight
  • Dimerized molecule dimerized molecule
  • FIGS. 12A and 12B show the chromatography traces and results of a size exclusion chromatography analysis (SEC) of purified trispecific antitumor antagonist TS- ZPT-3L(2Pl7) purified from the CHO stable pool and stored at 4 degrees C for 112 days.
  • SEC size exclusion chromatography analysis
  • FIG. 13A shows the heavy chain (SEQ ID NO:202) and light chain amino acid sequences (SEQ ID NO:201) for an exemplary trispecific antitumor antagonist with the trebananib long peptide, /. e.. TS-ZPT-3L (2P17), comprising (1) anti-PD-l of another checkpoint antagonist antibody 2P17 variable domain (VH1, VL1), aflibercept VEFG binding domain fused to the carboxyl-terminal end of each antagonist, and connected to the CH3 domain with a 3xG4S linker; and (3) a trebananib peptide (or other biological peptide) inserted within each of the two CH3 regions.
  • FIG. 13B depicts an exemplary molecule derived from these sequences.
  • FIG. 14A shows the heavy chain (SEQ ID N0:200) and light chains amino acid sequences (SEQ ID NO:201) for an exemplary trispecific antitumor antagonist with the trebananib short peptide, i.e., TS-ZPT-3S(2Pl7), comprising (1) anti-PD-l of another checkpoint antagonist antibody 2P17 variable domain (VH1, VL1), aflibercept VEFG binding domain fused to the carboxyl-terminal end of each antagonist, and connected to the CH3 domain with a 3xG4S linker; and (3) a trebananib peptide (or other biological peptide) inserted within each of the two CH3 regions; (i) a single copy of the trebananib blocking peptide.
  • FIG. 14B depicts an exemplary molecule derived from these sequences.
  • FIG. 15 show SDS-PAGE of various trispecific antitumor antagonists depicted in FIGS. 5F and 5G and FIGS. 13A-14B that were produced by transiently transfected HEK293 cells.
  • FIG. 16 shows the results from a size exclusion chromatography analysis of TS-ZPT-3L(2Pl7), TS-ZPT-3S(2Pl7), TS-ZPT-3L(2Pl6), and TS-ZPT-3S(2Pl6) produced from HEK293 cells.
  • FIG. 17 shows the size exclusion chromatography analysis of TS-ZPT- 3L(2Pl7) and TS-ZPT-3S(2Pl7) over time with storage at 4 degrees C.
  • FIG. 18A shows the results of a cell-based binding assay measuring the ability of TS-ZPT-3S(2Pl6 and 2P17) and TS-ZPT-3L(2Pl6 and 2P17) trispecific antitumor antagonists compared to parental antibody 2P17 to block the interaction between PD-l and PD-L1.
  • FIG. 18B shows the IC50 values (nM) obtained from this analysis.
  • FIG. 19A shows the results of a cell-based bioassay measuring the ability of TS-ZPT-3S(2Pl6 and 2P17) and TS-ZPT-3L(2Pl6 and 2P17) trispecific antitumor antagonists compared to the benchmark antibody bevacizumab and a non-specific control antibody to block the interaction between VEGF and VEGFR-2.
  • FIG. 19B shows the IC50 values (nM) obtained from this analysis.
  • FIG. 20A shows the results of a binding assay measuring the ability of TS- TS-ZPT-3S(2Pl6 and 2P17) and TS-ZPT-3L(2Pl6 and 2P17) trispecific antitumor antagonists compared to the benchmark molecule trebananib and a non-specific control antibody to block the interaction between Ang2 and Tie2.
  • FIG. 20B shows the IC50 values (nM) obtained from this analysis.
  • FIGS. 21A-21E show the results of PD-l binding to TS-ZPT-3S(2Pl6 and 2P17) and TS-ZPT-3L(2Pl6 and 2P17) trispecific antitumor antagonists compared to the benchmark anti-PD-l antibody as determined by bio-layer interferometry, along with their resultant binding affinity constants (FIG. 21F).
  • FIGS. 22A-22E show the results of VEGF165 binding to TS-ZPT-3S(2Pl6 and 2P17) and TS-ZPT-3L(2Pl6 and 2P17) trispecific antitumor antagonists compared to the benchmark antibody bevacizumab as determined by bio-layer interferometry, along with their resultant binding affinity constants (FIG. 22F).
  • FIGS. 23A-23E show the results of Ang2 binding to TS-ZPT-3S(2Pl6 and 2P17) and TS-ZPT-3L(2Pl6 and 2P17) trispecific antitumor antagonists compared to the benchmark molecule trebananib as determined by bio-layer interferometry, along with their resultant binding affinity constants (FIG. 23F).
  • FIG.24 shows the use of bio-layer interferometry to characterize the sequential binding of TS-ZPT-3S(2Pl6 and 2P17) and TS-ZPT-3L(2Pl6 and 2P17) trispecific antitumor antagonists to each of its three binding partners using the Octet RED96 System (ForteBio).
  • FIG. 25 shows pharmacokinetic profiles of the TS-ZPT-3L(2Pl7) trispecific antitumor antagonists in 4 separate mice.
  • FIGS. 26A-26B show certain trispecific antitumor checkpoint antagonist configurations, where: (1) the VH1 and VL1 regions correspond to checkpoint #1, for example anti-PD-l or anti-PD-Ll variable domains; (2) the VH2 and VL2 regions correspond to checkpoint #2, for example, anti-TIGIT, anti-LAG-3 variable domains; and (3) the circular region corresponds to trebananib (or any other biological peptide) inserted within the CH3 domain.
  • FIG. 27 depicts a trispecific antitumor antagonist, TS-A1BT-1, having amino- terminal anti-VEGF variable regions (VH1, VL1) containing two amino acid substitutions in the VH region (E6Q, L11V) from Avastin/bevacizumab in a mutant IgGl (K447A) scaffold; Trebananib short peptide inserted within the IgGl CH3 domain; and a carboxy -terminal TGF-b! RII ECD connected to the CH3 domain with a 4xG4S linker.
  • the amino acid sequences of the HC and LC of this trispecific antitumor antagonist is listed in SEQ ID
  • FIG. 28 shows a non-reducing SDS-PAGE analysis of TS-A1BT-1 produced by HEK293 transiently transfected cells compared to the control transfection antibody, 2P17, showing good expression levels of the trispecific antitumor antagonist.
  • FIG. 29 shows the size exclusion chromatography analysis of TS-AIBT-l.
  • FIGS. 30A-30F show six trispecific antitumor antagonist configurations comprising a TGF- RII extracellular domain (ECD): TS-ZPB-l, TS-ZPB-2, TS-ZPB-3, TS- ZPB-4, TS-ZPB-5, and TS-ZPB-6, respectively.
  • ECD extracellular domain
  • FIG. 31A shows a non-reducing SDS-PAGE analysis of trispecific antitumor antagonists TS-ZPB-l(2Pl7), TS-ZPB-2(2Pl7), TS-ZPB-3(2Pl7), TS-ZPB-5(2Pl7), and TS- ZPLB-l transiently expressed by HEK 293 cells, before and after purification.
  • FIG. 32A shows exemplary size exclusion chromatograph (SEC) profiles for TS-ZPB-l(2Pl7), TS-ZPB-2(2Pl7), TS-ZPB-3(2Pl7), TS-ZPB-5(2Pl7), and TS-ZPLB-l compared to TGF- -RII-Fc.
  • SEC size exclusion chromatograph
  • FIG. 32B is a diagrammatic representation of FIG. 32B.
  • FIG. 33A shows the results of a cell-based binding assay measuring the ability of TS-ZPB-l (2P 17), TS-ZPB-2(2Pl7), TS-ZPB-3(2Pl7), and TS-ZPB-5(2Pl7) to block the interaction between PD-l and PD-L1.
  • FIG. 33B shows the IC50 values (nM) obtained from this analysis.
  • FIG. 34A shows the results of a bioassay to measure the ability of TS-ZPB- 1(2R17), TS-ZPB-2(2Pl7), TS-ZPB-3(2Pl7), TS-ZPB-5(2Pl7), and TS-ZPLB-l to block TGF l signaling compared to the benchmark TGF-b! RII-Fc.
  • FIG. 34B shows the IC50 values (nM) obtained from this analysis.
  • FIG. 35A shows the results of an ELISA assay measuring the ability of TS- ZPB-l(2Pl7), TS-ZPB-2(2P 17), TS-ZPB-3(2Pl7), TS-ZPB-5(2Pl7), and TS-ZPLB-l to block the interaction between VEGF and VEGFR-2, as compared to the benchmark
  • FIG. 35B shows the IC50 values (nM) obtained from this analysis.
  • FIGS. 36A-36C show three trispecific antagonists: TS-ZPB-5, and variants TS- ZPB-5 A, with K447A mutation in the CH3 domain, and TS-ZPB-5B, additionally with mutations to eliminate N-linked glycosylation of the aflibercept VEGF binding domain.
  • FIG. 37 shows expression levels of the trispecific antitumor antagonists TS-ZPB- 5, TS-ZPB-5A and TS-ZPB-5B produced from transiently transfected HEK293 cells.
  • FIG. 38 shows the results of a cell-based assay measuring the ability of trispecific antitumor antagonists TS-ZPB-5, TS-ZPB-5A and TS-ZPB-5B to block the interaction between VEGF and VEGFR-2.
  • FIG. 39 shows the results of a bioassay measuring the ability of trispecific antitumor antagonists TS-ZPB-5, TS-ZPB-5A and TS-ZPB-5B to block TGF l signaling.
  • FIG. 40 shows the pharmacokinetic profiles of TS-ZPT-5A and the TS-ZPT- 5B in two mice each.
  • FIGS. 41A-41C show three different bispecific antitumor antagonist configurations, Bi-ZPL-l (or Bi-ZP-l) and Bi-ZPL-2 (or Bi-ZP-2) and Bi-ZPL-3 (or Bi-ZP- 3) each comprising (1) anti-PD-l or anti-PD-Ll variable regions and (2) an afilibercept VEGF binding domain (i) fused to the amino-terminus of the VH1; (ii) mutated to eliminate the N-linked glycosylation sites and fused to the amino-terminus of the VH1; (iii) mutated to eliminate the N-Linked glycosylation sites and fused to the carboxy -terminus of the CH3 with a 3xG4S linker.
  • FIG. 42A shows the results of a cell-based assay measuring the ability of Bi- ZPL-l molecules compared to the benchmark anti-PD-l antibody for the inhibition of the interaction between PD-L1 and PD-l.
  • FIG. 42B shows the resulting IC50 values (nM) obtained from this analysis.
  • FIG. 43A shows the results of a bioassay measuring the ability of Bi-ZPL-l molecules compared to the benchmark anti-PD-l antibody for the inhibition of the interaction between VEGF and VEGFR-2.
  • FIG. 43B shows the resulting IC50 values (nM).
  • FIG. 44 shows the results of a size exclusion chromatography analysis of protein A purified Bi-ZP-2 and Bi-ZPL-3.
  • FIG. 45 shows the pharmacokinetic assessment in mice of Bi-ZP-2 and Bi-
  • FIGS. 46A-46C show the sequences of the exemplary framework regions.
  • FIG. 46A shows the exemplary framework regions of anti-TIGIT mAbs.
  • FIG. 46B shows the exemplary framework regions of anti-PD-l and anti-PD-Ll mAbs.
  • FIG. 46C shows the exemplary framework regions of anti-LAG3 mAbs.
  • PD-l refers to any form of PD-l and variants thereof that retain at least part of the activity of PD-l. Unless indicated differently, such as by specific reference to human PD-l, PD-l includes all mammalian species of native sequence PD-l, e.g., human, canine, feline, equine, and bovine. An exemplary human PD-l amino acid sequence is listed below:
  • PD-L1 refers to any form of PD-L1 and variants thereof that retain at least part of the activity of PD-L 1.
  • PD-L1 includes all mammalian species of native sequence PD-L1, e.g., human, canine, feline, equine, and bovine.
  • An exemplary human PD-L1 amino acid sequence is listed below:
  • TIGIT refers to any form of TIGIT and variants thereof that retain at least part of the activity of TIGIT. Unless indicated differently, such as by specific reference to human TIGIT, TIGIT includes all mammalian species of native sequence TIGIT, e.g., human, canine, feline, equine, and bovine. The following is an exemplary human TIGIT amino acid sequence:
  • LAG-3 refers to any form of LAG-3 and variants thereof that retain at least part of the activity of lymphocyte-activation gene 3 (LAG-3).
  • LAG-3 includes all mammalian species of native sequence LAG-3, e.g., human, canine, feline, equine, and bovine.
  • LAG-3 amino acid sequence is an exemplary human LAG-3 amino acid sequence:
  • agonist refers to a substance which promotes (i.e., induces, causes, enhances, or increases) the biological activity or effect of another molecule.
  • agonist encompasses substances which bind receptor, such as an antibody, and substances which promote receptor function without binding thereto (e.g., by activating an associated protein).
  • the term“antagonist” or“inhibitor” refers to a substance that prevents, blocks, inhibits, neutralizes, or reduces a biological activity or effect of another molecule, such as a receptor or ligand.
  • An antagonist can be a mono-specific antibody, a bispecific antibody or a trispecific antibody.
  • antibody refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen through one or more
  • An antibody can be a whole antibody, an antigen binding fragment or a single chain thereof.
  • the term“antibody” encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as alpha, delta, epsilon, gamma, and mu, or a, d, e, g and m) with some subclasses among them (e.g., g1-g4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
  • immunoglobulin subclasses e.g., IgGl, IgG2, IgG3, IgG4, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discemable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
  • Antibodies or antibody antagonists of the present application may include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific, bispecific, trispecific, human, humanized, primatized, chimeric and single chain antibodies.
  • Antibodies disclosed herein may be from any animal origin, including birds and mammals.
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
  • the variable region may be condricthoid in origin (e.g., from sharks).
  • antibody fragment or“antigen-binding fragment” are used with reference to a portion of an antibody, such as F(ab')2, F(ab) 2 , Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library and anti -idiotypic (anti -Id) antibodies. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.
  • the term“antibody fragment” includes DARTs and diabodies.
  • antibody fragment also includes any synthetic or genetically engineered proteins comprising immunoglobulin variable regions that act like an antibody by binding to a specific antigen to form a complex.
  • A“single-chain fragment variable” or“scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of
  • the regions are connected with a short linker peptide of ten to about 25 amino acids.
  • the linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.
  • This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
  • a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration where the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
  • variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the numbering of the constant region domains in conventional antibodies increases as they become more distal from the antigen-binding site or amino-terminus of the antibody.
  • the N-terminal portion is a variable region and at the carboxy -terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy -terminus of the heavy and light chain, respectively.
  • Exemplary CHI-CH2-CH3 sequences described in the current application include the following wild type IgGl (SEQ ID NO:274), IgGl with a K447A mutation (SEQ ID NO:275), IgGl with the N297A mutation (SEQ ID NO:276), wild type IgG2 (SEQ ID NO:278), IgG2 with carboxy terminal lysine deleted (SEQ ID NO:279), IgG4 with the hinge S231P mutation (SEQ ID NO:280), IgG4 with S23P and K447A (SEQ ID NO:281), and IgG4 S231P and the carboxy -terminal lysine deleted (SEQ ID NO:282).
  • Exemplary CL sequence is SEQ ID NO:292.
  • Fc fragment or“Fc” are used with reference to a portion of an antibody contains no antigen-binding activity but was originally observed to crystallize readily, and for this reason was named the Fc fragment, for Fragment crystallizable.
  • This fragment corresponds to the paired CH2 and CH3 domains and is the part of the antibody molecule that interacts with effector molecules and cells.
  • the Fc fragments described herein may be derived from human IgGl, IgG2 and IgG4 antibodies with the modifications of a-glycosylation, hinge mutation and deletion of carboxy-terminal lysine.
  • Exemplary Fc sequences described in the current application include the following: wild type IgGl Fc (SEQ ID NO:210), a-glycosylated IgGl Fc (SEQ ID NO:211), IgG4 Fc with hinge mutation (SEQ ID NO:212), wild type IgG2 Fc (SEQ ID NO:213), IgGl Fc with deletion of carboxy-terminal lysine (SEQ ID NO:214), a- glycosylated IgGl Fc with deletion of carboxy-terminal lysine (SEQ ID NO:215), IgG4 Fc with hinge mutation and deletion of carboxy-terminal lysine (SEQ ID NO:216), and IgG2 Fc with deletion of carboxy-terminal lysine (SEQ ID NO:217).
  • variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen-binding site.
  • This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR 2 and LCDR 3).
  • immunoglobulin molecules are derived from camelid species or engineered based on camelid immunoglobulins.
  • an immunoglobulin molecule may consist of heavy chains only with no light chains or light chains only with no heavy chains.
  • each antigen-binding domain is short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the antigen-binding domains referred to as“framework” regions, show less inter-molecular variability.
  • the framework regions largely adopt a b-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the b-sheet structure.
  • framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
  • the amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined.
  • VH1 and VH2 refer to immunoglobulin heavy chain variable domains corresponding to two different binding specificities.
  • VL1 and“VL2” refer to light chain variable domains corresponding to two different binding specificities.
  • variable domain residues refers to variable domain residues other than the CDR residues.
  • Each variable domain typically has four FRs flanking the corresponding CDRs.
  • a VH domain typically has four HFRs, HFR1, HFR2, HFR3 and HFR4, flanking the three HCDRs in the configuration of HFR 1 -HCDR 1 -HFR2- HCDR2-HFR3-HCDR3-HFR4.
  • an LH domain typically has four LFR, LFR1, LFR2, LFR3 and LFR4, flanking the three LCDRs in the configuration of: LFR1-LCDR1-LFR2- LCDR2-LFR3-LCDR3-LFR4.
  • Exemplary FRs are summarized in FIGS. 46A-46C.
  • Light chains are classified as either kappa or lambda (K, l). Each heavy chain class may be bound with either a kappa or lambda light chain.
  • the light and heavy chains are covalently bonded to each other, and the“tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • the term“light chain constant region (CL)” includes amino acid sequences derived from antibody light chain CL (SEQ ID NO:292).
  • the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.
  • the term“heavy chain constant region (CH)” includes amino acid sequences derived from an immunoglobulin heavy chain.
  • a polypeptide comprising a heavy chain constant region comprises at least one of: a CH1 domain (SEQ ID NOS:290-291), a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • an antigen-binding polypeptide for use in the disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain.
  • a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain.
  • an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). It should be understood that the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
  • the CH3 domain can tolerate or accommodate significant insertions (e.g., greater than 100 aa) in the Fc loop of the CH3 domain (see, e.g., FIG. 3E). Therefore, in the present application, any of the disclosed inhibitor domains may be similarly inserted in the Fc loop in a manner analogous to the insertion of trebananib or the TGFP 1 RII ECD domain in the Fc loop.
  • the heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules.
  • a heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule.
  • a heavy chain constant region can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule.
  • a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
  • A“light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.
  • VH domain includes the amino terminal variable domain of an immunoglobulin heavy chain
  • CH1 domain includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain.
  • the CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
  • CH2 domain includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system).
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule.
  • the CH3 domain extends from the CH2 domain to the carboxy -terminal of the IgG molecule and comprises approximately 108 residues.
  • Hinge region includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains.
  • the term“disulfide bond” includes a covalent bond formed between two sulfur atoms.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are structurally linked by a disulfide bond and the two heavy chains are structurally linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
  • a“variant” of antibody, antibody fragment or antibody domain refers to antibody, antibody fragment or antibody domain that (1) shares a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with the original antibody, antibody fragment or antibody domain, and (2) binds specifically to the same target that the original antibody, antibody fragment or antibody domain binds specifically. It should be understood that where a measure of sequence identity is presented in the form of the phrase“at least x % identical” or“at least x % identity”, such an embodiment includes any and all whole number percentages equal to or above the lower limit.
  • amino acid sequence is presented in the present application, it should be construed as additionally disclosing or embracing amino acid sequences having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to that amino acid sequence.
  • humanized antibody refers to an antibody derived from a non-human antibody, typically a mouse monoclonal antibody.
  • a humanized antibody may be derived from a chimeric antibody that retains or substantially retains the antigen binding properties of the parental, non-human, antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans.
  • the phrase“chimeric antibody,” refers to an antibody where the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species.
  • the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.
  • multispecific antibodies of the present application include various compositions and methodologies, including asymmetric IgG-like antibodies (e.g., triomab/quadroma, Trion Pharma/Fresenius Biotech); knobs-into-holes antibodies (Genentech); Cross MAbs (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); strand exchange engineered domain (SEED) body (EMD Serono;
  • asymmetric IgG-like antibodies e.g., triomab/quadroma, Trion Pharma/Fresenius Biotech
  • knobs-into-holes antibodies (Genentech); Cross MAbs (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); strand exchange engineered domain (SEED) body (EMD Serono;
  • Fab-exchanged antibodies (Genmab); symmetric IgG-like antibodies (e.g. dual targeting (DT)-Ig (GSK/Domantis); two-in-one antibody (Genentech); crosslinked MAbs (Karmanos Cancer Center); mAh 2 (F-star); Cov X-body (Cov X/Pfizer); dual variable domain (DVD)-Ig fusions (Abbott); IgG-like bispecific antibodies (Eli Lilly); Ts2Ab (Medimmune/AZ); BsAb (ZymoGenetics); HERCULES (Biogen Idec,TvAb, Roche); scFv/Fc fusions; SCORPION (Emergent BioSolutions/Trubion, ZymoGenetics/BMS); dual affinity retargeting technology (Fc-DART); MacroGenics; dual (scFv)2-Fabs (National Research Center for Antibody
  • F(abf fusions Medarex/ AMGEN); dual-action or Bis-Fab (Genentech); Dock-and- Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); scFv- and diabody-based antibodies (e.g., bispecific T cell engagers (BiTEs, Micromet); tandem diabodies (Tandab, Affimed);
  • DARTs MicroGenics
  • AIT Receptor Logics
  • human serum albumin scFv fusion Merrimack
  • COMBODIES Epigen Biotech
  • IgG/non- IgG fusions e.g., immunocytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics).
  • “specifically binds” or“has specificity to” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope.
  • an antibody is said to“specifically bind” to an epitope when it binds to that epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
  • the term“specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
  • antibody“A” may be deemed to have a higher specificity for a given epitope than antibody“B,” or antibody“A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope“D”.
  • an antibody or an antibody fragment“has specificity to” an antigen if the antibody or antibody fragment forms a complex with the antigen with a dissociation constant (K C
  • dissociation constant
  • antigen antibody refers to an antibody that binds to a target and prevents or reduces the biological effect of that target.
  • the term can denote an antibody that prevents the target, e.g., TIGIT, to which it is bound from performing a biological function.
  • an“anti -PD- 1 antagonist antibody” refers to an antibody that is able to inhibit PD-l biological activity and/or downstream events(s) mediated by PD-l.
  • Anti- PD-l antagonist antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) PD-l biological activity, including downstream events mediated by PD-l, such as PD-l binding and downstream signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and inhibition of anti -tumor immune responses.
  • anti-PD-l antagonist antibody encompasses all the previously identified terms, titles, and functional states and characteristics whereby PD-l itself, a PD-l biological activity, or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree.
  • an anti-PD-l antagonist antibody binds PD-l and upregulates an anti -tumor immune response.
  • an“anti-PD-Ll antagonist antibody” refers to an antibody that is able to inhibit PD-L1 biological activity and/or downstream event(s) mediated by PD-L1.
  • Anti- PD-Ll antagonist antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) PD-L1 biological activity, including downstream events mediated by PD-L1, such as PD-L1 binding and downstream signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and inhibition of anti -tumor immune responses.
  • anti-PD-Ll antagonist antibody encompasses all the previously identified terms, titles, and functional states and characteristics whereby PD-L1 itself, a PD-L1 biological activity, or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree.
  • an anti- PD-L1 antagonist antibody binds PD-L1 and upregulates an anti -tumor immune response.
  • the phrase“immune checkpoint regulator” refers to a functional class of agents, which inhibit or stimulate signaling through an immune checkpoint.
  • An“immune checkpoint regulator” includes cell surface receptors and their associated ligands, which together provide a means for inhibiting or stimulating signaling pathways associated with T-cell activation.
  • immune checkpoint regulators include, but are not limited to PD-l and its ligands, PD-L1 and PD-L2; TIGIT and its CD 155 ligand, PVR; CTLA-4 and its ligands, B7-1 and B7-2; TIM-3 and its ligand, Galectin-9; LAG-3 and its ligands, including liver sinusoidal endothelial cell lectin (LSECtin) and Galectin-3; CD122 and its CD122R ligand; CD70, B7H3, B and T lymphocyte attenuator (BTLA), and VISTA.
  • PD-l and its ligands PD-L1 and PD-L2
  • TIGIT and its CD 155 ligand, PVR
  • CTLA-4 and its ligands B7-1 and B7-2
  • TIM-3 and its ligand Galectin-9
  • LAG-3 and its ligands including liver sinusoidal endothelial cell lectin (LSECtin)
  • phrases“checkpoint regulator antagonist”,“immune checkpoint binding antagonist” and“immune checkpoint antagonist” are used interchangeably herein with reference to a class of agents that interfere with (or inhibit) the activity of an immune checkpoint regulator so that, as a result of the binding to the checkpoint regulator or its ligand, signaling through the checkpoint regulator receptor is blocked or inhibited. By inhibiting this signaling, immune- suppression can be reversed so that T cell immunity against cancer cells can be re-established or enhanced.
  • Immune checkpoint regulator antagonists include antibody fragments, peptide inhibitors, dominant negative peptides and small molecule drugs, either in isolated forms or as part of a fusion protein or conjugate.
  • immune checkpoint binding agonist and“immune checkpoint agonist” are used interchangeably herein with reference to a class of agents that stimulate the activity of an immune checkpoint regulator so that, as a result of the binding to the checkpoint regulator or its ligand, signaling through the checkpoint regulator receptor is stimulated. By stimulating this signaling, T cell immunity against cancer cells can be re-established or enhanced.
  • exemplary immune checkpoint regulator agonists include, but are not limited to members of the tumor necrosis factor (TNF) receptor superfamily, such as CD27, CD40, 0X40 (CD 134), glucocorticoid-induced TNFR family-related protein (GITR), and 4-1BB (CD137) and their ligands. Additional checkpoint regulator agonists belong to the B7-CD28 superfamily, including CD28 and ICOS.
  • phrases“dominant-negative protein” or“dominant-negative peptide” refer to a protein or peptide derived from a wild type protein that has been genetically modified by mutation and/or deletion so that the modified protein or peptide interferes with the function of the endogenous wild-type protein from which it is derived.
  • VEGF binding antagonist refers to a functional class of agents that bind to VEGF- A or its receptor, VEGFR-2, so that, as a result of the binding, activation of VEGFR-2 by VEGF- A is blocked or inhibited.
  • the term“VEGF binding antagonists” include antibody fragments, peptide inhibitors, dominant negative peptides and small molecule drugs, either in isolated forms or as part of a fusion protein or conjugate.
  • Tie2 tyrosine kinase receptor binding antagonist refers to a functional class of agents that bind to a Tie2 tyrosine kinase receptor or one of its ligands so that, as a result of the binding, activation of the Tie2 tyrosine kinase receptor by one or more of its ligands (i.e., Angl, Ang2, Ang3 and Ang4) is blocked or inhibited.
  • Tie2 tyrosine kinase receptor binding antagonist include antibody fragments, peptide inhibitors, dominant negative peptides and small molecule drugs, either in isolated forms or as part of a fusion protein or conjugate.
  • small molecule drug refers to a molecular entity, often organic or organometallic, that is not a polymer, that has medicinal activity, and that has a molecular weight less than about 2 kDa, less than about 1 kDa, less than about 900Da, less than about 800Da or less than about 700Da.
  • the term encompasses most medicinal compounds termed “drugs” other than protein or nucleic acids, although a small peptide or nucleic acid analog can be considered a small molecule drug. Examples include chemotherapeutic anticancer drugs and enzymatic inhibitors.
  • Small molecules drugs can be derived synthetically, semi-synthetically (i.e., from naturally occurring precursors), or biologically.
  • immunoconjugate refers to an antibody which is fused by covalent linkage to an inhibitory peptide or small molecule drug.
  • the peptide or small molecule drug can be chemically linked to the C-terminus of a constant heavy chain or to the N-terminus of a variable light and/or heavy chain.
  • A“linker” may be used to link the peptide or small molecule drug, such as a maytansinoid, to the antitumor antagonists in a stable, covalent manner.
  • Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active.
  • Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.
  • Linkers also include charged linkers, and hydrophilic forms thereof as described herein and know in the art.
  • the immunoconjugate may further include a flexible 3-15 amino acid linker, for example, GGG (SEQ ID NO:283) or GGGGS (G4S) repetitive peptide linker (SEQ ID NOS:284-289), between an antitumor antagonist and the peptide and/or small molecule drug.
  • scaffold refers to any polymer of amino acids that exhibits properties desired to support the function of an antagonist, including addition of antibody specificity, enhancement of antibody function or support of antibody structure and stability.
  • a scaffold can be grafted with binding domains of a donor polypeptide to confer the binding specificity of the donor polypeptide onto the scaffold.
  • the phrase“multispecific inhibitor” refers to a molecule comprising at least two targeting domains with different binding specificities.
  • the multispecific inhibitor is a polypeptide comprising a scaffold and two or more immunoglobulin antigen binding domains targeting different antigens or epitopes.
  • the multispecific inhibitor is a bispecific antibody or antagonist. In other embodiments, the multispecific inhibitor is a trispecific antibody or antagonist.
  • the phrase“bispecific” refers to a molecule comprising at least two targeting domains with different binding specificities. Each targeting domain is capable of binding specifically to a target molecule and inhibiting a biological function of the target molecule upon binding to the target molecule.
  • the bispecific checkpoint regulator antagonist is a polymeric molecule having two or more peptides.
  • the targeting domain comprises an antigen binding domain or a CDR of an antibody.
  • the bispecific inhibitor is a bispecific antibody.
  • the terms“bispecific antibody,” and“bispecific antagonist” are used interchangeably herein with reference to an antibody that can specifically bind two different antigens (or epitopes).
  • the bispecific antibody is a full-length antibody that binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC).
  • the bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen it binds to.
  • the bispecific antibody is a full-length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (two pairs of HC/LC)
  • the bispecific antibody has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen it binds to.
  • the terms“trispecific antibody” and“trispecific antagonist” are used
  • the trispecific antagonist is a polymeric molecule having two or more peptides.
  • the targeting domain comprises an antigen binding domain or a CDR of an antibody.
  • the trispecific antagonist is a trispecific antibody.
  • Exemplary bispecific and trispecific antibodies may include asymmetric IgG-like antibodies (e.g., triomab/quadroma, Trion Pharma/Fresenius Biotech); knobs-into-holes antibodies (Genentech); Cross MAbs (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); strand exchange engineered domain (SEED) body (EMD Serono; biolonic, Merus); Fab-exchanged antibodies (Genmab), symmetric IgG-like antibodies (e.g., triomab/quadroma, Trion Pharma/Fresenius Biotech); knobs-into-holes antibodies (Genentech); Cross MAbs (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); strand exchange engineered domain (SEED) body (EMD Serono; biolonic, Merus); Fab-exchanged antibodies (Genmab), symmetric IgG-like antibodies (e.
  • F(ab)2 fusions Medarex/ AMGEN
  • dual-action or Bis-Fab Genetech
  • Dock-and- Lock DNL, ImmunoMedics
  • Fab-Fv UB-Celltech
  • scFv- and diabody-based antibodies e.g., bispecific T cell engagers (BiTEs, Micromet); tandem diabodies (Tandab, Affimed);
  • DARTs MicroGenics
  • AIT Receptor Logics
  • human serum albumin scFv fusion Merrimack
  • COMBODIES Epigen Biotech
  • IgG/non- IgG fusions e.g., immunocytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics).
  • the terms“treat” and“treatment” refer to the amelioration of one or more symptoms associated with a cell proliferative disorder; prevention or delay of the onset of one or more symptoms of a cell proliferative disorder; and/or lessening of the severity or frequency of one or more symptoms of cell proliferative disorder.
  • phrases“to a patient in need thereof’,“to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of the antitumor antagonist of the present disclosure for treatment of a cell proliferative disorder.
  • the terms“therapeutically effective amount”,“pharmacologically effective amount”, and“physiologically effective amount” are used interchangeably to mean the amount of an antitumor antagonist that is needed to provide a threshold level of active antagonist agents in the bloodstream or in the target tissue.
  • the precise amount will depend upon numerous factors, e.g., the particular active agent, the components and physical characteristics of the composition, intended patient population, patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein or otherwise available in the relevant literature.
  • the terms“improve”,“increase” or“reduce”, as used in this context, indicate values or parameters relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein.
  • A‘‘control individual” is an individual afflicted with the same cell proliferative disorder as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual(s) are comparable).
  • the individual (also referred to as“patient” or“subject”) being treated may be a fetus, infant, child, adolescent, or adult human with a cell proliferative disorder.
  • cell proliferative disorder refers to a disorder characterized by abnormal proliferation of cells.
  • a proliferative disorder does not imply any limitation with respect to the rate of cell growth, but merely indicates loss of normal controls that affect growth and cell division.
  • cells of a proliferative disorder can have the same cell division rates as normal cells but do not respond to signals that limit such growth.
  • Within the ambit of“cell proliferative disorder” is a neoplasm, cancer or tumor.
  • cancer refers to any one of a variety of malignant neoplasms characterized by the proliferation of cells that have the capability to invade surrounding tissue and/or metastasize to new colonization sites, and includes leukemia, lymphoma, carcinoma, melanoma, sarcoma, germ cell tumor and blastoma.
  • exemplary cancers for treatment with the methods of the instant disclosure include cancer of the brain, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, stomach and uterus, leukemia, and medulloblastoma.
  • leukemia refers to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • exemplary leukemias include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia,
  • hemocytoblastic leukemia histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and
  • carcinoma refers to the malignant growth of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma,
  • basosquamous cell carcinoma bronchioalveolar carcinoma
  • bronchiolar carcinoma basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,
  • bronchogenic carcinoma cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, ductal carcinoma in situ, invasive ductal carcinoma, lobular carcinoma, invasive lobular carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epithebale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-
  • sarcoma refers to a tumor made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sar
  • melanoma refers to a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas include, for example, acral -lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
  • Additional cancers include, for example, Hodgkin's Disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer.
  • One aspect of the present application relates to trispecific antitumor antagonists that comprise: a protein scaffold, a first targeting domain that binds specifically to an immune checkpoint regulator; a second targeting domain that binds specifically to VEGF and comprises one or more peptide domains derived from VEGFR, a third targeting domain comprising an inhibitor of the angiopoietin/Tie-2 signaling pathway, wherein the first targeting domain is located at an amino terminal of the protein scaffold and the second targeting domain is located at a carboxyl terminal of the protein scaffold.
  • the third targeting domain is inserted into a CH3 domain in a Fc loop of the protein scaffold.
  • Fusion proteins e.g., Fc-fusion proteins
  • Fc-fusion proteins produced by recombinant DNA technology often face a serious problem of fully or partially degradation in cell culture
  • Clipping because of the host-cell derived proteases. Clipping generates low molecular weight (LMW) species of the target protein and leads to inactive polypeptides. While not wishing to be bound by any particular theory, it has been hypothesized that the location of the biological peptide affects the degree of degradation in the resulting fusion protein. The inventors of the present application found that insertion of the second targeting domain into the CH3 region of the Fc loop significantly reduces clipping of the resulting trispecific antagonist.
  • LMW low molecular weight
  • Another aspect of the present application relates to trispecific antitumor antagonists that comprise: a protein scaffold, a first targeting domain that binds specifically to an immune checkpoint regulator; a second targeting domain that binds specifically to VEGF and comprises one or more peptide domains derived from VEGFR, and a third targeting domain comprising a TGF pathway inhibitor.
  • the second targeting domain is located at the carboxy - terminal of a CH3 domain of the protein scaffold
  • the third targeting domain is located at the carboxy -terminal of the second targeting domain.
  • the second targeting domain is structurally linked to the carboxy -terminal of the CH3 domain of the protein scaffold through a first linker.
  • the third targeting domain is structurally linked to the carboxy-terminal of the second targeting domain through a second linker.
  • the second targeting domain is structurally linked to the carboxy-terminal of the CH3 domain of the protein scaffold through a first linker and the third targeting domain is structurally linked to the carboxy-terminal of the second targeting domain through a second linker.
  • the second targeting domain comprises a VEGFR component with amino acid sequence of SEQ ID NO:185 (afilbercept).
  • the VEGFR component is a-glycosylated by replacing asparagine residues with glutamic acid residues.
  • An exemplary amino acid sequence of a-glycosylated VEGFR component is demonstrated in SEQ ID NO:207.
  • the trispecific antitumor antagonist variants with a-glycosylated VEGFR component retain the VEGFR bioactivity while demonstrating improved pharmacokinetics compared to the wild type afilbercept.
  • Another aspect of the present application relates to a trispecific antitumor antagonist that comprises a protein scaffold, a first targeting domain that binds specifically to an immune checkpoint regulator; a second targeting domain comprising a TGF-b pathway inhibitor; and a third targeting domain comprising a peptide inhibitor of the angiopoietin/Tie-2 signaling pathway.
  • the first targeting domain comprises bevacizumab or a variant thereof and the third targeting domain is inserted within each of the two CH3 regions of the Fc loop of the protein scaffold.
  • the first targeting domain comprising one or more anti -PD- 1 variable domains and the third targeting domain is inserted within each of the two CH3 regions of the Fc loop of the protein scaffold.
  • the first targeting domain comprising one or more anti-PD-Ll variable domains and the third targeting domain is inserted within each of the two CH3 regions of the Fc loop of the protein scaffold.
  • the trebananib peptide comprises the sequence:
  • the“trebananib peptide” and the designation“- long” or“-L” correspond to the sequence of SEQ ID NO: 182, which comprises two copies of the Ang-l/2 blocking peptide sequence, QEECEWDPWTCEHM (SEQ ID NO:194).
  • the trispecific antagonists of the present application comprise modified version of the trebananib peptide containing a single copy of the sequence corresponding to SEQ ID NO: 194 with the designation“-short” or“-S”.
  • Another aspect of the present application relates to a bispecific antitumor antagonist that comprises a protein scaffold, a first targeting domain that binds specifically to an immune checkpoint regulator; and a second targeting domain comprising a peptide inhibitor of the angiopoietin/Tie-2 signaling pathway.
  • the first targeting domain comprises one or more anti-PD- 1 or anti-PD-Ll variable domains
  • the second targeting domain is inserted within each of the two CH3 regions of the Fc loop of the protein scaffold.
  • Another aspect of the present application relates to a bispecific antitumor antagonist that comprises a protein scaffold, a first targeting domain that binds specifically to an immune checkpoint regulator; and a second targeting domain comprising a VEGFR component.
  • FIGS. 41A-40C depict exemplary bispecific anti-PD-l /VEGFR component and anti-PD-Ll /VEGFR component molecules, Bi-ZPL-l and Bi-ZPL-2, and Bi-ZPL-3, respectively that contain an aflibercept at the N-terminal end (FIG. 41A) or the carboxy -terminal end (FIG. 41B-C).
  • Bi-ZPL-l has an immunoglobulin heavy chain containing the sequence set forth in SEQ ID NO:218 and/or an immunoglobulin light chain containing the sequence set forth in SEQ ID NO:219.
  • Bi-ZP-l has an immunoglobulin heavy chain containing the sequence set forth in SEQ ID NO:218 and/or an immunoglobulin light chain containing the sequence set forth in SEQ ID NO:219.
  • Bi-ZP-l has an immunoglobulin heavy chain containing the sequence set forth in SEQ ID NO:218 and/or an immunoglobulin light chain containing the sequence set forth in
  • immunoglobulin heavy chain containing the sequence set forth in SEQ ID NO:220 and/or an immunoglobulin light chain containing the sequence set forth in SEQ ID NO:221.
  • Bi-ZP-2 has an immunoglobulin heavy chain containing the sequence set forth in SEQ ID NO:222 and/or an immunoglobulin light chain containing the sequence set forth in SEQ ID NO:223.
  • Bi-ZPL-2 has an immunoglobulin heavy chain containing the sequence set forth in SEQ ID NO:222 and/or an immunoglobulin light chain containing the sequence set forth in SEQ ID NO:223.
  • Bi-ZPL-2 has an immunoglobulin heavy chain containing the sequence set forth in SEQ ID NO:222 and/or an immunoglobulin light chain containing the sequence set forth in SEQ ID NO:223.
  • Bi-ZPL-2 has an immunoglobulin heavy chain containing the sequence set forth in SEQ ID NO:222 and/or an immunoglobulin light chain containing the sequence set forth in SEQ ID NO:223.
  • Bi-ZPL-3 has an immunoglobulin heavy chain containing the sequence set forth in SEQ ID NO:296 and/or an immunoglobulin light chain containing the sequence set forth in SEQ ID NO:297.
  • bispecific antitumor antagonists that comprise a protein scaffold, a first targeting domain comprising bevacizumab; and a second targeting domain comprising a peptide inhibitor of the angiopoietin/Tie-2 signaling pathway.
  • the second targeting domain is inserted within the CH3 domain in the Fc loop of the protein scaffold.
  • bispecific antitumor antagonists that comprise a protein scaffold comprising an Fc fragment, a first targeting domain comprising a VEGFR component, and a second targeting domain comprising a peptide inhibitor of the angiopoietin/Tie-2 signaling pathway.
  • the second targeting domain is inserted within the CH3 domain in the Fc loop of the protein scaffold.
  • the Fc fragment of protein scaffold comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:210-217 prior to the insertion of the second targeting domain.
  • bispecific antitumor antagonists that comprise a protein scaffold comprising an Fc fragment, a first targeting domain comprising a TGFBR2 ECD, and a second targeting domain comprising a peptide inhibitor of the angiopoietin/Tie-2 signaling pathway.
  • the second targeting domain is inserted within the CH3 domain in the Fc loop of the protein scaffold.
  • the Fc fragment of protein scaffold comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:210-217 prior to the insertion of the second targeting domain.
  • the bispecific antitumor antagonists of the present application may be constructed with an IgG backbone. More specifically, any of the bispecific antagonists of the present application may be constructed with an IgGl or IgG4 backbone.
  • IgGl backbone is preferable for cancer treatment where a target is present on antigen presenting cells that can mediate antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Use of an IgG4 backbone allows targeting of antigen where antigen binding alone is sufficient to generate the desired therapeutic benefits.
  • IgG4-based antagonists preclude undesirable effector functions associated with e.g., IgGl antibodies, including FcyR binding and complement activation.
  • Targeting Domains that bind specifically to immune checkpoint regulators include, but are not limited to, (1) anti-PD-l antibody and anti-PD-l antibody fragments, (2) anti-PD-Ll antibody and anti-PD-Ll antibody fragments, (3) Anti-TIGIT antibody and anti- TIGIT antibody fragments, and (4) Anti -LAG-3 antibody and anti -LAG-3 antibody fragments.
  • the checkpoint regulator antagonist is an anti-PD-l antibody or antibody fragment.
  • the PD-l inhibitor of the present application is an antibody, or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:48, 51, 54, 56 and 59, wherein HCDR2 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:49, 52, 57 and 60, and wherein HCDR3 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:50, 53, 55, 58 and 61; and (2) a light chain variable
  • the PD-l inhibitor of the present application is an antibody, or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:127, 129, 131, 133, 135 and 137; and (2) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:127, 129, 131, 133, 135 and 137; and (2) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:127, 129, 131, 133, 135 and 137; and (2) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:127, 129, 131, 133, 135 and 137;
  • the anti-PD-l antibody or antibody fragment comprises a heavy chain variable region that comprises (1) an HCDR1 of SEQ ID NO:59, an HCDR2 of SEQ ID NO:60 and an HCDR 3 of SEQ ID NO:61 and (2) an HFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:366, an HFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:360, an HFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:371, an HFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:357, and an immunoglobulin heavy chain variable region that comprises (1) an LCDR1 of SEQ ID NO:73, an LCDR2 of SEQ ID NO:74 and an LCDR 3 of SEQ ID NO:75 and (2) an LFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ
  • an LFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:375.
  • the anti-PD-Ll antibody or antibody fragment comprises a heavy chain variable region that comprises (1) an HCDR1 of SEQ ID NO:56, an HCDR2 of SEQ ID NO:57 and an HCDR 3 of SEQ ID NO:58 and (2) an HFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:366, an HFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:360, an HFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:367, an HFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:357, and an immunoglobulin heavy chain variable region that comprises (1) an LCDR1 of SEQ ID NO:70, an LCDR2 of SEQ ID NO:71 and an LCDR 3 of SEQ ID NO:72 and (2) an LFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of
  • an LFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:365.
  • the term“2P17” or“PD-06” refers to the PD-l inhibitor of the present application, comprising: (1) a heavy chain variable region having an amino acid sequence of SEQ ID NO:137, and (2) a light chain variable region having an amino acid sequence of SEQ ID NO:138.
  • the CDR and FR regions of 2P17 (or PD-06) are listed as SEQ ID NOS:59-61, 73-75, 357, 360, 366 and 371-375
  • the term“2P16” or“PD-05” refers to the PD-l inhibitor of the present application, comprising: (1) a heavy chain variable region having an amino acid sequence of SEQ ID NO:135, and (2) a light chain variable region having an amino acid sequence of SEQ ID NO:136.
  • the CDR and FR regions of 2P16 (or PD-05) are listed as SEQ ID NOS:56-58, 70-72, 357, 360, 366 and 365-370
  • the checkpoint regulator antagonist is an anti-PD-Ll antibody or antibody fragment.
  • the PD-L1 inhibitor of the present application is an antibody, or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:76, 79, 85 and 88, wherein HCDR2 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:77, 80, 82, 84, 86 and 89, and wherein HCDR3 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:78, 81, 83, 87 and 90
  • the anti-PD-Ll antibody or antibody fragment comprises a heavy chain variable region that comprises (1) an HCDR1 of SEQ ID NO:79, an HCDR2 of SEQ ID NO:82 and an HCDR 3 of SEQ ID NO:83 and (2) an HFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:366, an HFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:360, an HFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:383, an HFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:357, and an immunoglobulin heavy chain variable region that comprises (1) an LCDR1 of SEQ ID NO:91, an LCDR2 of SEQ ID NO:92 and an LCDR 3 of SEQ ID NO:93 and (2) an LFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:
  • an LFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:365.
  • the PD-L1 inhibitor of the present application is an antibody, or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:139, 141, 143, 145, 147, 149, 151 and 153; and (2) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 140, 142, 144, 146, 148, 150, 152 and 154, wherein the antibody, or the antigen-binding portion thereof, binds specifically to human PD-L1.
  • the anti-PD-Ll antibody heavy and light chains consist of SEQ ID NOs: 153-154.
  • PL-08 refers to the PD-L1 inhibitor of the present application, comprising: (1) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 153, and (2) a light chain variable region having an amino acid sequence of SEQ ID NO: 154.
  • the CDR and FR regions of PL-08 are listed as SEQ ID NOS:79, 82, 83, 91-93, 357, 360, 365, 366, 377-379 and 383
  • the checkpoint regulator antagonist is an anti-TIGIT antibody or antibody fragment.
  • the TIGIT inhibitor of the present application is an antibody, or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:l, 6, 11, 15, 17, 20 and 23, wherein HCDR2 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 7, 9, 12, 13, 16, 18, 21 and 24, and wherein HCDR3 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:3, 5, 8, 10, 14, 19, 22 and 25; and (2) a light chain variable region, wherein HCDR1 has an amino acid sequence that is
  • the anti-TIGIT antibody or antibody fragment comprises a heavy chain variable region that comprises (1) an HCDR1 of SEQ ID NO:23, an HCDR2 of SEQ ID NO:24 and an HCDR 3 of SEQ ID NO:25 and (2) an HFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:343, an HFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:330, an HFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:344, an HFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:319, and an immunoglobulin heavy chain variable region that comprises (1) an LCDR1 of SEQ ID NO:45, an LCDR2 of SEQ ID NO:46 and an LCDR 3 of SEQ ID NO:47 and (2) an LFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ
  • an LFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:345.
  • the TIGIT inhibitor of the present application is an antibody, or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:107, 109, 111, 113, 115, 117, 119, 121, 123 and 125; and (2) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 108, 110, 112, 114, 116, 118, 120, 122, 124 and 126, wherein the antibody, or the antigen-binding portion thereof, binds specifically to human TIGIT.
  • the anti-TIGIT antibody has heavy and light chain sequences consisting of SEQ ID NOs: 125 and 126.
  • B21-35 or“T-10” refers to the TIGIT inhibitor of the present application, comprising: (1) a heavy chain variable region having an amino acid sequence of SEQ ID NO:125, and (2) a light chain variable region having an amino acid sequence of SEQ ID NO:126.
  • the CDR and FR regions of B21-35 (or T-10) are listed as SEQ ID NOS:23-25, 45-47, 303, 311, 317, 319, 330 and 343-345
  • the checkpoint regulator antagonist is an anti-LAG-3 antibody or antibody fragment.
  • the LAG-3 inhibitor of the present application is an antibody, or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 155-157, wherein HCDR2 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:158-160, and wherein HCDR3 has an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 161-163; and (2) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): HCDR1, HCDR2
  • the anti-LAG-3 antibody or antibody fragment comprises a heavy chain variable region that comprises (1) an HCDR1 of SEQ ID NO:156, an HCDR2 of SEQ ID NO:158 and an HCDR 3 of SEQ ID NO:161 and (2) an HFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:399, an HFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID N0:400, an HFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:401, an HFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:402, and an immunoglobulin heavy chain variable region that comprises (1) an LCDR1 of SEQ ID NO: 164, an LCDR2 of SEQ ID NO: 167 and an LCDR 3 of SEQ ID NO: 169 and (2) an LFR1 having at least 80%, 85% or 90% identity to the amino
  • the LAG-3 inhibitor of the present application is an antibody, or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:171-174; and (2) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 175-178, wherein the antibody, or the antigen-binding portion thereof, binds specifically to human LAG-3.
  • the anti-LAG-3 antibody has heavy and light chain sequences consisting of SEQ ID NOS: 171 and 175.
  • the term“2L2A.1” refers to the LAG-3 inhibitor of the present application, comprising: (1) a heavy chain variable region having an amino acid sequence of SEQ ID NO:171, and (2) a light chain variable region having an amino acid sequence of SEQ ID NO:175.
  • the CDR and FR regions of 2L2A.1 are listed as SEQ ID NOS:156, 158, 161, 164, 167, 169 and 399-406
  • Angiogenesis the development of new blood vessels from pre-existing vessels, is essential for tumor growth and metastasis.
  • Angiogenesis inhibition presents a potentially valuable strategy for treating diseases, such as cancer, in which progression (e.g., metastasis) is dependent on neovascularization.
  • Inhibition of angiogenesis leads to tumor cell death, which may feed tumor antigen into host antigen presentation pathways.
  • VEGF vascular endothelial growth factor
  • VEGF pathway antagonists and inhibitors 1.
  • VEGF-R2 The principal VEGF pathway is mediated by the transmembrane tyrosine kinase VEGF-R2.
  • VEGF-A Various isoforms of VEGF, particularly VEGF-A, bind to VEGF-R1 and VEGFF- R2, resulting in dimerization and activation through phosphorylation of various downstream tyrosine kinases.
  • the VEGF pathway antagonist binds to VEGF-A or its receptors VEGFR-l and VEGFR-2 so that, as a result of the binding, activation of VEGFR-l and VEGFR-2 by VEGF-A is blocked or inhibited.
  • Angiogenesis inhibitors may be in the form of e.g., antibodies, variable domain fragments, or dominant negative fusion protein fragments.
  • An exemplary dominant negative anti-VEGFR antagonist is a protein fragment corresponding to the extracellular domain (ECD) of human VEGF receptor 1 or 2.
  • the dominant negative anti-VEGFR antagonist is aflibercept (Zaltrap®), a recombinant fusion protein containing VEGF-A binding portions from the extracellular domains of human VEGF receptors 1 and 2 fused to the human IgGl or IgG4 Fc fragment.
  • VEGFR ECDs, such as aflibercept act as soluble receptor decoys for VEGF-A.
  • Aflibercept also known as Zaltrap® is a recombinant VEGF fusion protein consisting of vascular endothelial growth factor (VEGF)-binding portions from the extracellular domains of human VEGF receptors 1 and 2 that are fused to the Fc portion of the human IgGl immunoglobulin.
  • VEGF vascular endothelial growth factor
  • Aflibercept binds multiple ligands involved in angiogenesis, including VEGF- A, VEGF-B, anti-placental growth factor (PIGF)-l and PIGF-2, including soluble ligands in circulation.
  • the aflibercept domain of the trispecific antitumor antagonists described herein is a-glycosylated by replacing asparagine (Asn, N) residues with glutamic acid (Glu, E) residues.
  • N-linked glycosylation is the attachment of a glycan, to a nitrogen atom (the amide nitrogen) of an Asn residue of a protein.
  • the Asn residue to be glycosylated must be located in a specific consensus sequence in the primary structure (Asn-X- Ser or Asn-X-Thr, where X refers to any amino acid except proline).
  • Glu residues or any other amino acid residues
  • Asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2) is transmembrane proteins that play a critical role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N- acetylgalactosamine residues. In some cases, glycosylation of proteins can lead to increased clearance by ASGR1 and ASGR2.
  • ASGR1 and ASGR2 To improve the pharmacokinetics (i.e., achieve longer half- life) of the trispecific antitumor antagonists with VEGFR component domains, the inventors of the present application created a-glycosylated variant of the VEGFR component for limiting protein clearance by ASGR1 and ASGR2.
  • the trispecific antitumor antagonist variant with a- glycosylated VEGFR component retains the VEGFR bioactivity while improves the alpha phase of pharmacokinetics by about five fold compared to the glycosylated VEGFR component. See FIG. 37.
  • An exemplary amino acid sequence of aflibercept VEGF binding domain is demonstrated in SEQ ID NO:185, i.e.,
  • CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQG LYTCAASSGLMTKKNSTFVRVHEK an exemplary amino acid sequence of a- glycosylated aflibercept VEGF binding domain is demonstrated in SEQ ID NO:207, /. e..
  • An exemplary VEGF antibody antagonist is bevacizumab (AVASTINTM), a humanized antibody.
  • Bevacizumab comprises mutated human IgGl framework regions (FRs) and antigen-binding complementarity-determining regions from the murine anti-hVEGF monoclonal antibody A4.6.1 that blocks binding of human VEGF-A to VEGFR-2.
  • Bevacizumab Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgGl, and about 7% of the sequence is derived from the murine antibody A4.6.1. Bevacizumab has a molecular mass of about 149,000 Daltons and is glycosylated. In certain embodiments, amino acid substitutions may be included in a bevacizumab/ AVASTIN antibody as described in U.S. Patent No. 7,575,893 (SEQ ID
  • amino acid substitutions include, but are not limited to E1Q, E6Q, L11V, Q13K, L18V, R19K, A23K, or combinations thereof.
  • Additional anti -VEGF antibodies include ranibizumab (trade name
  • LucentisTM (SEQ ID NOS:294-295), a monoclonal antibody fragment derived from the same parent murine antibody as bevacizumab; the G6 or B20 series antibodies (e.g., G6-23, G6-31, B20-4.1) described in U.S. Publication No. 2006/0280747, 2007/0141065 and/or 2007/0020267, as well as the antibodies described in U.S. Patent Nos. 7,297,334, 7,060,269, 6,884,879, 6,582,959, 6,703,020, 6,054,297; U.S. Patent Application Publication Nos. U.S. 2007/059312, U.S. 2006/009360, U.S. 2005/0186208, U.S. 2003/0206899, U.S. 2003/0190317, and U.S. 2003/0203409.
  • An exemplary anti -VEGFR-2 antibody antagonist is the humanized IgGl monoclonal antibody, ramucirumab, which binds to the extracellular domain of VEGFR-2, thereby blocking its interaction with VEGF-A. Additional anti -VEGFR-2 antibodies are described in U.S. Patent Nos. 7,498,414, 6,448,077 and 6,365,157.
  • Exemplary small molecule antagonists of the VEGF pathway include multikinase inhibitors of VEGFR-2, including sunitinib, sorafenib, cediranib, pazonpanib and nintedanib.
  • the Tie2 pathway is another angiogenesis pathway for which therapeutic antibodies and small molecule drugs have been developed.
  • the Tie2 tyrosine kinase receptor activates angiogenesis in response to binding by its angiopoietin (Ang) ligands (i.e., Angl, Ang2, Ang3 (mouse) and Ang4).
  • Ang angiopoietin
  • a Tie2 pathway antagonist binds to the Tie2 tyrosine kinase receptor or one of its angiopoietin (Ang) ligands (i.e., Ang-l, Ang-2, Ang-3 and Ang-4) so that, as a result of the binding, activation of the Tie2 receptor by one or more of its ligands is blocked or inhibited.
  • Ang angiopoietin
  • the Tie2 receptor binding antagonist is an inhibitory peptide.
  • the inhibitory peptide comprises the amino acid sequence in SEQ ID NO: 182, i.e. ,
  • the Tie2 receptor binding antagonist comprises the peptide of SEQ ID NO: 182 fused to the C-terminus of an Fc fragment, i.e. ,
  • the inhibitory peptide comprises a single copy of the amino acid sequence of QEECEWDPWTCEHM (SEQ ID NO: 194).
  • the inhibitory peptide is incorporated within each of the two CH3 regions in the Fc loops.
  • the loop region is an irregular secondary structure in proteins that is not a-helix or b-sheet, while it connects together b-sheets to b-sheets, b-sheets to a- helices, or a-helices to a-helices.
  • the inhibitory peptide may be added by insertion (i.e., between amino acids in the previously existing Fc loop) or by replacement of amino acids in the previously existing Fc loop (i.e., removing amino acids in the previously existing Fc loop and adding peptide amino acids).
  • An exemplary Fc fragment comprises the amino acid sequence in SEQ ID NO: 1
  • the inhibitory peptide is inserted into the Fc loop defined as the sequence EEMTK, between Fc residues Met and Lys and includes two Gly residues as linkers flanking either side of the inserted peptide, as shown in SEQ ID NO: 209, i.e.,
  • the Gly linkers are in italics, and the inhibitory peptide insertion is underlined.
  • the insertion site is also demonstrated in FIGS.13A-14B of TS-ZPT-3.
  • the first underlined section corresponds to the anti-PD-l variable domain
  • the second underlined section corresponds to the trebananib peptide inserted in the Fc loop
  • the third underlined section corresponds to the aflibercept domain fused to the carboxy -terminal end.
  • peptide inhibitors of Tie2 activation include A-l 1 (Compugen), which comprises the amino acid sequence ETFLSTNKLENQ (SEQ ID NO:184); a peptide having the amino acid sequence NSLSNASEFRAPY (SEQ ID NO:195); a peptide having the amino acid sequence NLLMAAS (SEQ ID NO: 196); the CVX-060 peptide (Pfizer); the CVX-037 peptide (Pfizer); and CGEN-25017 (Compugen). Additional peptide inhibitors of Tie2 activation are described in U.S. Patent No. 7,138,370. Exemplary peptide inhibitors of angiopoietin -1 or -2 are described in U.S. Patent Nos. 7,138,370, 7,521,053, 7,658,924, and 8,030,025.
  • Antibody inhibitors of Tie2 activation include AMG-780 (Amgen), MEDI-3617 (Medlmmune/ AstraZeneca), DX-2240 (Dyax/Sanofi-Aventis), REGN- 910 (Sanofi/Regeneron), RG7594 (Roche), LC06 (Roche), TAvi6 (Roche), AT-006
  • Tie2 binding antagonists also include the small molecule inhibitors, CGI-1842 (CGI Pharmaceuticals), LP-590 (Locus Pharmaceuticals), ACTB-1003 (Act Biotech/Bayer AG), CEP-11981 (Cephalon/Teva), MGCD265 (Methylgene), Regorafenib (Bayer),
  • TGF-b includes a multifunctional set of peptides that control cell proliferation and differentiation, migration and adhesion, extracellular matrix modification including tumor stroma and immunosuppression, angiogenesis and desmoplasia, apoptosis, and other functions in many cell types.
  • TGF-b is a potent inducer of angiogenesis, which provides a critical support system for solid tumors, as well as a mechanism for tumor cell dissemination. Many cells synthesize TGF-b and almost all of them have specific receptors for these peptides.
  • TGF-b!, TGF ⁇ 2, and TGF ⁇ 3 all function through the same receptor signaling systems.
  • the active form of TGF-b is a dimer that signals through the formation of a membrane bound heterotetramer composed of the serine threonine type 1 and type 2 receptors, TGF-b RI and TORb RII, respectively.
  • TGF-b pathway inhibitors have been developed in the form of e.g., antibodies or binding fragments directed against TGF-b! or TGF-b! RII, such as dominant negative fusion protein fragments containing the extracellular domain (ECD) of TGF-b! RII.
  • ECD extracellular domain
  • T ⁇ RbI RII ECD has been further combined with additional binding agents targeting additional checkpoint regulator pathways or angiogenesis pathways, which are central to tumor growth and dissemination.
  • An exemplary human TGF-b! RII ECD (wild-type) has the amino acid sequence set forth in SEQ ID NO: 186.
  • a TGF-b pathway inhibitor may be in the form of e.g., antibodies or variable domain fragments directed against TGF-b! or a TGF-b! RII, a TGF binding peptide, or dominant negative fusion protein fragment, such as the extracellular domain (ECD) of TGF-b! RII.
  • a bispecific or trispecific antitumor antagonist comprises a TGF- b ⁇ RII ECD.
  • a bispecific or trispecific antitumor antagonist comprises a TGF-b! RII ECD.
  • the TGF-b! the TGF-b!
  • RII ECD is fused to the carboxy- terminus of an IgG in a bispecific or trispecific antitumor antagonist, as depicted in e.g., FIGS. 30A and 30E.
  • the TGF-b! RII ECD is fused to the amino-terminus of an IgG in a bispecific or trispecific antitumor antagonist, as depicted in e.g., FIGS. 30B and 30D.
  • the TGF-b! RII ECD is inserted within the IgG Fc fragments (i.e., CH2 or CH3 regions) of an IgG in a bispecific or trispecific antitumor antagonist, as depicted in e.g, FIGS. 30C and 30F
  • Exemplary anti-TGF-b! antibodies are described in U.S. Patent Nos. 7,067,637, 7,494,651, 7,527,791, and 7,619,069.
  • Exemplary anti-TGF-b! RII antibodies are described in U.S. Patent No. 7,579,186.
  • An exemplary peptide inhibitor of TGF-b! is
  • any one of the anti-PD-l, anti-PD-Ll, anti-TIGIT, anti-LAG-3, anti-VEGF, anti- VEGFR, anti-angiopoietin-l/2, and/or anti-Tie2 receptor antagonists can be in the form of a monoclonal antibody, chimeric antibody, humanized antibody, scFv or multi-specific antagonists, such as bispecific and trispecific antagonists.
  • any of the antagonists described herein may include multiple binding specificities targeting PD-l, PD-L1, TIGIT, LAG-3, VEGF, VEGFR, angiopoietin, and/or Tie2 receptor.
  • any of the antibody antagonists may be engineered to target multiple epitopes in a given target.
  • the checkpoint antagonist and/or angiogenesis specificity may be included in the form of a dominant negative fusion protein, such as an extracellular domain (ECD) from a corresponding receptor.
  • ECD extracellular domain
  • the HCVRs and LCVRs described herein may be structurally linked to a naturally-occurring CH1-CH2-CH3 region or a non-naturally occurring or mutated Fc (CH2- CH3) region, e.g., an effectorless or mostly effectorless Fc (e.g., human IgG2 or IgG4) or, alternatively, an Fc with enhanced binding to one or more activating Fc receptors (FcyRI, FcyRIIa or FcyRIIIa) so as to enhance Treg depletion in the tumor environment.
  • an effectorless or mostly effectorless Fc e.g., human IgG2 or IgG4
  • FcyRIIa activating Fc receptors
  • the anti-PD-l, anti-PD-Ll, anti-TIGIT, anti-LAG-3, anti-VEGF, anti- VEGFR HCVRs and LCVRs described herein may be structurally linked to an CH1-CH2-CH3 comprising one or more modifications, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen- dependent cellular cytotoxicity.
  • an antibody described herein may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or it may be modified to alter its glycosylation, to alter one or more functional properties of the antibody.
  • the antibodies in the present application may include modifications in the Fc region in order to generate an Fc variant with (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement mediated cytotoxicity (CDC), (c) increased or decreased affinity for Clq and/or (d) increased or decreased affinity for a Fc receptor relative to the parent Fc.
  • Fc region variants will generally comprise at least one amino acid modification in the Fc region. Combining amino acid modifications is thought to be particularly desirable.
  • the variant Fc region may include two, three, four, five, etc. substitutions therein, e.g., of the specific Fc region positions identified herein.
  • IgG4 antibodies may be used, or antibodies or fragments lacking the Fc region or a substantial portion thereof can be devised, or the Fc may be mutated to eliminate glycosylation altogether (e.g., N297A).
  • a hybrid construct of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 and CH3 domains) may be generated that is devoid of effector function, lacking the ability to bind FcyRs (like IgG2) and activate complement (like IgG4).
  • substitution S228P which mimics the hinge sequence in IgGl and thereby stabilizes IgG4 molecules, reducing Fab-arm exchange between the therapeutic antibody and endogenous IgG4 in the patient being treated.
  • the anti-PD-l, anti-PD-Ll, anti-TIGIT, anti -LAG-3, anti-VEGF, anti-VEGFR, anti-angiopoietin, and anti-Tie2R antibodies or fragments thereof may be modified to increase its biological half-life.
  • Various approaches may be employed, including e.g., that increase the binding affinity of the Fc region for FcRn.
  • the antibody is altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022.
  • the numbering of residues in the Fc region is that of the EU index. Sequence variants disclosed herein are provided with reference to the residue number followed by the amino acid that is substituted in place of the naturally occurring amino acid, optionally preceded by the naturally occurring residue at that position. Where multiple amino acids may be present at a given position, e.g., if sequences differ between naturally occurring isotypes, or if multiple mutations may be substituted at the position, they are separated by slashes (e.g.,“X/Y/Z”).
  • pharmacokinetic properties include substitutions at positions 259, 308, and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M.
  • Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et ak, 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356),
  • combination Fc variants comprising M252Y-M428L, M428L- N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, and M428L-N434S variants have also been shown to extend half-life (U.S. 2006/173170). Further, a combination Fc variant comprising M252Y, S254T and T256E was reported to increase half-life-nearly 4- fold. Dall’Acqua et al. (2006) J. Biol. Chem. 281:23514.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:127, 129, 131, 133, 135 and 137; and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:128, 130, 132, 134, 136 and 138; (2) a second targeting domain conferred by one or more anti-VEGF antagonist and/or one or more anti-VEGFR antagonist as further described herein and (3) a third targeting domain comprising a biological peptide that binds specifically to Tie2 tyrosine kinase receptor, Ang-l, Ang-2, Ang-3 or Ang-4 as further described herein.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:137, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:138; (2) a second targeting domain conferred by one or more anti-VEGF antagonist and/or one or more anti-VEGFR antagonist as further described herein and (3) a third targeting domain comprising a biological peptide that binds specifically to Tie2 tyrosine kinase receptor, Ang-l, Ang-2, Ang-3 or Ang-4 as further described herein.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted within the CH3 domain.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2) a second targeting domain conferred by one or more anti-VEGF antagonist and/or one or more anti-VEGFR antagonist as further described herein and (3) a third targeting domain comprising a biological peptide that binds specifically to Tie2 tyrosine kinase receptor, Ang-l, Ang-2, Ang-3 or Ang-4 as further described herein.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted within the CH3 domain.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:48, 51, 54, 56 and 59, wherein HCDR2 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:49, 52, 57 and 60, and wherein HCDR3 has an amino acid sequence that is about 80%, about 90% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:50, 53, 55, 58 and 61, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% or about 100% identity to an amino acid of SEQ ID NO:59, wherein HCDR2 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95% or about 100% identity to an amino acid sequence of SEQ ID NO:60, and wherein HCDR3 has an amino acid sequence that is about 80%, about 90% or about 100% identity to an amino acid sequence of SEQ ID NO:61, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence that is about 80%, about 90% or about 100% identity to an amino acid sequence of SEQ ID NO:
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% or about 100% identity to an amino acid of SEQ ID NO:56, wherein HCDR2 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95% or about 100% identity to an amino acid sequence of SEQ ID NO:57, and wherein HCDR3 has an amino acid sequence that is about 80%, about 90% or about 100% identity to an amino acid sequence of SEQ ID NO:58, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence that is about 80%, about 90% or about 100% identity to an amino acid sequence of SEQ ID NO:
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain conferred by one or more variable regions comprising one or more anti-PD-l variable domains or one or more anti-PD-Ll variable domains; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 1;
  • a third targeting domain comprising a biological peptide that binds specifically to Tie2 tyrosine kinase receptor, Ang-l, Ang-2, Ang-3 or Ang-4 as further described herein.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted within the CH3 domain.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:137, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:138; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprising a biological peptide that binds specifically to Tie2 tyrosine kinase receptor, Ang-l, Ang-2, Ang-3 or Ang-4 as further described herein.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted within the CH3 domain.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprising a biological peptide that binds specifically to Tie2 tyrosine kinase receptor, Ang-l, Ang-2, Ang-3 or Ang-4 as further described herein.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted within the CH3 domain.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:137, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:138; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprises only one of the amino acid sequence of SEQ ID NO:194.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted within the CH3 domain.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprises only one of the amino acid sequence of SEQ ID NO:194.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted within the CH3 domain.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:137, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:138; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprises the amino acid sequence of SEQ ID NO:182.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted within the CH3 domain.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprises the amino acid sequence of SEQ ID NO:182.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted within the CH3 domain.
  • the trispecific antitumor antagonists comprises: a heavy chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 198, 200, 202, 203 and 204; and a light chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 199 and 201.
  • the trispecific antitumor antagonists comprises: a heavy chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO: 198; and a light chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:199.
  • the trispecific antitumor antagonists comprises: a heavy chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID N0:200; and a light chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:201.
  • the trispecific antitumor antagonists comprises: a heavy chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:202; and a light chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:201.
  • FIGS. 3A-FIG. 4C show a variety of different trispecific antitumor antagonists, where: (1) the VH1 and VL1 regions correspond to anti-PD-l variable domains or other checkpoint antibodies; (2) the double ovals correspond to aflibercept fusion protein domains; and (3) the circles correspond to trebananib peptide.
  • FIGS. 5A-5E show a variety of different trispecific antagonists where (1) the VH1 and VL1 regions correspond to anti-PD-l variable domains or another checkpoint antibody; (2) VH and VL of bevacizumab or another anti-VEGF- A antibody (3) the circles correspond to trebananib peptide. As shown in these figures, these components can be rearranged in multiple configurations.
  • the trebananib peptides are arranged in the following locations: (i) fused to the carboxy -terminal end of each IgG4 CH3 region (FIGS. 3A-3D); (ii) inserted within each of the two CH3 regions (FIG. 3E, FIG. 4A, and FIG. 5A); (iii) fused to the carboxy-terminal end of each CH1 region (FIG. 3F, FIG. 4C, FIG. 5C-5E); (iv) fused to the carboxy-terminal end of each aflibercept fusion protein domain (FIG. 3G); (v) fused to the carboxy-terminal end of each CL region (FIG. 3H, FIG. 5B); or (vi) fused to the amino-terminal end of the CH2 domain (FIG. 4B).
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:127, 129, 131, 133, 135 and 137; and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:128, 130, 132, 134, 136 and 138; (2) a second targeting domain comprising an anti-VEGF antagonist or an anti-VEGFR antagonist; and (3) a third targeting domain comprising a TGF-b pathway inhibitor.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist
  • the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:137, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:138; (2) a second targeting domain comprising an anti-VEGF antagonist or an anti-VEGFR antagonist; and (3) a third targeting domain comprising a TGF-b pathway inhibitor.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2) a second targeting domain comprising an anti-VEGF antagonist or an anti-VEGFR antagonist; and (3) a third targeting domain comprising a TGF-b pathway inhibitor.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:48, 51, 54, 56 and 59, wherein HCDR2 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:49, 52, 57 and 60, and wherein HCDR3 has an amino acid sequence that is about 80%, about 90% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:50, 53, 55, 58 and 61, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% or about 100% identity to an amino acid of SEQ ID NO:59, wherein HCDR2 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95% or about 100% identity to an amino acid sequence of SEQ ID NO:60, and wherein HCDR3 has an amino acid sequence that is about 80%, about 90% or about 100% identity to an amino acid sequence of SEQ ID NO:61, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence that is about 80%, about 90% or about 100% identity to an amino acid sequence of SEQ ID NO:
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% or about 100% identity to an amino acid of SEQ ID NO:56, wherein HCDR2 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95% or about 100% identity to an amino acid sequence of SEQ ID NO:57, and wherein HCDR3 has an amino acid sequence that is about 80%, about 90% or about 100% identity to an amino acid sequence of SEQ ID NO:58, and (b) a light chain variable region, wherein the light chain variable
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:137, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:138; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprising a TGF-b pathway inhibitor.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprising a TGF-b pathway inhibitor.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:137, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:138; (2) a second targeting domain conferred by an a-glycosylated aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprising a TGF-b pathway inhibitor.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2) a second targeting domain conferred by an a-glycosylated aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprising a TGF-b pathway inhibitor.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:137, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:138; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprises a T ⁇ RbI RII extracellular domain (EAD) with amino acid sequence of SEQ ID NO: 186.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2) a second targeting domain conferred by an aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO: 185; and (3) a third targeting domain comprises a TGF l RII extracellular domain (ECD) with amino acid sequence of SEQ ID NO: 186.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:137, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:138; (2) a second targeting domain conferred by an a-glycosylated aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO:185; and (3) a third targeting domain comprises a TGFP 1 RII extracellular domain (EAD) with amino acid sequence of SEQ ID NO:186.
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonist comprises: (1) a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2) a second targeting domain conferred by an a-glycosylated aflibercept (or Zaltrap®) domain with amino acid sequence of SEQ ID NO:185; and (3) a third targeting domain comprises a TGFP 1 RII extracellular domain (ECD) with amino acid sequence of SEQ ID NO:186.
  • a first targeting domain comprises: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:136; (2)
  • the second targeting domain is inserted downstream of the CH3 domain at the carboxy -terminal end of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxy -terminal end.
  • the trispecific antitumor antagonists comprise: a heavy chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:187, 189, 190, 192, 205 and 206; and a light chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 188, 191 and 193.
  • the trispecific antitumor antagonists comprise: a heavy chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO: 192; and a light chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:193.
  • the trispecific antitumor antagonists comprise: a heavy chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:205; and a light chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:193.
  • the trispecific antitumor antagonists comprise: a heavy chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:206; and a light chain having an amino acid sequence that is about 80% to about 100% identity to an amino acid sequence of SEQ ID NO:193.
  • the trispecific antitumor antagonists of the present application may be constructed with an IgG backbone. More specifically, any of the trispecific antagonists of the present application may be constructed with an IgGl or IgG4 backbone.
  • an IgGl backbone is preferable for cancer treatment where a target is present on antigen presenting cells that can mediate antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Use of an IgG4 backbone allows targeting of antigen where antigen binding alone is sufficient to generate the desired therapeutic benefits.
  • IgG4-based antagonists preclude undesirable effector functions associated with e.g., IgGl antibodies, including FcyR binding and complement activation.
  • antitumor antagonists of the present application such as anti-PD-l antagonists, anti-PD-l antibody fragments, anti-PD-Ll antibodies, anti-PD-Ll antibody fragments, anti-TIGIT antibodies, anti-TIGIT antibody fragments, anti-LAG-3 antibodies, anti- LAG-3 antibody fragments, angiogenesis pathway inhibitors, TGF pathway inhibitors, as well as bispecific antitumor antagonists and trispecific antitumor antagonists that bind specifically to PD-l, PD-L1, TIGIT, LAG-3, VEGF/VEGFR, TGF /TGF receptor, have numerous in vitro and in vivo utilities including, for example, enhancement of immune responses and treatment of cancers, infectious diseases or autoimmune diseases.
  • the antitumor antagonists of the present application are administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of diseases.
  • methods of modifying an immune response in a subject comprising administering to the subject an antibody or antigen-binding fragment thereof as described herein such that the immune response in the subject is enhanced, stimulated or up-regulated.
  • Preferred subjects include human patients in whom enhancement of an immune response would be desirable.
  • the methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting an immune response (e.g., the T-cell mediated immune response).
  • the methods are particularly suitable for treatment of cancer or chronic infections in vivo.
  • the anti-PD-l, anti- PD-L1, anti-TIGIT or anti -LAG-3 compositions may be administered together with an antigen of interest or the antigen may already be present in the subject to be treated (e.g., a tumor bearing or virus-bearing subject) to enhance antigen-specific immunity.
  • anti-TIGIT antibodies are administered together with another agent, the two can be administered separately or simultaneously.
  • the checkpoint regulator antagonist used in the above- described method is an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, anti- LAG-3 antibody, a fragment thereof, or combination thereof.
  • the checkpoint regulator antagonist is a bispecific or trispecific antibody of the present application.
  • the checkpoint regulator antagonist or antitumor antagonist is in the form of an antibody or antibody fragment.
  • the antibodies described herein are human or humanized antibodies.
  • Also encompassed are methods for detecting and/or measuring the presence of human PD-l, human PD-L1, human TIGIT or human LAG-3, in a sample comprising contacting the sample and a control sample, with a human monoclonal antibody thereof, or an antigen binding fragment thereof, which specifically binds to human PD-l, human PD-L1, human TIGIT or human LAG-3 under conditions that allow for formation of a complex between the antibody or fragment thereof and human PD-l, human PD-L1, human TIGIT or human LAG-3.
  • the formation of a complex is then detected, wherein a difference in complex formation between the sample compared to the control sample is indicative the presence of human PD-l, human PD-L1, human TIGIT or human LAG-3 antigen in the sample.
  • anti-PD-l, anti-PD-Ll, anti-TIGIT and anti-LAG-3 antibodies are in vitro and in vivo methods of using the antibodies described herein to stimulate, enhance or upregulate antigen-specific T cell responses, e.g., anti-tumor T cell responses.
  • CD3 stimulation is also provided (e.g., by co-incubation with a cell expressing membrane CD3), which stimulation can be provided at the same time, before, or after treatment with an anti -PD- 1, anti-PD-Ll, anti-TIGIT or anti-LAG-3 antibody.
  • an antigen-specific T cell response comprising contacting said T cell with an anti-PD- 1, anti-PD-Ll, anti-TIGIT or anti-LAG-3 antibody described herein, and optionally with CD3, such that an antigen-specific T cell response is enhanced, e.g., by removal of a PD-l, PD-L1, TIGIT or LAG-3 mediated inhibitory effect.
  • Any suitable indicator of an antigen-specific T cell response can be used to measure the antigen-specific T cell response.
  • suitable indicators include increased T cell proliferation in the presence of the antibody and/or increase cytokine production in the presence of the antibody.
  • interleukin-2 and/or interferon-gamma production by the antigen-specific T cell is enhanced.
  • an immune response e.g., an antigen-specific T cell response
  • a subject comprising administering an anti-PD-l antibody, an anti-PD-Ll antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, or a bispecific or trispecific antitumor antagonist described herein to the subject such that an immune response (e.g., an antigen-specific T cell response) in the subject is enhanced.
  • the subject is a tumor-bearing subject and an immune response against the tumor is enhanced.
  • a tumor may be a solid tumor or a liquid tumor, e.g., a hematological malignancy.
  • a tumor is an immunogenic tumor.
  • a tumor is non- immunogenic.
  • a tumor is PD-L1 positive.
  • a tumor is PD-L1 negative.
  • a subject may also be a virus-bearing subject in whom an immune response against the virus is enhanced as a consequence of administering an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, anti-LAG-3 antibody, bispecific antitumor antagonist or trispecific antitumor antagonist as described herein.
  • a method for inhibiting the growth of tumor cells in a subject comprises administering to the subject an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, anti-LAG-3 antibody, bispecific antitumor antagonist or trispecific antitumor antagonist described herein such that growth of the tumor is inhibited in the subject.
  • methods of treating chronic viral infection in a subject comprising administering to the subject an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, anti-LAG-3 antibody, bispecific antitumor antagonist or trispecific antitumor antagonist as described herein such that the chronic viral infection is treated in the subject.
  • An Fc may, e.g., be an Fc with effector function or enhanced effector function, such as binding or having enhanced binding to one or more activating Fc receptors.
  • Treg depletion occurs without significant depletion or inhibition of Teff in the tumor microenvironment, and without significant depletion or inhibition of Teff cells and Treg cells outside of the tumor microenvironment.
  • the subject has higher levels of TIGIT on Treg cells than on Teff cells, e.g., in the tumor microenvironment.
  • anti-TIGIT antibodies or antagonists may deplete Tregs in tumors and/or Tregs in tumor infiltrating lymphocytes (TILs).
  • an anti-mouse TIGIT antibody formatted as a mouse IgG2a (which exhibits effector function) partially depleted both Treg and CD8+ T cells, but did not deplete CD4+ T cells.
  • TIGIT signaling e.g., using an antagonist anti-TIGIT antibody of the present invention
  • blocking of TIGIT signaling might also enhance anti-tumor activity.
  • an antagonist anti-TIGIT antibody lacking effector function which: i) blocks TIGIT signaling in Tregs thus reducing their immunesuppresive activity; ii) activates anti-tumor CD8+ T cells by blocking TIGIT’ s inhibitory effects, while at the same time avoiding their effector-function-mediated depletion; and iii) enhances DNAM-mediated activation by allowing DNAM to bind to PVR (CD155, the TIGIT ligand) that would otherwise have been bound by TIGIT (and by reducing direct TIGIT-DNAM interactions) (Johnston et al. (2014) Cancer Cell 26:923).
  • PVR CD155, the TIGIT ligand
  • an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, anti-LAG-3 antibody, bispecific antitumor antagonist or trispecific antitumor antagonist described herein is given to a subject as an adjunctive therapy.
  • Treatment of cancer patient with an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, anti-LAG-3 antibody, bispecific antitumor antagonist or trispecific antitumor antagonist according to the present application may lead to a long-term durable response relative to the current standard of care; long term survival of at least 1, 2, 3, 4, 5, 10 or more years, recurrence free survival of at least 1, 2, 3, 4, 5, or 10 or more years.
  • treatment of a cancer patient with an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, anti-LAG-3 antibody, bispecific antitumor antagonist or trispecific antitumor antagonist prevents recurrence of cancer or delays recurrence of cancer by, e.g., 1, 2, 3, 4, 5, or 10 or more years.
  • An anti-PD-l, anti-PD- Ll, anti-TIGIT, and/or anti-LAG-3 treatment can be used as a primary or secondary line of treatment.
  • the subject has a cell proliferative disease or cancer.
  • Blocking of PVR/Nectin-2 signaling through TIGIT by anti-TIGIT antibodies can enhance the immune response to cancerous cells in the patient.
  • blocking of Provided herein are methods for treating a subject having cancer, comprising administering to the subject an anti-PD-l, anti-PD-Ll, anti-TIGIT, anti-LAG-3, bispecific antitumor antagonist or trispecific antitumor antagonist thereof as described herein, such that the subject is treated, e.g., such that growth of cancerous tumors is inhibited or reduced and/or that the tumors regress.
  • An anti-PD- 1, anti-PD-Ll, anti-TIGIT, anti-LAG-3, bispecific antitumor antagonist or trispecific antitumor antagonist thereof as described herein can be used alone to inhibit the growth of cancerous tumors.
  • any of these antitumor antagonists can be used in conjunction with another agent, e.g., other anti-cancer targets, immunogenic agents, standard cancer treatments, or other antibodies, as described below.
  • methods of treating cancer e.g., by inhibiting growth of tumor cells, in a subject, comprising administering to the subject a therapeutically effective amount of an anti-PD-l, anti-PD-Ll, anti-TIGIT, anti-LAG-3, bispecific antitumor antagonist or trispecific antitumor antagonist as described herein.
  • the antibody is a human anti-PD-l, anti-PD-Ll, anti-TIGIT or anti-LAG-3 antibody comprising the anti-PD-l, anti-PD-Ll, anti-TIGIT or anti-LAG-3 HCVRs and LCVR described herein, or it may be a chimeric or humanized non-human anti-hu PD-l, anti-PD-Ll antibody, anti-hu TIGIT or anti- LAG-3 antibody, e.g., a chimeric or humanized anti-PD-l, anti-PD-Ll, anti-TIGIT or anti-LAG- 3 antibody that competes for binding with, or binds to the same epitope as, at least one of the anti-PD-l, anti-PD-Ll, anti-TIGIT or anti-LAG-3 antibodies described herein.
  • Cancers whose growth may be inhibited using the antibodies of the application include cancers typically responsive to immunotherapy.
  • cancers for treatment include squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g.
  • prostate adenocarcinoma thyroid cancer
  • neuroblastoma pancreatic cancer
  • glioblastoma glioblastoma multiforme
  • cervical cancer stomach cancer
  • bladder cancer hepatoma
  • breast cancer colon carcinoma
  • head and neck cancer gastric cancer
  • gastric cancer germ cell tumor
  • pediatric sarcoma sinonasal natural killer
  • melanoma e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma
  • bone cancer skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra
  • lymphoblastic leukemia diffuse large B-cell lymphoma, Burkit s lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mes
  • the methods described herein may also be used for treatment of metastatic cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, e.g., with a blocking CTLA-4 or PD-l antibody), and recurrent cancers.
  • refractory cancers e.g., cancers refractory to previous immunotherapy, e.g., with a blocking CTLA-4 or PD-l antibody
  • recurrent cancers e.g., metastatic cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, e.g., with a blocking CTLA-4 or PD-l antibody)
  • refractory cancers e.g., cancers refractory to previous immunotherapy, e.g., with a blocking CTLA-4 or PD-l antibody
  • An anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT, anti-LAG-3 antibody, bispecific antitumor antagonist or trispecific antitumor antagonist can be administered alone, in combination with another antitumor antagonist, or concurrently with another antitumor antagonist.
  • An anti-PD-l antibody, anti-TIGIT, anti-LAG-3 antibody, bispecific antitumor antagonist or trispecific antitumor antagonist can also be administered in combination, or concurrently with, an immunogenic agent, such as cancerous cells, tumor vaccines, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells transfected with genes encoding immune stimulating cytokines, in a cancer vaccine strategy (He et al. (2004) J. Immunol. 173:4919-28), or an oncolytic virus.
  • a vaccine is prepared using autologous or allogeneic tumor cells.
  • GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 3539- 43).
  • Cancer vaccines have been shown to enhance effector T-cell infiltration into the tumors in preclinical models.
  • the major types of cancer vaccines include peptide vaccines, vector-based antigen specific vaccines, whole-cell vaccines, and dendritic cell vaccines.
  • All vaccine-based therapies are designed to deliver either single or multiple antigenic epitopes or antigens from the whole cells to the patients and induce tumor-specific effector T cells.
  • a vaccine-based therapy may be the most efficient way to induce T-cell infiltration into the tumor.
  • tumor specific antigens are differentiation antigens expressed in the tumors and in the cell from which the tumor arose, for example melanocyte antigens gplOO, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be the targets of tumor specific T cells found in the host.
  • PD-l, PD-L1, TIGIT and/or LAG-3 inhibition may be used in conjunction with a collection of recombinant proteins and/or peptides expressed in a tumor in order to generate an immune response to these proteins.
  • proteins may be viewed by the immune system as self antigens and are therefore tolerant to them.
  • the tumor antigen can include the protein telomerase, which is required for the synthesis of telomeres of chromosomes and which is expressed in more than 85% of human cancers and in only a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013).
  • Tumor antigens can also be“neo-antigens” expressed in cancer cells because of somatic mutations that alter protein sequence or create fusion proteins between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), or idiotype from B cell tumors.
  • Non-limiting examples of tumor vaccines include sipuleucel-T (Provenge®), an FDA-approved tumor vaccine for metastatic prostate cancer; tumor cells transfected to express the cytokine granulocyte macrophage colony -stimulating factor (GM-CSF), such as the whole cell GM-CSF-secreting irradiated, allogeneic pancreatic cancer vaccine (GVAX; Johns
  • a multi-peptide vaccine consisting of immunogenic peptides derived from breast cancer antigens, neu, legumain, and b-catenin, which prolonged the vaccine-induced progression-free survival of breast tumor-bearing mice when administered in combination with anti-PD-l antibody (Karyampudi L. et al. (2014) Cancer Res 74:2974-2985); peptides of melanoma antigens, such as peptides of gplOO, MAGE antigens, Trp-2, MARTI and/or tyrosinase.
  • tumor vaccines include proteins from viruses implicated in human cancers such as human papilloma viruses (HPV)(e.g., Gardasil®, Gardasil 9®, and Cervarix®; hepatitis B virus (e.g., Engerix-B and Recombivax HB); hepatitis C virus (HCV), Kaposi’s sarcoma associated herpes sarcoma virus (KSHV).
  • HPV human papilloma viruses
  • HSP hepatitis C virus
  • KSHV Kaposi’s sarcoma associated herpes sarcoma virus
  • HSP purified heat shock proteins isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins from the tumor cells and these HSPs are highly efficient at delivery to antigen presenting cells for eliciting tumor immunity.
  • Talimogene laherparepvec T-VEC, or Imlygic® is an FDA-approved oncolytic virus for the treatment of some patients with metastatic
  • DC Dendritic cells
  • DC are potent antigen presenting cells that can be used to prime antigen-specific responses.
  • DC’s can be produced ex vivo and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328- 332).
  • DCs can also be transduced by genetic means to express these tumor antigens as well.
  • DCs have also been fused directly to tumor cells for the purposes of immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As a method of vaccination, DC immunization may be effectively combined with TIGIT blocking to activate (unleash) more potent anti-tumor responses.
  • PD-l, PD-L1, TIGIT and/or LAG-3 inhibition can also be combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy).
  • standard cancer treatments e.g., surgery, radiation, and chemotherapy
  • PD-l, PD-L1, TIGIT and/or LAG-3 inhibition can be effectively combined with chemotherapeutic regimes.
  • it may be possible to reduce the dose of chemotherapeutic reagent administered Mokyr et al. (1998) Cancer Research 58: 5301-5304).
  • An example of such a combination is an antitumor antagonist in combination with decarbazine for the treatment of melanoma.
  • IL-2 interleukin-2
  • the scientific rationale behind the combined use of PD-l, PD-L1, TIGIT and/or LAG-3 inhibition and chemotherapy to promote cell death is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway.
  • Other combination therapies that may result in synergy with TIGIT, PD-l, PD-L1 and/or LAG-3 inhibition through cell death are radiation, surgery, and hormone deprivation. Each of these protocols creates a source of tumor antigen in the host.
  • Angiogenesis inhibitors can also be combined with TIGIT, PD-l, PD-L1 and/or LAG-3 inhibition. Inhibition of angiogenesis leads to tumor cell death, which may feed tumor antigen into host antigen presentation pathways.
  • anti-TIGIT antibodies, anti-PD-l antibodies, anti-PD-Ll antibodies, bispecific antitumor antagonists and trispecific antitumor antagonists described herein may also be used in combination with bispecific antibodies that target Fca or Fey receptor-expressing effectors cells to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243).
  • Bispecific antibodies can be used to target two separate antigens.
  • anti-Fc receptor/antitumor antigen e.g., Her-2/neu
  • bispecific antibodies have been used to target macrophages to sites of tumor. This targeting may more effectively activate tumor specific responses.
  • T cell arm of these responses would be augmented by the inhibition of TIGIT, PD-l, PD-L1 and/or LAG-3.
  • antigen may be delivered directly to DCs by the use of bispecific antibodies that bind to tumor antigen and a dendritic cell specific cell surface marker.
  • Tumors evade host immune surveillance by a large variety of mechanisms. Many of these mechanisms may be overcome by the inactivation of immunosuppressive proteins expressed by the tumors. These include among others TGF-b, IL-10, and Fas ligand.
  • Antibodies to each of these entities can be used in combination with the antitumor antagonists described herein to counteract the effects of the immunosuppressive agent and favor tumor immune responses by the host.
  • T cell costimulatory molecules such as OX-40 (Weinberg et al. (2000) Immunol 164: 2160- 2169), CD137/4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff et al. (1999) Nature 397: 262-266) may also provide for increased levels of T cell activation.
  • inhibitors of other immune checkpoint regulators may also be used in conjunction with other antitumor antagonists described herein, as further described below.
  • Bone marrow transplantation is currently being used to treat a variety of tumors of hematopoietic origin. While graft versus host disease is a consequence of this treatment, TIGIT inhibition may be used to increase the effectiveness of the donor engrafted tumor specific T cells by reducing graft vs. tumor responses.
  • an antitumor antagonist described herein may be administered to a subject with an infectious disease, especially chronic infections.
  • antibody-mediated PD-l, PD-L1, TIGIT and/or LAG-3 inhibition can be used alone, or as an adjuvant, in combination with vaccines, to enhance immune responsiveness to pathogens, toxins, and self-antigens.
  • pathogens for which this therapeutic approach can be applied include, but are not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa.
  • PD-l, PD-L1, TIGIT and/or LAG-3 inhibition is particularly useful against established infections by agents such as HIV that present novel or altered antigens over the course of the infections.
  • Administration of the anti-PD-l antibodies, anti-PD-Ll antibodies, anti-TIGIT antibodies, bispecific antitumor antagonists and trispecific antitumor antagonists can allow for recognition of these antigens as foreign so as to provoke an appropriate T cell response.
  • viruses causing infections treatable by the methods described herein include HIV, hepatitis (A, B, or C), herpesvirus infections (e.g., VZV, HSV-l, HAV-6, HSV-II, and CMV, Epstein Barr virus), and infections caused by an adenovirus, influenza virus, flavivirus, echoviruses, rhinoviruses, coxsackie viruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,
  • herpesvirus infections e.g., VZV, HSV-l, HAV-6, HSV-II, and CMV, Epstein Barr virus
  • infections caused by an adenovirus e.g., influenza virus, flavivirus, echoviruses, rhinoviruses, coxsackie viruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotavirus, me
  • HTLV virus dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, or combination thereof.
  • Exemplary pathogenic bacteria or diseases caused therefrom which may be treatable by the methods described herein include Chlamydia, Rickettsia, Mycobacteria, Staphylococci, Streptococci, Pneumonococci, Meningococci and Gonococci, Klebsiella,
  • Proteus Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli, Cholera,
  • Leptospirosis tetanus Leptospirosis tetanus, botulism, anthrax, plague, and Lyme disease.
  • Exemplary pathogenic fungi causing infections treatable by the methods described herein include Candida (e.g., albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger, etc.), Mucorales (e.g., mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
  • Candida e.g., albicans, krusei, glabrata, tropicalis, etc.
  • Cryptococcus neoformans e.g., Aspergillus, e., fumigatus, niger, etc.
  • Mucorales e.g., mucor, absidia, rhizopus
  • Exemplary pathogenic parasites causing infections treatable by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia Zambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasibensis.
  • PD-l, PD-L1, TIGIT and/or LAG-3 inhibition can be combined with other forms of immunotherapy such as cytokine treatment (e.g., interferons, GM- CSF, G-CSF, IL-2), or bispecific antibody therapy using two different binding specificities to provide enhanced presentation of tumor antigens.
  • cytokine treatment e.g., interferons, GM- CSF, G-CSF, IL-2
  • bispecific antibody therapy using two different binding specificities to provide enhanced presentation of tumor antigens.
  • Anti-PD-l antibodies, anti-PD-Ll antibodies, anti-TIGIT antibodies, anti-LAG-3 antibodies, bispecific antitumor antagonists and trispecific antitumor antagonists described herein can be used to enhance antigen-specific immune responses by co-administration of one or more of any of these antibodies with an antigen of interest (e.g., a vaccine).
  • an antigen of interest e.g., a vaccine
  • an immune response to an antigen in a subject comprising administering to the subject: (i) the antigen; and (ii) anti-PD-l antibody, anti-PD-Ll antibody, an anti-TIGIT antibody, anti-LAG-3 antibody, bispecific antitumor antagonists, trispecific antitumor antagonist, or combination thereof, such that an immune response to the antigen in the subject is enhanced.
  • the antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen.
  • Non-limiting examples of such antigens include those discussed in the sections above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from the viruses, bacteria or other pathogens described above.
  • a peptide or fusion protein comprising the epitope to which an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, LAG-3 antibody, bispecific antitumor antagonists, trispecific antitumor antagonist binds may be used as a vaccine instead of, or in addition to, the antitumor antagonist(s).
  • Suitable routes of administering the antibody compositions e.g., human monoclonal antibodies, multi-specific antibodies or antagonists and immunoconjugates
  • the antibody compositions can be administered by injection (e.g., intravenous or subcutaneous).
  • Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.
  • the pharmaceutical composition comprises one or more antitumor antagonists of the present application.
  • the antitumor antagonist(s) comprise one or more checkpoint regulator antagonists, such as PD-l inhibitors, PD-L1 inhibitors, anti-TIGIT inhibitors, LAG-3 inhibitors; one or more angiogenesis inhibitors, such as VEGF inhibitors, VEGFR2 inhibitors,
  • angiopoietin-l/2 inhibitors, and Tie2R inhibitors one or more antitumor inhibitors, such as TGF-b! inhibitors and TGF-b! RII inhibitors; or bispecific and trispecific antitumor antagonists thereof.
  • the antagonist(s) are formulated together with a pharmaceutically acceptable carrier.
  • Pharmaceutical composition of the present application may include one or more different antibodies, one or more multispecific antibodies, one or more immunoconjugates, or a combination thereof as described herein.
  • methods for using the pharmaceutical compositions described herein comprise administering to a subject in need thereof an effective amount of the pharmaceutical composition according to the present disclosure.
  • Any suitable route or mode of administration can be employed for providing the patient with a therapeutically or prophylactically effective dose of the antibody or antagonist.
  • routes or modes of administration include parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intratumoral), oral, topical (nasal, transdermal, intradermal or intraocular), mucosal (e.g., nasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, enteral, intrapulmonary, intralymphatic, intracavital, intraorbital, intracapsular and transurethral, as well as local delivery by catheter or stent.
  • parenteral e.g., intravenous, intraarterial, intramuscular, subcutaneous, intratumoral
  • oral topical (nasal, transdermal, intradermal or intraocular), mucosal (e.
  • a pharmaceutical composition comprising an antibody or antagonist in accordance with the present disclosure may be formulated in any pharmaceutically acceptable carrier(s) or excipient(s).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions may comprise suitable solid or gel phase carriers or excipients.
  • exemplary carriers or excipients include but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • exemplary pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, poly alcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the therapeutic agents.
  • auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the therapeutic agents.
  • the antitumor antagonist can be incorporated into a pharmaceutical composition suitable for parenteral administration.
  • Suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate.
  • Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form).
  • Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%).
  • cryoprotectants include trehalose and lactose.
  • Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 2-4%).
  • Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM).
  • Other suitable bulking agents include glycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).
  • Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants.
  • Therapeutic antitumor antagonist preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol preservative) or in sterile water prior to injection.
  • Pharmaceutical composition may be formulated for parenteral administration by injection e.g., by bolus injection or continuous infusion.
  • the therapeutic agents in the pharmaceutical compositions may be formulated in a“therapeutically effective amount” or a“prophylactically effective amount”.
  • therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the recombinant vector may vary depending on the condition to be treated, the severity and course of the condition, the mode of administration, whether the antibody or agent is administered for preventive or therapeutic purposes, the bioavailability of the particular agent(s), the ability of the antitumor antagonist to elicit a desired response in the individual, previous therapy, the age, weight and sex of the patient, the patient’s clinical history and response to the antibody, the type of the antitumor antagonist used, discretion of the attending physician, etc.
  • a therapeutically effective amount is also one in which any toxic or detrimental effect of the recombinant vector is outweighed by the therapeutically beneficial effects.
  • A“prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
  • the polypeptide domains in the antitumor antagonist are derived from the same host in which they are to be administered in order to reduce inflammatory responses against the administered therapeutic agents.
  • the antitumor antagonist is suitably administered to the patent at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards.
  • the antitumor antagonist may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.
  • a therapeutically effective amount or prophylactically effective amount of the antitumor antagonist will be administered in a range from about 1 ng/kg body weight/day to about 100 mg/kg body weight/day whether by one or more administrations.
  • each antitumor antagonist is administered in the range of from about 1 ng/kg body weight/day to about 10 mg/kg body weight/day, about 1 ng/kg body weight/day to about 1 mg/kg body weight/day, about 1 ng/kg body weight/day to about 100 pg/kg body weight/day, about 1 ng/kg body weight/day to about 10 pg/kg body weight/day, about 1 ng/kg body weight/day to about 1 pg/kg body weight/day, about 1 ng/kg body weight/day to about 100 ng/kg body weight/day, about 1 ng/kg body weight/day to about 10 ng/kg body weight/day, about 10 ng/kg body weight/day to about 100 mg/kg body weight/day, about 10 ng/kg body weight/day to about 10 mg/kg body weight/day, about 10 ng/kg body weight/day to about 1 mg/kg body weight/day, about 10 ng/kg body weight/day to about 100 pg/kg weight/day, about
  • the antitumor antagonist is administered at a dose of 500 pg to 20 g every three days, or 25 mg/kg body weight every three days.
  • each antitumor antagonist is administered in the range of about 10 ng to about 100 ng per individual administration, about 10 ng to about 1 pg per individual administration, about 10 ng to about 10 pg per individual administration, about 10 ng to about 100 pg per individual administration, about 10 ng to about 1 mg per individual administration, about 10 ng to about 10 mg per individual administration, about 10 ng to about 100 mg per individual administration, about 10 ng to about 1000 mg per injection, about 10 ng to about 10,000 mg per individual administration, about 100 ng to about 1 pg per individual administration, about 100 ng to about 10 pg per individual administration, about 100 ng to about 100 pg per individual administration, about 100 ng to about 1 mg per individual administration, about 100 ng to about 10 mg per individual administration, about 100 ng to about 100 mg per mg per mg per mg per mg per
  • administration about 1 pg to about 100 pg per individual administration, about 1 pg to about 1 mg per individual administration, about 1 pg to about 10 mg per individual administration, about 1 pg to about 100 mg per individual administration, about 1 pg to about 1000 mg per injection, about 1 pg to about 10,000 mg per individual administration, about 10 pg to about 100 pg per individual administration, about 10 pg to about 1 mg per individual administration, about 10 pg to about 10 mg per individual administration, about 10 pg to about 100 mg per individual administration, about 10 pg to about 1000 mg per injection, about 10 pg to about 10,000 mg per individual administration, about 100 pg to about 1 mg per individual administration, about 100 pg to about 10 mg per individual administration, about 100 pg to about 100 mg per individual administration, about 100 pg to about 1000 mg per injection, about 100 pg to about 10,000 mg per individual administration, about 1 mg to about 10 mg per individual administration, about 1 mg to about 100 mg per individual administration, about 1 mg to about 1000 mg per injection
  • the amount of the antitumor antagonist may be administered at a dose of about 0.0006 mg/day, 0.001 mg/day, 0.003 mg/day, 0.006 mg/day,
  • the coding sequences for an antitumor antagonist are incorporated into a suitable expression vector (e.g., viral or non-viral vector) for expressing an effective amount of the antitumor antagonist in patient with a cell proliferative disorder.
  • a suitable expression vector e.g., viral or non-viral vector
  • the pharmaceutical composition may comprise the rAAVs in an amount comprising at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 genome copies (GC) or recombinant viral particles per kg, or any range thereof.
  • the pharmaceutical composition comprises an effective amount of the recombinant virus, such as rAAV, in an amount comprising at least 1010, at least 1011, at least 1012, at least 1013, at least 1014, at least 1015 genome copies or recombinant viral particles genome copies per subject, or any range thereof.
  • the recombinant virus such as rAAV
  • Dosages can be tested in several art-accepted animal models suitable for any particular cell proliferative disorder.
  • Delivery methodologies may also include the use of poly cationic condensed DNA linked or unlinked to killed viruses, ligand linked DNA, liposomes, eukaryotic cell delivery vehicles cells, deposition of photopolymerized hydrogel materials, use of a handheld gene transfer particle gun, ionizing radiation, nucleic charge neutralization or fusion with cell membranes, particle mediated gene transfer and the like.
  • the present application provides combination therapies for enhancing an antigen-specific T cell response in a subject.
  • the method includes contacting a T cell with an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, anti-LAG-3 antibody, antibody fragment thereof, bispecific antitumor antagonist or trispecific antitumor antagonist in combination with a second antibody, antibody fragment, antagonist or drug such that an antigen-specific T cell response or apoptotic pathway is enhanced.
  • a method of reducing or depleting regulatory T cells in a tumor of a subject in need thereof includes administering an effective amount of an antibody or antibody fragment in combination with a second antibody, antibody fragment, antagonist or drug such that the number of regulatory T cells in the subject is reduced.
  • the subject has a cell proliferative disease or cancer as described herein.
  • the subject has a chronic viral infection, inflammatory disease or autoimmune disease as described herein.
  • T-cells lymphocyte activation of resting T lymphocytes by antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • the primary signal, or antigen specific signal is transduced through the T-cell receptor (TCR) following recognition of foreign antigen peptide presented in the context of the major histocompatibility- complex (MHC).
  • MHC major histocompatibility- complex
  • the second or co-stimulatory signal is delivered to T-cells by co-stimulatory molecules expressed on antigen-presenting cells (APCs). This induces T-cells to promote clonal expansion, cytokine secretion and effector function. In the absence of co-stimulation, T-cells can become refractory to antigen stimulation, which results in a tolerogenic response to either foreign or endogenous antigens.
  • T-cells receive both positive co-stimulatory and negative co-inhibitory signals.
  • the regulation of such positive and negative signals is critical to maximize the host's protective immune responses, while maintaining immune tolerance and preventing autoimmunity.
  • Negative signals seem necessary for induction of T-cell tolerance, while positive signals promote T-cell activation.
  • Both co-stimulatory and co-inhibitory signals are provided to antigen-exposed T cells, and the interplay between co-stimulatory and co-inhibitory signals is essential to controlling the magnitude of an immune response. Further, the signals provided to the T cells change as an infection or immune provocation is cleared, worsens, or persists, and these changes powerfully affect the responding T cells and re-shape the immune response.
  • the mechanism of co-stimulation is of therapeutic interest because the manipulation of co-stimulatory signals has shown to provide a means to either enhance or terminate cell-based immune response.
  • T cell dysfunction or energy can occur concurrently with an induced and sustained expression of immune checkpoint regulators, such as PD-l and its ligands, PD-L1 and PD-L2.
  • PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Thompson R H et al, Cancer Res 2006, 66(7):338l).
  • the majority of tumor infiltrating T lymphocytes predominantly express PD-l, in contrast to T lymphocytes in normal tissues and peripheral blood T
  • lymphocytes indicating that up-regulation of PD-l 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-L1 signaling mediated by PD-L1 expressing tumor cells interacting with PD- 1 expressing T cells to result in attenuation of T cell activation and evasion of immune surveillance. Inhibition of the PD-L1/PD-1 interaction provides a means to enhance T cell immunity, including CD8+ T cell-mediated killing of cancer cells and tumors. Similar enhancements to T cell immunity have been observed by inhibiting the binding of PD-L1 to the binding partner B7-1. Consequently, therapeutic targeting of PD-l and other immune checkpoint regulators are an area of intense interest.
  • B7 family which includes CTLA-4 and its ligands, B7-1 and B7-2; PD-l and its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC); B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6.
  • Additional immune checkpoint antagonists include, but are not limited to TIM-3 and its ligand, Galectin-9; LAG-3 and its ligands, including liver sinusoidal endothelial cell lectin (LSECtin) and Galectin-3; CD 122 and its CD122R ligand; CD70, B7H3, B and T lymphocyte attenuator (BTLA), and VISTA (Le Mercier et al. (2015) Front. Immunol., (6), Article 418).
  • a number of checkpoint regulator antagonists have been identified and tested in various clinical and pre-clinical models and/or approved by the FDA (Kyi et al., FEBS Letters, 588:368-376 (2014).
  • inhibitory receptor blockade also known as immune checkpoint blockade
  • immune checkpoint blockade has been validated by virtue of e.g., the FDA approval of the PD-l inhibitors, nivolumab and pembrolizumab, as well as the anti-CTLA-4 antibody, ipilimumab for metastatic melanoma.
  • An immune checkpoint antagonist modulates or interferes with the activity of the immune checkpoint regulator so that, as a result of the binding to the checkpoint regulator or its ligand, signaling through the checkpoint regulator receptor is blocked or inhibited. By inhibiting this signaling, immune-suppression can be reversed so that T cell immunity against cancer cells can be re-established or enhanced.
  • an immune checkpoint agonist (of e.g., a costimulatory molecule) stimulates the activity of an immune checkpoint regulator so that, as a result of the binding to the checkpoint regulator or its ligand, signaling through the checkpoint regulator receptor is stimulated. By stimulating this signaling, T cell immunity against cancer cells can be re-established or enhanced.
  • a method for stimulating an immune response in a subject comprises administering to the subject an anti-PD-l antibody, anti -PD-L 1 antibody, anti-TIGIT antibody, anti-LAG-3 antibody, antibody fragment(s) thereof (e.g., anti-TIGIT HCVR and/LCVRs), bispecific antitumor antagonist or trispecific antitumor antagonist described herein in combination with another immune checkpoint regulator described herein above, such that an immune response is stimulated in the subject, for example to inhibit tumor growth or to stimulate an anti-viral response.
  • an anti-PD-l antibody anti-PD-L 1 antibody
  • anti-TIGIT antibody anti-LAG-3 antibody
  • antibody fragment(s) thereof e.g., anti-TIGIT HCVR and/LCVRs
  • bispecific antitumor antagonist or trispecific antitumor antagonist described herein in combination with another immune checkpoint regulator described herein above, such that an immune response is stimulated in the subject, for example to inhibit tumor growth or to stimulate an anti-viral response.
  • an anti -PD- 1 antibody, anti-PD-Ll antibody, anti -TI GIT antibody, anti-LAG-3 antibody, antibody fragment(s) thereof, bispecific antitumor antagonist or trispecific antitumor antagonist according to the present application is administered in combination with another immune checkpoint regulator, either as separate antibodies or in multi-specific antibody comprising binding specificities to multiple products.
  • an anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody, anti-LAG-3 antibody, bispecific antitumor antagonist or trispecific antitumor antagonist described herein can be combined to stimulate an immune response with (i) an antagonist of the IgSF family protein, B7 family or TNF family that inhibit T cell activation, or antagonist of a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-b, VEGF, or other immunosuppressive cytokines) and/or (ii) an agonist of a stimulatory receptors of the IgSF family, B7 family or TNF family or of cytokines to stimulate T cell activation, for stimulating an immune response.
  • an antagonist of the IgSF family protein, B7 family or TNF family that inhibit T cell activation or antagonist of a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-b, VEGF, or other immunosuppressive
  • only subjects with a cancer exhibiting high expression of a ligand for an immune checkpoint regulator are selected for combination treatment with the anti-PD-l, anti-PD-Ll, anti-TIGIT, and/or anti-LAG-3 antibody, fragment thereof, or any of the bispecific or trispecific antagonists of the present application.
  • a subject with a cancer exhibiting high expression of PVR (CD155) and/or Nectin- 2 (CD112) and/or low expression PD-L1 may be selected for monotherapy with anti-TIGIT antibodies, fragments thereof, or TIGIT antagonists of the present application, or combination therapy with a PD-l antagonist or other immune checkpoint regulator.
  • the anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT antibody may be administered separately from the second antibody, antibody fragment or antagonist, or a multispecific antibody or antagonist may be administered comprising at least one binding specificity for TIGIT and a second binding specificity for the other targeted product.
  • anti-PD-l antibody, anti-PD-Ll antibody, anti-TIGIT or bispecific or trispecific antagonists in accordance with the present application may be co-administered with one or more additional agents, e.g., antibodies, antagonists, or drugs in amount(s) effective in stimulating an immune response and/or apoptosis so as to further enhance, stimulate or up-regulate an immune response and/or apoptosis in a subject.
  • additional agents e.g., antibodies, antagonists, or drugs in amount(s) effective in stimulating an immune response and/or apoptosis so as to further enhance, stimulate or up-regulate an immune response and/or apoptosis in a subject.
  • the anti-PD-l, anti-PD-Ll, anti-TIGIT or anti-LAG-3 antibody or fragment(s) thereof is administered subsequent to treatment with a different antitumor antagonist.
  • anti-PD-l, anti-PD-Ll, anti-TIGIT or anti-LAG-3 antibodies may be administered only after treatment with a PD-1/PD-L1 antagonist has failed, has led to incomplete therapeutic response, or there has been recurrence of the tumor or relapse (or“PD-l failure”).
  • cancers exhibiting such failures may be screened for expression of e.g., PVR and/or Nectin-2 and only those having high level expression are treated with an anti-PD-l, anti-PD-Ll, anti-TIGIT or anti-LAG-3 antibody, fragment or antagonist of the present application.
  • anti-PD-l antibodies include, but are not limited to, nivolumab (BMS- 936558, MDX-1106, OPDIVOTM), a humanized immunoglobulin G4 (IgG4) mAh (Bristol- Myers Squibb); pembrolizumab (MK-3475, lambrolizumab, KeytrudaTM)(Merck); pidilizumab (CT-0l l)(Medivation); and AMP -224 (Merck).
  • Anti-PD-l antibodies are commercially available, for example from ABCAM (AB137132), BIOLEGENDTM (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, J116, MIH4).
  • anti-PD-Ll antibodies include atezolizumab (MPDL3280A, RG7446), a fully human IgG4 mAb Genentech/Roche); BMS-936559 (MDX-l 105), a fully humanized IgG4 mAb (Bristol-Myers Squibb); MEDI4736, a humanized IgG antibody
  • Exemplary anti-CTLA-4 antibodies for use in accordance with the present methods include ipilimumab, trevilizumab and tremelimumab.
  • the antitumor antagonist is a dominant negative protein of the immune checkpoint regulator.
  • the dominant negative protein comprises an extracellular domain derived from a member selected from the group consisting of PD-L1, PD-L2, PD-l, B7-1, B7-2, B7H3, CTLA-4, LAG-3, TIM-3, TIGIT, BTLA, VISTA, CD70, and combinations thereof.
  • these extracellular domains are fused to an immunoglobulin constant region or Fc receptor in the presently described antibodies. Such mutants can bind to the endogenous receptor so as to form a complex that is deficient in signaling.
  • the extracellular domain is fused to an immunoglobulin constant region or Fc fragment or to a monomer in the oligomeric protein complex.
  • a dominant negative PD-L1 antagonist comprises the extracellular domain of PD-L1, PD-L2 or PD-L
  • a dominant-negative PD-l antagonist is employed, which has a mutation so that it is no longer able to bind PD-L1.
  • An exemplary dominant negative protein is AMP -224 (co-developed by Glaxo Smith Kline and Amplimmune), a recombinant fusion protein comprising the extracellular domain of PD-L2 and the Fc region of human IgG.
  • Exemplary immune checkpoint regulator agonists include, but are not limited to members of the tumor necrosis factor (TNF) receptor superfamily, such as CD27, CD40, 0X40, GITR and 4-1BB (CD 137) and their ligands, or members of the B7-CD28 superfamily, including CD28 and ICOS (CD278).
  • Additional checkpoint regulator agonists include CD2, CDS, ICAM-l, LFA-l (CDl la/CDl8), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83 ligand.
  • Immune checkpoint antagonists can include antibodies or soluble fusion protein agonists comprising one or more co-stimulatory domains.
  • Agonist antibodies include, but are not limited to anti-CD40 mAbs, such as CP-870,893, lucatumumab, and dacetuzumab; anti-CDl37 mAbs, such as BMS-663513 urelumab, and PF- 05082566; anti-OX40 mAbs; anti-GITR mAbs, such as TRX518; anti-CD27 mAbs, such as CDX-1127; and anti-ICOS mAbs.
  • Exemplary GITR agonists include, e.g., GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as, e.g., a GITR fusion protein described in U.S. Patent Nos. 6,111,090 and 8,586,023; European Patent No.: 090505B1, U.S. Patent No.
  • Anti-GITR antibodies are described in, e.g., in U.S. Patent Nos. 7,025,962, 7,618,632, 7,812,135, 8,388,967, and 8,591,886;
  • An exemplary anti-GITR antibody is TRX518.
  • TNF family of molecules that bind to cognate TNF receptor family members which include CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4- 1BBL, CD137/4-1BB, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fnl4, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LT R, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, Lymphotoxin a/TNF g, TNFR2, TNF a, LT R, Lymphotoxin a 1
  • Immune checkpoint agonists or co-stimulatory molecules include cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response, and include, but are not limited to MHC class I molecules, MHC class II molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-l, LFA-l
  • CDl la/CDl8 4-1BB (CD137), B7-H3, CDS, ICAM-l, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46,
  • CD 19 CD4, CD 8 alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-l, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, LFA-l, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,
  • CD 100 SEMA4D
  • CD69 SLAMF6
  • NTB-A SLAMF6
  • SLAM SLAMF1, CD150, IPO-3
  • BLAME SLAMF8
  • SELPLG CD 162
  • LTBR LAT
  • GADS GADS
  • SLP-76 PAG/Cbp
  • CDl9a a ligand that specifically binds with CD83.
  • T cell responses can be stimulated by a combination of the anti- PD-l, anti-PD-Ll, anti-TIGIT or anti-LAG-3 mAbs of the present application and one or more of (i) an antagonist of a protein that inhibits T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-l, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-l, BTLA, CD69, Galectin-l, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD-1H, LAIR1, TIM-l, CD96 and TIM-4, and (ii) an agonist of a protein that stimulates T cell activation such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40, ICOS-L, 0X40, OX40L, GITR, GITRL, CD
  • Exemplary agents that modulate one of the above proteins and may be combined with the anti-PD-l antibodies, anti-PD-Ll antibodies, anti-TIGIT antibodies and/or anti-LAG-3 antibodies of the present application for treating cancer include: YERVOYTM/ipilimumab or tremelimumab (to CTLA-4), galiximab (to B7.1), OPDIVOTM/nivolumab/BMS-936558 (to PD- 1), pidilizumab/CT-Ol l (to PD-l), KEYTRUDATM/pembrolizumab/MK-3475 (to PD-l), AMP224 (to B7-DC/PD-L2), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3), IMP321 (to LAG-3
  • antagonists of inhibitory receptors on NK cells include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells.
  • antagonist anti-PD-l, anti-PD-Ll and/or anti-TIGIT antibodies can be combined with antagonists of KIR (e.g., lirilumab), CSF-1R antagonists, such as RG7155.
  • KIR e.g., lirilumab
  • CSF-1R antagonists such as RG7155.
  • Tumors evade host immune surveillance by a large variety of mechanisms. Many of these mechanisms may be overcome by the inactivation of immunosuppressive proteins expressed by the tumors. These include among others TGF-b, IL-10, and Fas ligand.
  • Antibodies to each of these entities can be used in combination with the antitumor antagonists described herein to counteract the effects of the immunosuppressive agent and favor tumor immune responses by the host.
  • T cell costimulatory molecules such as OX-40, CD137/4-1BB, and ICOS may also provide for increased levels of T cell activation.
  • the antitumor antagonists described herein can be co administered with one or other more therapeutic agents, e.g., anti-cancer agents, radiotoxic agents or an immunosuppressive agent.
  • therapeutic agents e.g., anti-cancer agents, radiotoxic agents or an immunosuppressive agent.
  • co-administration can solve problems due to development of resistance to drugs, changes in the antigenicity of the tumor cells that would render them unreactive to the antibody, and toxicities (by administering lower doses of one or more agents).
  • the antitumor antagonists described herein can be chemically linked to the agent (as an immuno-complex) or can be administered separate from the agent. In the latter case (separate administration), the antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation.
  • the antitumor antagonists described herein may be co-administered with one or more anti-cancer agents so as to provide two anti-cancer agents operating synergistically via different mechanisms to yield a cytotoxic effect in human cancer cells.
  • the antitumor antagonists described herein may be combined with an anti-cancer agent, such an alkylating agent; an anthracy cline antibiotic; an anti-metabolite; a detoxifying agent; an interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor; a histone deacetylase inhibitor; a hormone; a mitotic inhibitor; a phosphatidylinositol-3-kinase (PI3K) inhibitor; an Akt inhibitor; a mammalian target of rapamycin (mTOR) inhibitor; a proteasomal inhibitor; a poly(ADP-ribose) polymerase (PARP) inhibitor; a Ras/MAPK pathway inhibitor; a centrosome declustering agent; a multi-kinase inhibitor; a serine/threonine kinase inhibitor; a tyrosine kinase inhibitor; a VEGF/VEGFR
  • alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa
  • anthracycline antibiotics include, but are not limited to, doxorubicin (Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone); bleomycin
  • Exemplary anti-metabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine (Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine (Arranon); cladribine (Cladribine Novaplus);
  • clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal (DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex);
  • Trexall methotrexate
  • thioguanine Tabloid
  • TS-l or cytarabine Tarabine PFS
  • Exemplary detoxifying agents include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).
  • interferons include, but are not limited to, interferon alfa-2b (Intron A) or interferon alfa-2a (Roferon-A).
  • Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab/iodinel3l tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab (Zevalin; In-l l l; Y-90 Zevalin); gemtuzumab
  • Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; canertinib (0-1033); matuzumab (Emd7200) or EKB-569.
  • HER2 inhibitors include, but are not limited to, trastuzumab
  • Exemplary histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza), valproic acid, romidepsin, entinostat abexinostat, givinostat, and mocetinostat.
  • Exemplary hormones include, but are not limited to, tamoxifen (Soltamox;
  • Nolvadex Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera);
  • estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon);
  • nilutamide (Nilandron); abarelix (Plenaxis); or testolactone (Teslac).
  • Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone
  • Exemplary phosphatidyl-inositol-3 kinase (PI3K) inhibitors include wortmannin an irreversible inhibitor of PI3K, demethoxyviridin a derivative of wortmannin, LY294002, a reversible inhibitor of PI3K; BKM120 (Buparlisib); Idelalisib (a PBK Delta inhibitor); duvelisib (IPI-145, an inhibitor of PI3K delta and gamma); alpelisib (BYL719), an alpha-specific PI3K inhibitor; TGR 1202 (previously known as RP5264), an oral PI3K delta inhibitor; and copanlisib (BAY 80-6946), an inhibitor RI3Ka,d isoforms predominantly.
  • PI3K phosphatidyl-inositol-3 kinase
  • Exemplary Akt inhibitors include, but are not limited to miltefosine, AZD5363, GDC-0068, MK2206, Perifosine, RX-0201, PBI-05204, GSK2141795, and SR13668.
  • Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afmitor) or temsirolimus (Torisel); rapamune, ridaforolimus; deforolimus (AP23573), AZD8055
  • Exemplary proteasomal inhibitors include, but are not limited to, bortezomib (PS- 34!), ixazomib (MLN 2238), MLN 9708, delanzomib (CEP-18770), carfilzomib (PR-171), YU101, oprozomib (ONX-0912), marizomib (NPI-0052), and disufiram.
  • Exemplary PARP inhibitors include, but are not limited to, olaparib, iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP- 9722, PJ-34, Tiq-A, Phen, PF-01367338 and combinations thereof.
  • Exemplary Ras/MAPK pathway inhibitors include, but are not limited to, trametinib, selumetinib, cobimetinib, 0-1040, PD0325901, AS703026, R04987655,
  • centrosome declustering agents include, but are not limited to, griseofulvin; noscapine, noscapine derivatives, such as brominated noscapine (e.g., 9- bromonoscapine), reduced bromonoscapine (RBN), N-(3-brormobenzyl) noscapine,
  • noscapine noscapine derivatives, such as brominated noscapine (e.g., 9- bromonoscapine), reduced bromonoscapine (RBN), N-(3-brormobenzyl) noscapine,
  • Exemplary multi-kinase inhibitors include, but are not limited to, regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.
  • Exemplary serine/threonine kinase inhibitors include, but are not limited to, ruboxistaurin; eril/easudil hydrochloride; flavopiridol; seliciclib (CYC202; Roscovitrine); SNS- 032 (BMS-387032); Pkc4l2; bryostatin; KAI-9803; SF1126; VX-680; Azdl l52; Arry-l42886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.
  • Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent);
  • trastuzumab Herceptin; bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afmitor); alemtuzumab (Campath); gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient); dasatinib (Spry cel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX- 322; Azd0530; BMS-354825; SKI-606 CP-690; AG-490; WHI-P154; WHI-P131; AC -220; or AMG888.
  • VEGF/VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; pegaptanib; or vandetinib.
  • microtubule targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilones and navelbine.
  • topoisomerase poison drugs include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.
  • Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.
  • Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferative agents include, but are not limited to, altretamine (Hexalen); isotretinoin (Accutane; Amnesteem;
  • lenalidomide lenalidomide
  • bexarotene Targretin
  • thalidomide Thalomid
  • temsirolimus Torisel
  • arsenic trioxide Trisenox
  • verteporfm Visudyne
  • mimosine Leucenol
  • the antitumor antagonists described herein are administered at a subtherapeutic dose, another anti-immune checkpoint regulator antibody or antagonist is administered at a subtherapeutic dose, the angiogenesis antagonist is administered at a subtherapeutic dose, or any antagonist in a combination thereof is each administered at a subtherapeutic dose.
  • PD-l, PD-L1, TIGIT and/or LAG-3 inhibition is combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy).
  • standard cancer treatments e.g., surgery, radiation, and chemotherapy.
  • PD-l, PD-L1, TIGIT and/or LAG-3 inhibition can be effectively combined with chemotherapeutic regimes.
  • An example of such a combination is an anti-TIGIT, anti-PD-l, anti-PD-Ll or anti-LAG-3 antibody in combination with decarbazine for the treatment of melanoma.
  • Another example of such a combination is an anti-PD-l, anti-PD-Ll, anti-TIGIT or anti-LAG-3 antibody in combination with interleukin-2 (IL-2) for the treatment of melanoma.
  • IL-2 interleukin-2
  • PD-l, PD-L1, TIGIT and/or LAG-3 inhibition and chemotherapy can enhance apoptosis and increase tumor antigen presentation for cytotoxic immunity.
  • Other synergistic combination therapies include PD-l, PD-L1, TIGIT and/or LAG-3 inhibition through cell death when used in combination with radiation, surgery or hormone deprivation. Each of these protocols creates a source of tumor antigen in the host.
  • checkpoint regulator antagonists described herein can be used in multi-specific antagonists or in combination with bispecific antibodies targeting Fca or Fey receptor-expressing effector cells to tumor cells (see, e.g., U.S. Patent Nos.
  • Bispecific antibodies can be used to target two separate antigens.
  • anti-Fc receptor/anti -tumor antigen e.g., Her-2/neu
  • bispecific antibodies have been used to target macrophages to cancer cells or tumors. This targeting may more effectively activate tumor specific responses.
  • the T cell arm of these responses would be augmented by the inhibition of TIGIT, PD-l, PD-L1 and/or LAG-3.
  • antigen may be delivered directly to DCs by the use of bispecific antibodies that bind to tumor antigen and a dendritic cell specific cell surface marker.
  • the present application provides nucleic acids encoding the antitumor antagonist of the present application, and expression vectors comprising such nucleic acids.
  • nucleic acids encodes an HCVR and/or LCVR fragment of an antibody or fragment in accordance with the embodiments described herein, or any of the other antibodies and antibody fragments described herein.
  • DNA encoding an antigen binding site in a monoclonal antibody can be isolated and sequenced from the hybridoma cells 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 monoclonal antibodies).
  • amino acid sequences from immunoglobulins of interest may be determined by direct protein sequencing, and suitable encoding nucleotide sequences can be designed according to a universal codon table.
  • nucleotide and amino acid sequences of antigen binding sites or other immunoglobulin sequences, including constant regions, hinge regions and the like may be obtained from published sources well known in the art.
  • Expression vectors encoding a particular monospecific, bispecific or trispecific antitumor antagonist may be used to synthesize the antitumor antagonists of the present disclosure in cultured cells in vitro or they may be directly administered to a patient to express the antitumor antagonist in vivo or ex vivo.
  • an“expression vector” refers to a viral or non-viral vector comprising a polynucleotide encoding one or more polypeptide chains corresponding to the monospecific, bispecific or trispecific antitumor antagonists of the present disclosure in a form suitable for expression from the polynucleotide(s) in a host cell for antibody preparation purposes or for direct administration as a therapeutic agent.
  • a nucleic acid sequence is“operably linked” to another nucleic acid sequence when the former is placed into a functional relationship with the latter.
  • a DNA for a presequence or signal peptide is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a signal peptide, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional practice.
  • Nucleic acid sequences for expressing the antitumor antagonists typically include an amino terminal signal peptide sequence, which is removed from the mature protein. Since the signal peptide sequences can affect the levels of expression, the polynucleotides may encode any one of a variety of different N-terminal signal peptide sequences. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the above described“regulatory sequences” refer to DNA sequences necessary for the expression of an operably linked coding sequence in one or more host organisms.
  • regulatory sequences is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells or those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • Expression vectors generally contain sequences for transcriptional termination, and may additionally contain one or more elements positively affecting mRNA stability.
  • the expression vector contains one or more transcriptional regulatory elements, including promoters and/or enhancers, for directing the expression of antitumor antagonists.
  • a promoter comprises a DNA sequence that functions to initiate transcription from a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may operate in conjunction with other upstream elements and response elements.
  • promoter is to be taken in its broadest context and includes transcriptional regulatory elements (TREs) from genomic genes or chimeric TREs therefrom, including the TATA box or initiator element for accurate transcription initiation, with or without additional TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) which regulate activation or repression of genes operably linked thereto in response to developmental and/or external stimuli, and trans-acting regulatory proteins or nucleic acids.
  • TREs transcriptional regulatory elements from genomic genes or chimeric TREs therefrom, including the TATA box or initiator element for accurate transcription initiation, with or without additional TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) which regulate activation or repression of genes operably linked thereto in response to developmental and/or external stimuli, and trans-acting regulatory proteins or nucleic acids.
  • a promoter may contain a genomic fragment or it may contain a chimera of one
  • Preferred promoters are those capable of directing high-level expression in a target cell of interest.
  • the promoters may include constitutive promoters (e.g., HCMV, SV40, elongation factor-la (EF-la)) or those exhibiting preferential expression in a particular cell type of interest.
  • Enhancers generally refer to DNA sequences that function away from the transcription start site and can be either 5’ or 3' to the transcription unit.
  • enhancers can be within an intron as well as within the coding sequence. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase and/or regulate transcription from nearby promoters.
  • Preferred enhancers are those directing high-level expression in the antibody producing cell.
  • TREs tissue-specific transcriptional regulatory elements
  • Pol III promoters Hl or U6 are particularly useful for expressing shRNAs from which siRNAs are expressed.
  • An expression vector may be designed to facilitate expression of the antitumor antagonist in one or more cell types.
  • one or more expression vectors may be engineered to express both the antitumor antagonist and one or more siRNA targeting the Tie2 pathway, the VEGF pathway or an immune checkpoint regulator.
  • siRNA is a double-stranded RNA that can be engineered to induce sequence- specific post-transcriptional gene silencing of mRNAs. Synthetically produced siRNAs structurally mimic the types of siRNAs normally processed in cells by the enzyme Dicer. When expressed from an expression vector, the expression vector is engineered to transcribe a short double-stranded hairpin-like RNA (shRNA) that is processed into a targeted siRNA inside the cell. Synthetic siRNAs and shRNAs may be designed using well known algorithms and synthesized using a conventional DNA/RNA synthesizer.
  • a suitable splice donor and splice acceptor sequences may be incorporated for expressing both products.
  • an internal ribosome binding sequence (IRES) or a 2A peptide sequence, may be employed for expressing multiple products from one promoter.
  • IRES provides a structure to which the ribosome can bind that does not need to be at the 5' end of the mRNA. It can therefore direct a ribosome to initiate translation at a second initiation codon within a mRNA, allowing more than one polypeptide to be produced from a single mRNA.
  • a 2A peptide contains short sequences mediating co-translational self-cleavage of the peptides upstream and downstream from the 2A site, allowing production of two different proteins from a single transcript in equimolar amounts.
  • CHYSEL is a non-limiting example of a 2A peptide, which causes a translating eukaryotic ribosome to release the growing polypeptide chain that it is synthesizing without dissociating from the mRNA. The ribosome continues translating, thereby producing a second polypeptide.
  • An expression vector may comprise a viral vector or a non- viral vector.
  • a viral vectors may be derived from an adeno-associated virus (AAV), adenovirus, herpesvirus, vaccinia virus, poliovirus, poxvirus, a retrovirus (including a lentivirus, such as HIV-l and HIV- 2), Sindbis and other RNA viruses, alphavirus, astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picomavirus, togaviruses and the like.
  • a non-viral vector is simply a“naked” expression vector that is not packaged with virally derived components (e.g., capsids and/or envelopes).
  • these vectors may be engineered to target certain diseases or cell populations by using the targeting characteristics inherent to the virus vector or engineered into the virus vector.
  • Specific cells may be“targeted” for delivery of polynucleotides, as well as expression.
  • targeting in this case, may be based on the use of endogenous or heterologous binding agents in the form of capsids, envelope proteins, antibodies for delivery to specific cells, the use of tissue-specific regulatory elements for restricting expression to specific subset(s) of cells, or both.
  • expression of the antibody chains is under the control of the regulatory element such as a tissue specific or ubiquitous promoter.
  • a ubiquitous promoter such as a CMV promoter, CMV-chicken beta-actin hybrid (CAG) promoter, a tissue specific or tumor-specific promoter to control the expression of a particular antibody heavy or light chain or single-chain derivative therefrom.
  • Non-viral expression vectors can be utilized for non-viral gene transfer, either by direct injection of naked DNA or by encapsulating the antitumor antagonist-encoding polynucleotides in liposomes, microparticles, microcapsules, virus-like particles, or erythrocyte ghosts. Such compositions can be further linked by chemical conjugation to targeting domains to facilitate targeted delivery and/or entry of nucleic acids into desired cells of interest.
  • plasmid vectors may be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, and chemically linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose or transferrin.
  • synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, and chemically linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose or transferrin.
  • naked DNA may be employed. Uptake efficiency of naked DNA may be improved by compaction or by using biodegradable latex beads. Such delivery may be improved further by treating the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.
  • the present application provides host cells transformed with the anti-PD-l, anti-PD-Ll, anti-TIGIT, and/or anti-LAG-3 HCVRs and/or LCVRs, encoding nucleic acids or expression vectors, or nucleic acids/expression vectors encoding the bi-specific and/or trispecific antitumor antagonist of the present application.
  • the host cells can be any bacterial or eukaryotic cell capable of expressing the anti-PD-l, anti-PD-Ll, anti-TIGIT and/or anti-LAG-3 HCVRs and/or LCVRs encoding nucleic acids or expression vectors or any of the other co administered antibodies or antagonists described herein.
  • a method of producing an antitumor antagonist comprises culturing a host cell transformed with one or more anti-PD-l, anti-PD-Ll anti-TIGIT, and/or anti-LAG-3 HCVRs and/or LCVRs encoding nucleic acids or expression vectors under conditions that allows production of the antibody or fragment, and purifying the antibody from the cell.
  • the present application provides a method for producing an antibody comprising culturing a cell transiently or stably expressing one or more constructs encoding one or more polypeptide chains in the antibody; and purifying the antibody from the cultured cells. Any cell capable of producing a functional antibody may be used.
  • the antibody-expressing cell is of eukaryotic or mammalian origin, preferably a human cell. Cells from various tissue cell types may be used to express the antibodies. In other embodiments, the cell is a yeast cell, an insect cell or a bacterial cell. Preferably, the antibody- producing cell is stably transformed with a vector expressing the antibody.
  • One or more expression vectors encoding the antibody heavy or light chains can be introduced into a cell by any conventional method, such as by naked DNA technique, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, virus-mediated infection, physical or chemical agents or treatments, electroporation, etc.
  • cells may be transfected with one or more expression vectors for expressing the antibody along with a selectable marker facilitating selection of stably transformed clones expressing the antibody.
  • the antibodies produced by such cells may be collected and/or purified according to techniques known in the art, such as by centrifugation, chromatography, etc.
  • Suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • neomycin neomycin analog G418, hydromycin
  • puromycin puromycin.
  • These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, or hygromycin.
  • the three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puromycin.
  • Exemplary antibody-expressing cells include human Jurkat, human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO) cells, mouse WEHI fibrosarcoma cells, as well as unicellular protozoan species, such as Leishmania tarentolae.
  • stably transformed, antibody producing cell lines may be produced using primary cells immortalized with c-myc or other immortalizing agents.
  • the cell line comprises a stably transformed Leishmania cell line, such as Leishmania tarentolae.
  • Leishmania are known to provide a robust, fast-growing unicellular host for high level expression of eukaryotic proteins exhibiting mammalian-type glycosylation patterns.
  • a commercially available Leishmania eukaryotic expression kit is available (Jena Bioscience GmbH, Jena, Germany).
  • the cell line expresses at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or at least 500 mg of the antibody /liter of culture.
  • the antibodies in the present application may be isolated from antibody expressing cells following culture and maintenance in any appropriate culture medium, such as RPMI, DMEM, and AIM V®.
  • the antibodies can be purified using conventional protein purification methodologies (e.g., affinity purification, chromatography, etc.), including the use of Protein-A or Protein-G immunoaffinity purification.
  • antibodies are engineered for secretion into culture supernatants for isolation therefrom.
  • An alternative approach is based on charged residues with ionic interactions or steric complementarity. This includes altering the charge polarity in the CH3 interface so that co-expression of electrostatically matched Fc domains support favorable attractive interactions and heterodimer formation while retaining the hydrophobic core, whereas unfavorable repulsive charge interactions suppress homodimerization. See Table 1.
  • the amino acid numbering in Table 1 follows the Kabat numbering scheme and can be applied to heavy chain amino acid sequences of the antibodies described herein.
  • leucine zipper (LZ) domains may be incorporated into a protein scaffold.
  • a leucine zipper is a common three-dimensional structural motif in proteins, typically as part of a DNA-binding domain in various transcription factors.
  • a single LZ typically contains 4-5 leucine residues at approximately 7-residue intervals, which forms an amphipathic alpha helix with a hydrophobic region running along one side.
  • a heterodimeric protein scaffold comprises a LZ from the c-jun transcription factor associated with a LZ from the c-fos transcription factor.
  • a leucine zipper domain may be incorporated in place of CH2-CH3 sequences in the protein scaffold or it may be placed at the carboxy terminal end of the two heavy chains in the bispecific or trispecific antitumor antagonist.
  • a furin cleavage site may be introduced between the carboxy terminal end of CH3 and the amino terminal end of the leucine zipper. This can facilitate furin-mediated cleavage of the leucine zipper following the heterodimerization step when co-expressing the heavy and light chains of the bispecific or trispecific antitumor antagonist in an appropriate mammalian cell expression system (see Wranik et al, J. Biol. Chem, 287(5):43331-43339, 2012).
  • the amino acid numbering in Table 1 follows the Kabat numbering scheme and can be applied to heavy chain amino acid sequences of the antibodies described herein.
  • the mutations described in Table 1 may be applied to the sequence (published or otherwise) of any immunoglobulin IgGl heavy chain, as well as other immunoglobulin classes, and subclasses (or isotypes) therein.
  • portions of the heavy chain, light chain or both may be modified relative to the“wild-type” antibody chains from which they are derived to prevent or reduce mispairing of both heavy chain constant regions to one another, as well mispairing of light chain constant regions to their heavy chain counterparts.
  • the light chain mispairing problem can be addressed in several ways.
  • sterically complementary mutations and/or disulfide bridges may be incorporated into the two VL/VH interfaces.
  • mutations can be incorporated based on ionic or electrostatic interactions.
  • light chain mispairing may be prevented or reduced by employing a first arm with an S183E mutation in the CH1 domain of the heavy chain and an S176K mutation in the CL domain of the light chain.
  • a second arm may include an S183K mutation in the in the CH1 domain of the heavy chain and an S176E mutation in the CL domain of the light chain.
  • a“CrossMab” approach is employed, where one arm in the bispecific or trispecific antitumor antagonist (e.g., Fab) is left untouched, but in the other arm containing the other binding specificity, one or more domains in the light chain are swapped with one or more domains in the heavy chain at the heavy chaimlight chain interface.
  • Fab bispecific or trispecific antitumor antagonist
  • Methods, immunoglobulin domain sequences, including specific mutations for preventing mispairing of heavy and light chains as disclosed above are further described in U.S. Patent Application Publication Nos. 2014/0243505, 2013/0022601.
  • the antitumor antagonists of the present application are chemically conjugated to one or more peptides and/or small molecule drugs.
  • the peptides or small molecule drug can be the same or different.
  • the peptides or small molecule drugs can be attached, for example to reduced SH groups and/or to carbohydrate side chains. Methods for making covalent or non-covalent conjugates of peptides or small molecule drugs with antibodies are known in the art and any such known method may be utilized.
  • the peptide or small molecule drug is attached to the hinge region of a reduced antibody component via disulfide bond formation.
  • such agents can be attached using a heterobifunctional cross-linkers, such as N-succinyl 3-(2- pyridyldithiojpropionate (SPDP).
  • SPDP N-succinyl 3-(2- pyridyldithiojpropionate
  • the peptide or small molecule drug is conjugated via a carbohydrate moiety in the Fc region of the antibody.
  • the carbohydrate group can be used to increase the loading of the same agent that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different therapeutic or diagnostic agent.
  • the method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
  • a carrier polymer that has at least one free amine function.
  • the antitumor antagonists in the present disclosure retain certain desirable characteristics and pharmacokinetic properties of antibodies, including a desirable in vitro and in vivo stability (e.g., lone half-life and shelf-life stability), efficient delivery into desired target cells, increased affinity for binding partners, desirable antibody-dependent cell- mediated cytotoxicity and complement-dependent cytotoxicity, and reduced renal clearance or excretion. Accordingly, careful attention to size and need for particular constant region effector functions may be considered in the design of the antitumor antagonists.
  • the anti-PD-l, anti-PD-Ll and anti-TIGIT, inhibitors may range in size from 50 kD to 300 kD, from 50 kD to 250 kD, from 60 kD to 250 kD, from 80 kD to 250 kD, from 100 kD to 250 kD, from 125 kD to 250 kD, from 150 kD to 250 kD, from 60 kD to 225 kD, from 75 kD to 225 kD, from 100 kD to 225 kD, from 125 kD to 225 kD, from 150 kD to 225 kD, from 60 kD to 200 kD, from 75 kD to 200 kD, from 100 kD to 125 kD to 200 kD, from 150 kD to 200 kD, from 60 kD to 150 kD, from 75 kD to 200 kD, from 100 kD to 125 kD to 200 kD,
  • Monoclonal antibodies (mAbs) of the present application are generated and screened using techniques well known in the art, see, e.g., Harlow and Lane ⁇ 1988) Antibodies ,
  • Example 2 Trispecific antagonists having a PD-1 targeting domain and multiple angiogenesis targeting moieties
  • FIG. 1 shows VH and VL sequences of anti-PD-l, anti-PD-Ll, anti-LAG-3 and anti-TIGIT mAbs.
  • FIG. 2 shows other functional domains including VH and VL of
  • ranibizumab ranibizumab, bevacizumab and a mutant variant of the VH, the VEGF binding domain of aflibercept, trebananib-short peptide and the trebananib-long peptide, and extracellular domain of TGF R-II (TGF R-II ECD).
  • the aflibercept fusion protein domain comprises vascular endothelial growth factor (VEGF)-binding portions from the extracellular domains of human VEGF receptors 1 and 2 and prevents the binding of VEGF-A, VEGF-B and PLGF to their receptors, VEGFR-l and VEGFR-2.
  • VEGF vascular endothelial growth factor
  • the trebananib peptide targets and binds to Angl and Ang2, thereby preventing the interaction of Angl and/or Ang2 with their cognate Tie2 receptors.
  • the antitumor antagonists may be constructed with an IgGl, IgGlagly, or IgG4 backbone.
  • FIGS. 3A-3H show eight different trispecific antitumor antagonists, TS-ZPT-l. l, -1.2, -1.3 and -2 to -6, respectively (1) anti-PD-l variable region (VH1, VL1) (FIGS. 3A-3H); (2) an aflibercept fusion protein domain: (i) at the amino terminal end of one or both IgG arms (FIGS.
  • FIGS. 4A-4C show five different trispecific antitumor antagonists, TS-ZPT-7, TS-ZPT-8, TS-ZPT-9, respectively: (1) anti-PD-l or other checkpoint antibody Fab domains (VH1-CH1, VL1-CL1) fused to the carboxy-terminus of the CH3 domain; (2) an aflibercept fusion protein domain at the amino-terminal end of the CH2 domain fused to the amino- terminus of the CH2 domain; (3) a trebananib peptide (or other biological peptide): (i) inserted within each of the two CH3 domains (FIG. 4A); (ii) fused to the amino-terminus of the CH2 domain (FIG. 4B); (iii) fused to the carboxy -terminal end of the CHI domain (FIG. 4C)
  • FIGS. 5A-5G show seven different trispecific antitumor antagonist, TS-LPT-l, TS-LPT-2, TS-LPT-3, TS-LPT-4, TS-LPT-5, TS-M3, TS-M4, respectively; (1) anti-PD-l or other checkpoint antibody variable domains (VH1, VL1) (i) fused to the amino-terminus of the CH1 and CL1, respectively (FIG. 5F); (ii) fused to the amino-terminus of the VH1 and VL1, respectively (FIG.
  • VH1, VL1 anti-PD-l or other checkpoint antibody variable domains
  • VH1, VL1 Lucentis, bevacizumab or other anti-VEGF antibody variable domains
  • VH1, VL1 Lucentis, bevacizumab or other anti-VEGF antibody variable domains
  • VH1, VL1 Lucentis, bevacizumab or other anti-VEGF antibody variable domains
  • VH1, VL1 Lucentis, bevacizumab or other anti-VEGF antibody variable domains
  • the amino acid sequences demonstrated in SEQ ID NOS:200 and 201 shows exemplary sequences of TS-ZPT-3S.
  • the amino acid sequences demonstrated in SEQ ID NOS:201 and 202 show exemplary sequences of TS-ZPT-3L.
  • the amino acid sequences demonstrated in SEQ ID NOS:187 and 188 show exemplary sequences of TS-LPT-l.
  • the amino acid sequences demonstrated in SEQ ID NOS:201 and 203 show exemplary sequences of TS-ZPT-2.
  • the amino acid sequences demonstrated in SEQ ID NOS:201 and 204 show exemplary sequences of TS-ZPT-5.
  • the amino acid sequences demonstrated in SEQ ID NOS:234 and 235 show exemplary sequences for TS-ZPT-6.
  • Example 3 Characterization of trispecific antitumor antagonists made with the anti-PD-1 antibody 2P16.
  • TS-ZPT-l.2 (SEQ ID NOS:238- 239), TS-ZPT-l.3(SEQ ID NOS:240-241), TS-ZPT-2(SEQ ID NOS:242-243), TS-ZPT-3(SEQ ID NOS:201-202), TS-ZPT-5(SEQ ID NOS:244 and 298), TS-ZPT-6 were constructed and compared to TS-LPT-l(SEQ ID NOS:234-235), a trispecific antitumor antagonist that utilized the 2P16 antibody, trebananib and the Fab portion of Lucentis.
  • FIGS. 6A and 6B show non-reducing SDS-PAGE analysis of the trispecific antitumor antagonists expressed by transiently transfected HEK293 cells. Samples were assessed before and after purification of the molecules. The results show all the molecules expressed and could be purified, and that TS-ZPT-2(2Pl6), TS-ZPT-3(2Pl6), TS-ZPT-5(2Pl6) expressed better than TS-ZPT-6(2Pl6), and TS-LPT-l(2Pl6).
  • a blocking assay was carried out to calculate the IC50 for selected trispecific molecules. Briefly, 2 or 3 fold serial dilutions of anti -human PD-l mAh were prepared. Human PD-l transfected CHO-K1 cells were washed with FACS buffer (0.5%BSA 2mM EDTA in PBS) and re-suspended at a concentration of 10 6 cells/ml. FITC labeled human PD-Ll-Fc protein was added to the human PD-l transfected CHO-K1 cells at a final concentration of 7 pg/ml and mixed well.
  • FIGS. 7A-B show the IC50s for the exemplary trispecific antitumor antagonists, TS-ZPT-l. l(2Pl6), TS-ZPT-l.2(2Pl6), TS-ZPT-l.3(2Pl6), TS-ZPT-2(2Pl6), TS-ZPT-3(2Pl6), TS-ZPT-5(2Pl6), TS-ZPT-6(2Pl6) along with an anti-PD-l mAh and a positive control or benchmark (“BM”) antibody (nivolumab).
  • BM positive control or benchmark
  • the IC50s were comparable to the benchmark antibodies in blocking this interaction.
  • TS-ZPT-2(2Pl6), TS-ZPT- 3(2Pl6), TS -ZPT-5 (2P 16), TS-ZPT-6(2Pl6) was determined.
  • 96-well assay plates were coated with 0.5ug/ml of recombinant human VEGF 165 (R&D) in carbonate-bicarbonate buffer (pH 9.6) at 4oC overnight followed by blocking with 1% BSA/PBS for 1 hour at room temperature. Serially diluted antibodies were then added to the plate and incubated for 30 minutes at room
  • FIGS. 8A and 8B show the IC50 values calculated from the VEGF-VEGFR2 blocking assays using the trispecific antagonists TS-ZPT-l.2(2Pl6), TS-ZPT-2(2Pl6), TS-ZPT- 5(2Pl6), TS-ZPT-6(2Pl6) and TS-LPT-l(2Pl6), which were compared to positive controls corresponding to the aflibercept peptide and the bevacizumab antibody.
  • the results of these analyses showed that the trispecific antagonists exhibited comparable or lower (i.e., better) IC50 values than the corresponding controls.
  • FIG. 9 shows the IC50 values calculated from the Tie2-Ang2 blocking assay testing the trispecific antagonists TS-ZPT-l.2(2Pl6), TS-ZPT-2(2Pl6), TS-ZPT-5(2Pl6), TS- ZPT-6(2Pl6) and TS-LPT-l(2Pl6) which were compared to positive controls corresponding to an antagonist, bevacizumab-trebananib.
  • the results of this analysis showed that the trispecific antagonists exhibited comparable or lower (i.e., better) IC50 values than the corresponding controls.
  • Example 4 Characterization of TS-ZPT-2, TS-ZPT-3L and TS-ZPT-5 molecules constructed with the anti-PD-1 antibody 2P17
  • TS-ZPT-2, TS-ZPT-3L and TS-ZPT-5 trispecific antitumor antagonists were made with the anti-PD-l antibody 2P17 (SEQ ID NOS:203 and 201, 233 and 201, 204 and 201, respectively) and characterized for homogeneity and stability by size exclusion
  • FIG. 10 shows the results of size exclusion chromatography analysis of TS-ZPT- 2(2Pl7), TS-ZPT-3L(2Pl7) and TS-ZPT-5(2Pl7), and parental 2P17 antibody produced by HEK293 transiently transfected cells.
  • the percentages of high molecular weight (HWM) species and low molecular weight (LMW) species in comparison to dimerized molecule (Dimer) indicate that of the three trispecific antagonists, TS-ZPT-3L(2Pl7) has the lowest percentage of LMW species and the highest percentage of Dimers.
  • FIG. 11 shows the relative amounts of HWM and LMW species in comparison to Dimers of three trispecific antagonists: TS-ZPT-2(2Pl7), TS-ZPT-3L (2Pl7)(purified from two pools) and TS-ZPT-5(2Pl7) produced from stable CHO cell pools.
  • TS-ZPT-2(2Pl7) TS-ZPT-2(2Pl7)
  • TS-ZPT-3L (2Pl7) purified from two pools
  • TS-ZPT-5(2Pl7) produced from stable CHO cell pools.
  • the results show that of the three trispecific antagonists, TS-ZPT-3L(2Pl7) has the lowest percentage of LMW species and the highest percentage of Dimers.
  • TS-ZPT-3L(2Pl7) exhibited lowest amount of clipping, indicating that inserting the trebananib peptide into the CH3 region protects the aflibercept fusion protein, and reduces clipping.
  • FIGS. 12A and 12B shows that the Dimer, HMW and LMW species of TS-ZPT- 3L(2Pl7) exhibit good stability for at least 112 days at 4° C.
  • TS-ZPT-3 versions were made using the anti-PD-l antibodies 2P16 and 2P17 with both the full length trebananib peptide, TS-ZPT-3L, and a single copy, or trebananib-short, peptide for TS-ZPT-3S. These four molecules were compared to trispecific molecules utilizing the VH and VL of bevacizumab, TS-M3 and TS-M4 (depicted in FIG. 5F), utilizing anti-PD-l antibodies nivolumab, 2P16 and 2P17 whose variable domain sequences are shown in FIG. 1 with sequences SEQ ID NOS:247-254.
  • FIG. 13A shows the heavy- and light chain amino acid sequences for an exemplary trispecific antitumor antagonist with the trebananib peptide, i.e., TS-ZPT-3L(2Pl7).
  • FIG. 13B depicts an exemplary molecule derived from these sequences.
  • FIG. 14A shows the heavy and light chains amino acid sequences for an exemplary trispecific antitumor antagonist with the trebananib Short peptide, i.e., TS-ZPT- 3S(2Pl7).
  • FIG. 14B depicts an exemplary molecule derived from these sequences. [0400] FIG.
  • FIG. 16 Shows the results from a size exclusion chromatography analysis of TS- ZPT-3L(2Pl7), TS-ZPL-3S(2Pl7), TS-ZPT-3L(2Pl6), TS-ZPT-3S(2Pl6) after purification by protein A chromatography.
  • the percentages of high molecular weight (HWM) species and low molecular weight (LMW) species in comparison to dimerized molecule (Dimer) indicate the molecules comprising the 2P17 antibody have higher levels of the preferred dimer species.
  • FIG. 17 shows the size exclusion chromatography analysis of TS-ZPT-3S(2Pl7) and TS-ZPT-3L(2Pl7) stored at 4 degrees C for 48 days, indicating both molecules are stable when stored.
  • the PD-1-PDL1 cell-based blocking assay in Example 5 was used to evaluate the ability of four variations of the trispecific antitumor antagonist, TS-ZPT-3S(2Pl6), TS-ZPT- 3L(2Pl6), TS-ZPT-3 S(2P 17) and TS-ZPT-3L(2Pl7), to block the interaction between PD-l and its ligand, PD-L1.
  • the results were used to calculate the corresponding IC50s along with 2P17 and a benchmark antibody (nivolumab).
  • FIGS. 18A-18B show that the IC50s were comparable to the benchmark antibodies in blocking this interaction.
  • the VEGF-VEGFR-2 bioassay was used to evaluate the ability of TS-ZPT- 3S(2Pl6), TS-ZPT-3L(2Pl6), TS-ZPT-3S(2Pl7) and TS-ZPT-3L(2Pl7) to block the interaction between VEGF and its receptor, VEGFR-2 and to calculate the corresponding IC50s along with a benchmark antibody (bevacizumab).
  • Recombinant Cell Line (BPS Bioscience Catalog #: 79387) was plated at a density of 30k cells per well into a white clear-bottom 96-well microplate. After 24 hours serial dilutions of indicated molecules and human VEGF 165 were added to the wells. After 4 hours of incubation, 100 pl of ONE-StepTM Luciferase reagent was added, the plates were rocked at room temperature for ⁇ l5minutes, and the plates read for the luminescence signal.
  • FIGS. 19A-19B show that the IC50s were comparable to the benchmark antibodies in blocking this interaction.
  • Example 5 The Ang2-Tie2 blocking assay in Example 5 was used to evaluate the ability of TS-ZPT-3S (2P 16), TS-ZPT-3L(2Pl6), TS-ZPT-3 S(2P 17) and TS-ZPT-3L(2Pl7) to block the interaction between Tie2 and its ligand, Ang-2 and to calculate the corresponding IC50s along with a benchmark antibody (trebananib).
  • 20A-20B shows that the IC50s of TS-ZPT- 3L(2Pl6) and TS-ZPT-3L(2Pl7) with two Ang-2 blocking peptides were comparable to the benchmark, while TS-ZPT-3S(2Pl6) and TS-ZPT-3S(2Pl7) with only one Ang-2 blocking peptide have higher IC50s than the benchmark.
  • FIGS. 21A-21F show the binding and the resultant binding affinity constants. The results show that TS-ZPT-3S(2Pl6), TS-ZPT-3L(2Pl6), TS-ZPT-3S(2Pl7) and TS-ZPT-3L(2Pl7) exhibited higher affinity than the benchmark (nivolumab).
  • FIGS. 22A-22F show the binding and the resultant binding affinity constants. The results show that TS-ZPT-3S(2Pl6), TS-ZPT-3L(2Pl6), TS-ZPT-3S(2Pl7) and TS-ZPT- 3L(2Pl7) exhibited higher affinity than the benchmark (bevacizumab).
  • FIGS. 23A-23F show the binding and the resultant binding affinity constants. The results show that TS-ZPT-3S(2Pl6), TS-ZPT-3L(2Pl6), TS-ZPT-3S(2Pl7) and TS-ZPT- 3L(2Pl7) exhibited similar affinity than the benchmark (trebananib).
  • FIG.24 shows the use of bio-layer interferometry to characterize the sequential binding of TS-ZPT-3S(2Pl6), TS-ZPT-3L(2Pl6), TS-ZPT-3S(2Pl7) and TS-ZPT-3L(2Pl7) to each of its three binding partners using the Octet RED96 System (ForteBio), largely as described in Example 11 above. Briefly, 20nM of TS-ZPT-3 trispecific antibodies were loaded onto the anti-human IgG capture biosensors. To monitor sequential binding of the three antigens to the four TS-ZPT-3 molecules, biosensors were placed into wells containing saturating
  • TS- ZPT-3S(2Pl6), TS -ZPT-3L(2P 16), TS-ZPT-3S(2Pl7) and TS-ZPT-3L(2Pl7) are able to bind to VEGF, Ang-2 and PD-l simultaneously.
  • TS-ZP17T-3 were detected following incubation with TMB-ELISA substrate.
  • the results of this analysis showed that the half-life (Tl/2) of TS-ZP17T-3 is about 2 days (FIG. 25).
  • trebananib allows for the creation of a stable and potent trispecific therapeutic molecule.
  • FIGS. 26A-26B show two additional trispecific molecules utilizing two checkpoint antibodies and the trebananib peptide inserted into the CH3 domain. These molecules comprise sequences shown in FIG. 1 with (1) the VH and VL from the first checkpoint antibody, for example anti-PD-l or anti-PD-Ll, (2) trebananib inserted into the CH3, (3) the VH and VL with a 3-6xG4S linker from a second checkpoint inhibitor, for example anti- TIGIT or anti-LAG3.
  • FIG. 27 shows an additional trispecific molecule, TS-A1BT-1 (SEQ ID NO:
  • FIGS:229-230 show a size exclusion chromatography analysis of protein A chromatography purified TS-A1BT-1.
  • TGF-P-RII ECD TGF-b RII extracellular domain
  • FIGS. 30A-30F show six trispecific antitumor antagonists, TS-ZPB-l, TS-ZPB- 2, TS-ZPB-3, TS-ZPB-4, TS-ZPB-5 and TS-ZPB-6, respectively. These antagonists comprise:
  • VH1, VL1 anti-PD-l or another checkpoint antibody variable domains (FIGS. 30A-30F);
  • an aflibercept fusion protein domain (i) fused to the amino-terminal end of each heavy chain VH1 region (FIGS. 30A, 30D, 30F) or (ii) fused to the carboxy -terminal end of each heavy chain CH3 region (FIGS. 30B, 30C, 30E) each carboxy -terminal end; and (3) a TGF-b RII extracellular domain (ECD): (i) fused to the carboxy-terminal end of each heavy chain CH3 region (FIG. 30A), (ii) fused to the amino-terminal end of each heavy chain VH1 region (FIG. 30B), (iii) inserted within the Fc loop in each heavy chain CH3 region (FIGS.
  • ECD TGF-b RII extracellular domain
  • the amino acid sequences demonstrated in SEQ ID NOS: 187 and 188 show exemplary sequences of TS-ZPLB-l.
  • the amino acid sequences demonstrated in SEQ ID NOS:270 and 271 show exemplary sequences of TS-ZPB-l.
  • the amino acid sequences demonstrated in SEQ ID NOS:272 and 273 show exemplary sequences for TS-ZPB-2.
  • amino acid sequences demonstrated in SEQ ID NOS: 188 and 189 show exemplary sequences of TS-ZPLB-3.
  • the amino acid sequences demonstrated in SEQ ID NOS: 190 and 191 show exemplary sequences of TS-ZPB-3.
  • the amino acid sequences demonstrated in SEQ ID NOS: 192 and 193 show exemplary sequences of TS-ZPB-5.
  • Example 8 Transient expression of TS-ZPB and TS-ZPLB molecules
  • TS-ZPB-l(2Pl7), TS-ZPB-2(2Pl7), TS-ZPB-3(2Pl7), TS-ZPB-5(2Pl7) and TS-ZPLB molecules were transiently expressed in human embryonic kidney HEK293 cells, which were cultured for 7 days. Titers were quantified using a POROS A 20 pm column,
  • FIG. 31A an SDS-PAGE analysis of TS-ZPB- l(2P 17), TS-ZPB- 2(2Pl7), TS-ZPB-3(2Pl7), TS-ZPB-5(2Pl7) and TS-ZPLB produced by transiently transfected HEK293 cells.
  • the results of this analysis all of the molecules express well (FIG. 31B).
  • FIG. 32A shows exemplary size exclusion chromatograph (SEC) profiles for TS- ZPB-l(2Pl7), TS-ZPB-2(2Pl7), TS-ZPB-3(2Pl7), TS-ZPB-5(2Pl7) and TS-ZPLB using a Tosoh TSKgel UP-G3000SWXL column with detection at 214 nm.
  • SEC size exclusion chromatograph
  • Example 9 Functional characterization of the TS-ZPB trispecific antagonists relative to controls
  • FIG. 33A shows the results of a cell-based assay measuring the ability of TS- ZPB-l(2Pl7), TS-ZPB-2(2Pl7), TS-ZPB-3(2Pl7), and TS-ZPB-5(2Pl7) to block the interaction between PD-l and PD-L1.
  • 3 fold serial dilutions of the TS-ZPB molecules (as indicated; highest Ab concentration: 300nM; triplicates for each mAh) were prepared;
  • human PD-l transfected CHOK1 cells were washed with FACS buffer (0.5% BSA 2mM EDTA in PBS) and re-suspended at a concentration of 10 6 cells/ml; FITC-labeled human PD-Ll-Fc protein was added to human PD-l transfected CHOK1 cells at a final concentration of 7pg/ml; mixed well; without incubation, 20xl0 3 of the transfected CHOK1 cells (with PD-L1 Protein) were immediately added to 20m1 FACS buffer in a 96-well round bottom plate; 20m1 3-fold serial dilutions of the TS-ZPB molecules were then immediately added to the cells, which were then incubated at 4°C for 30 mins; cells were then washed and re-suspended in 30m1 7AAD to which 35m1 of 10% neutral buffered formalin solution was added.
  • FACS buffer 0.5% BSA 2mM EDTA in PBS
  • FIG. 33B shows the IC50 values (nM) obtained from this analysis indicating all of the molecules are able to inhibit the interaction of PD-l with PD-L1.
  • FIG. 34A shows the results of a cell-based assay using a TGF /SMAD Signaling Pathway SBE Reporter -HEK293 Cell Line (BPS Bioscience, Inc., San Diego, CA, Catalog #: 60653) to measure the ability of TS-ZPB-l(2Pl7), TS-ZPB-2(2Pl7), TS-ZPB-3(2Pl7), TS- ZPB-5(2Pl7) and TS-ZPLB and a benchmark, TGF l RII-Fc to block TGF l signaling.
  • TGF /SMAD Signaling Pathway SBE Reporter -HEK293 Cell Line BPS Bioscience, Inc., San Diego, CA, Catalog #: 60653
  • a luciferase assay was conducted using the ONE-StepTM Luciferase Assay System (BPS Bioscience, Inc.) according to the protocol provided (i.e., 100 m ⁇ of ONE-StepTM Luciferase reagent was added per well and rocked at room temperature for -15 to 30 minutes before reading luminescence signal.
  • ONE-StepTM Luciferase Assay System BPS Bioscience, Inc.
  • 34B shows the IC50 values (nm) obtained from this analysis and indicates all of the molecules are able to block the bioactivity of TGF l.
  • FIG. 35A shows the results of an ELISA assay measuring the ability of TS-ZPB- 1(2R17), TS-ZPB-2(2Pl7), TS-ZPB-3(2Pl7), TS-ZPB-5(2Pl7) and TS-ZPLB to block the interaction between VEGF and VEGFR-2, as compared to the benchmark antibody,
  • bevacizumab 0.5ug/ml of recombinant human VEGF-165 (R&D #293-VE-050/CF) in carbonate-bicarbonate buffer was coated to the wells in a 96-well assay plate and incubated overnight at 4°C. The wells were then washed 3 times with 0.05% Tween 20 in PBS (wash buffer), blocked with 1% BSA in PBS for 1 hour at room temperature, and washed 3 times with wash buffer. 50m1 5-fold serially diluted antibodies were then added to wells in the plate and incubated for 30 minutes at room temperature.
  • VEGFR-2 binding was detected by measuring light absorbance at 650 nm after addition of 3,3',5,5'-Tetramethylbenzidine to each well in the plate.
  • FIG. 35B shows the IC50 values (nM) obtained from this analysis and indicates all of the molecules are able to inhibit the binding of VEGF with VEGFR-2.
  • Example 10 Functional characterization of TS-ZPT-5 variant with a-glycosylated VEGFR (aflibercept)
  • TS-ZPB-5 Two variants of TS-ZPB-5 were produced to improve the pharmacokinetics of TS-ZPB-5.
  • the heavy chain of TS-ZPB-5 A (SEQ ID NO:205), was mutated at codon for the carboxy -terminal lysine of the CH3 domain to reduce potential proteolytic activity by proteases present in the blood.
  • the heavy chain of TS-ZPB-5B (SEQ ID NO:206) additionally has mutations that eliminate the glycosylation sites in aflibercept, to reduce clearance by ASGRI and ASGRII.
  • FIGS. 36A-36C depict the three trispecific antitumor antagonists TS-ZPB-5, TS- ZPB-5A and TS-ZPB-5B.
  • FIG. 37 shows transient HEK293 cell expression levels of TS-TPB-5, TS-ZPB- 5A and TS-ZPB-5B are similar to the parental 2P17 antibody.
  • the VEGF -VEGFR-2 blocking assay in Example 10 was used to evaluate the ability of TS-TPB-5, TS-ZPB-5 A and TS-ZPB-5B to block the interaction between VEGF and VEGFR-2 (FIG. 38). The results show that binding and bioactivity of the VEGFR component are retained after mutation of aflibercept glycosylation sites from the Asn to Glu.
  • the TGFB signaling assay described in Example 10 was used to evaluate the ability of TS-TPB-5, TS-ZPB- 5A and TS-ZPB-5B to block the TGF signaling (FIG. 39). The results show that binding and bioactivity of TGF R-II are retained after mutation of aflibercept glycosylation sites from the Asn to Glu.
  • FIG. 39 shows the TS-ZPB-5B variant with the mutation of aflibercept glycosylation sites from the Asn to Glu improved the pharmacokinetics by approximately five fold.
  • Example 11 Production and characterization of Bispecific inhibitors of PD-l/PD- L1 and VEGF
  • FIGS. 41A-41C show bispecific molecules TS-ZPL-l (or TS-ZP-l), TS-ZPL-2 (or TS-ZP-2) and TS-ZPL-3 (or TS-ZP-3) comprising (1) a PD-l, PD-L1 or other checkpoint antibody variable domains (VH and VL) (2) aflibercept fuse (i) to the amino-terminus of the VH domain (FIGS. 40A and 40B); (ii) to the carboxy -terminus of the CH3 domain (FIG. 40C).
  • Exemplary sequences for these molecules are SEQ ID NOS:218-225.
  • FIG. 42A shows the results of a cell-based binding assay measuring the ability of Bi-ZPL-l molecules to block the interaction between PD-L1 and PD-l. Briefly, 2 or 3 fold serial dilutions of Anti -human PD-L1 mAh or bispecific Ab (Highest Ab concentration: 128hM;
  • FIG. 43A the bioassay of example 10 testing the ability of Bi-ZP-2 and Bi-ZPL-3 to block the bioactivity of VEGF165 on VEGFR-2 expressing cells.
  • the tabular results are shown in FIG. 43B, and indicate the molecules retain bioactivity relative the benchmark antibody, bevacizumab.
  • FIG. 45 shows the pharmacokinetics of Bi-ZP-2 and Bi-ZPL-3 in two mice each.
  • Bi-ZP-2 exhibits a longer half-life in vivo than Bi-ZPL-3.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022059800A1 (en) * 2020-09-15 2022-03-24 Santen Pharmaceutical Co., Ltd. Bispecific binding molecules against vegf and ang2
WO2022174451A1 (zh) * 2021-02-22 2022-08-25 浙江道尔生物科技有限公司 一种具有抗癌活性的多结构域融合蛋白

Families Citing this family (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9840553B2 (en) 2014-06-28 2017-12-12 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
AU2016381964B2 (en) 2015-12-30 2024-02-15 Kodiak Sciences Inc. Antibodies and conjugates thereof
WO2018128939A1 (en) * 2017-01-05 2018-07-12 Gensun Biopharma Inc. Checkpoint regulator antagonists
WO2019169341A1 (en) 2018-03-02 2019-09-06 Kodiak Sciences Inc. Il-6 antibodies and fusion constructs and conjugates thereof
WO2020006511A1 (en) * 2018-06-29 2020-01-02 Gensun Biopharma, Inc. Trispecific antagonists
TWI760751B (zh) * 2019-05-29 2022-04-11 美商美國禮來大藥廠 Tigit及pd-1/tigit-結合分子
US10851157B2 (en) * 2019-07-01 2020-12-01 Gensun Biopharma, Inc. Antagonists targeting the TGF-β pathway
CN114786731A (zh) 2019-10-10 2022-07-22 科达制药股份有限公司 治疗眼部病症的方法
JP7495489B2 (ja) * 2019-10-21 2024-06-04 ナンチン リーズ バイオラブス カンパニー，リミティド ＰＤ－１およびＴＧＦβを標的化する組換えタンパク質
AU2020375161A1 (en) 2019-11-01 2022-05-19 Ares Trading S.A. Combined inhibition of PD-1, TGFβ and ATM together with radiotherapy for the treatment of cancer
WO2021089704A1 (en) 2019-11-05 2021-05-14 Merck Patent Gmbh Combined inhibition of pd-1, tgfb and tigit for the treatment of cancer
BR112022013403A2 (pt) * 2020-01-10 2022-10-11 Shanghai Henlius Biotech Inc Anticorpos anti-tigit, anticorpos multiespecíficos que compreendem os mesmos e métodos de uso dos mesmos
JP7489468B2 (ja) * 2020-03-20 2024-05-23 レメゲン シーオー．，エルティーディー． 二重特異性融合タンパク質及びその応用
WO2021209458A1 (en) 2020-04-14 2021-10-21 Ares Trading S.A. Combination treatment of cancer
JP2023521465A (ja) 2020-04-14 2023-05-24 グラクソスミスクライン、インテレクチュアル、プロパティー、ディベロップメント、リミテッド ＩＣＯＳ抗体とＰＤ－Ｌ１抗体ＴＧＦ－β－受容体融合タンパク質とに基づく癌の併用治療
MX2022013620A (es) * 2020-04-29 2023-01-30 Chia Tai Tianqing Pharmaceutical Group Co Ltd Proteina bifuncional frente a pd-1 y tgf-beta.
TW202216778A (zh) 2020-07-15 2022-05-01 美商安進公司 Ｔｉｇｉｔ及ｃｄ１１２ｒ阻斷
KR20230166150A (ko) 2020-08-19 2023-12-06 젠코어 인코포레이티드 항-cd28 조성물
US20230372319A1 (en) 2020-11-02 2023-11-23 Ares Trading S.A. Combination Treatment of Cancer
CN116801906A (zh) 2020-11-02 2023-09-22 阿雷斯贸易股份有限公司 癌症的联合治疗
CN114539415B (zh) * 2020-11-24 2024-02-02 普米斯生物技术(珠海)有限公司 一种抗PD-L1/VEGF/TGF-β多特异性抗体及其用途
WO2022132932A1 (en) * 2020-12-15 2022-06-23 The Trustees Of Columbia University In The City Of New York Method of enhancing immune response and cancer immunotherapy by targeting the cd58:cd2 axis
CN112940134B (zh) * 2021-05-11 2021-09-03 正大天晴药业集团南京顺欣制药有限公司 针对PD-1和TGF-β的双功能蛋白
US20240279324A1 (en) * 2021-06-03 2024-08-22 Gensun Biopharma Inc. Multispecific antagonists
JP2024520764A (ja) 2021-06-07 2024-05-24 アレス トレーディング ソシエテ アノニム 癌の組み合わせ処置
JP2024523838A (ja) * 2021-06-09 2024-07-02 シャンハイ エピムアブ バイオセラピューティクス カンパニー リミテッド Ｏｘ４０及び／又はｐｄ－ｌ１に結合する抗体及び二重特異性結合タンパク質
WO2023001987A2 (en) 2021-07-22 2023-01-26 University Of Dundee Therapeutic muteins
TW202317644A (zh) * 2021-09-15 2023-05-01 大陸商江蘇恆瑞醫藥股份有限公司 一種含抗ｐｖｒｉｇ／ｔｉｇｉｔ雙特異性抗體的醫藥組成物
CN113773401B (zh) * 2021-09-15 2023-06-20 宜明昂科生物医药技术（上海）股份有限公司 靶向cd47和pd-l1的重组融合蛋白及其制备和用途
TW202328198A (zh) * 2021-10-21 2023-07-16 大陸商杭州阿諾生物醫藥科技有限公司 一種融合多肽及其用途
WO2023116911A1 (zh) * 2021-12-24 2023-06-29 百奥泰生物制药股份有限公司 抗FRα抗体及其抗体药物偶联物和用途
GEAP202416581A (en) * 2022-01-13 2024-12-10 Ingenia Therapeutics Inc Vegf and tie2-binding fusion protein and uses thereof
CN116496407B (zh) * 2022-01-25 2025-08-15 上海菩莳医药科技有限公司 一种双功能融合蛋白及其制备方法和应用
CA3252619A1 (en) 2022-02-23 2023-08-31 Xencor, Inc. Anti-CD28 and anti-PSMA antibodies
CN116836281A (zh) * 2022-03-25 2023-10-03 英诺湖医药(杭州)有限公司 B7h3抗体及包含其的双功能抗体
CN119546633A (zh) * 2022-07-20 2025-02-28 明慧医药（杭州）有限公司 多特异性抗体及其用途
WO2024041477A1 (zh) * 2022-08-22 2024-02-29 浙江道尔生物科技有限公司 多结构域融合蛋白的用途
WO2024218509A1 (en) * 2023-04-19 2024-10-24 Centessa Pharmaceuticals (Uk) Limited Activatable bispecific anti-cd28 and anti pd-l1 proteins and uses thereof
WO2025067442A1 (zh) * 2023-09-28 2025-04-03 南京维立志博生物科技股份有限公司 抗fgfr2抗体和包含其的多特异性结合分子
WO2025076440A1 (en) * 2023-10-04 2025-04-10 Agenus Inc. TGFβRII EXTRACELLULAR REGION VARIANTS AND USES THEREOF
WO2025111816A1 (en) * 2023-11-28 2025-06-05 Eluminex Biosciences Limited Multispecific fusion proteins targeting angiogenic, inflammatory, and/or fibrotic factors

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060099150A1 (en) * 2000-10-02 2006-05-11 Houston L L Compositions and methods for the transport of biologically active agents across cellular barriers
WO2016187594A1 (en) * 2015-05-21 2016-11-24 Harpoon Therapeutics, Inc. Trispecific binding proteins and methods of use
US20170275353A1 (en) * 2016-03-24 2017-09-28 Gensun Biopharma Inc Trispecific inhibitors for cancer treatment

Family Cites Families (104)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3030025A (en) 1962-04-17 Thermostatic gas valve
GB2116183B (en) 1982-03-03 1985-06-05 Genentech Inc Human antithrombin iii dna sequences therefore expression vehicles and cloning vectors containing such sequences and cell cultures transformed thereby a process for expressing human antithrombin iii and pharmaceutical compositions comprising it
US20030206899A1 (en) 1991-03-29 2003-11-06 Genentech, Inc. Vascular endothelial cell growth factor antagonists
US6582959B2 (en) 1991-03-29 2003-06-24 Genentech, Inc. Antibodies to vascular endothelial cell growth factor
WO1994004679A1 (en) 1991-06-14 1994-03-03 Genentech, Inc. Method for making humanized antibodies
US7067637B1 (en) 1992-02-12 2006-06-27 Biopharm Gesellschaft Zur Biotechnologischen Entwicklung Von Pharmaka Mbh Antibody or antibody fragments specific for a protein of the TGF-β family
US5861499A (en) 1994-02-10 1999-01-19 Imclone Systems Incorporated Nucleic acid molecules encoding the variable or hypervariable region of a monoclonal antibody that binds to an extracellular domain
US6448077B1 (en) 1994-02-10 2002-09-10 Imclone Systems, Inc. Chimeric and humanized monoclonal antibodies specific to VEGF receptors
US6054287A (en) 1994-05-27 2000-04-25 Methodist Hospital Of Indiana, Inc. Cell-type-specific methods and devices for the low temperature preservation of the cells of an animal species
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
US6410690B1 (en) 1995-06-07 2002-06-25 Medarex, Inc. Therapeutic compounds comprised of anti-Fc receptor antibodies
US5922845A (en) 1996-07-11 1999-07-13 Medarex, Inc. Therapeutic multispecific compounds comprised of anti-Fcα receptor antibodies
US6111090A (en) 1996-08-16 2000-08-29 Schering Corporation Mammalian cell surface antigens; related reagents
ATE397660T1 (de) * 1996-08-16 2008-06-15 Schering Corp Zelloberflächen-antigen aus säugetieren und verwandte reagenzien
WO1998023289A1 (en) 1996-11-27 1998-06-04 The General Hospital Corporation MODULATION OF IgG BINDING TO FcRn
US6884879B1 (en) 1997-04-07 2005-04-26 Genentech, Inc. Anti-VEGF antibodies
ATE293640T1 (de) 1997-04-07 2005-05-15 Genentech Inc Anti-vegf antikörper
US7365166B2 (en) 1997-04-07 2008-04-29 Genentech, Inc. Anti-VEGF antibodies
US20020032315A1 (en) 1997-08-06 2002-03-14 Manuel Baca Anti-vegf antibodies
AU1102399A (en) 1997-10-21 1999-05-10 Human Genome Sciences, Inc. Human tumor necrosis factor receptor-like proteins tr11, tr11sv1, and tr11sv2
AU2521999A (en) 1998-02-04 1999-08-23 Desire Jose Collen Identification, production and use of staphylokinase derivatives with reduced immunogenicity and/or reduced clearance
EP1053321A1 (en) 1998-02-09 2000-11-22 Genentech, Inc. Novel tumor necrosis factor receptor homolog and nucleic acids encoding the same
US6376653B1 (en) 1998-09-28 2002-04-23 Smithkline Beecham Plc Tie2 antagonist antibodies
US6703020B1 (en) 1999-04-28 2004-03-09 Board Of Regents, The University Of Texas System Antibody conjugate methods for selectively inhibiting VEGF
CA2378179A1 (en) 1999-07-12 2001-01-18 Genentech, Inc. Promotion or inhibition of angiogenesis and cardiovascularization by tumor necrosis factor ligand/receptor homologs
JP2001206899A (ja) 1999-11-18 2001-07-31 Japan Tobacco Inc ＴＧＦ−βＩＩ型受容体に対するヒトモノクローナル抗体及びその医薬用途
US6528959B2 (en) 2000-07-19 2003-03-04 Honda Giken Kogyo Kabushiki Kaisha Driving force control system for front-and-rear wheel drive vehicles
US7658924B2 (en) 2001-10-11 2010-02-09 Amgen Inc. Angiopoietin-2 specific binding agents
US7521053B2 (en) 2001-10-11 2009-04-21 Amgen Inc. Angiopoietin-2 specific binding agents
US7138370B2 (en) * 2001-10-11 2006-11-21 Amgen Inc. Specific binding agents of human angiopoietin-2
US6874679B2 (en) 2001-10-12 2005-04-05 Innovative Packaging Designs, L.P. Container having sliding corner support
AR039067A1 (es) 2001-11-09 2005-02-09 Pfizer Prod Inc Anticuerpos para cd40
ATE510561T1 (de) 2002-03-04 2011-06-15 Imclone Llc Kdr-spezifische menschliche antikörper und ihre verwendung
CA2384922C (en) 2002-05-03 2008-09-02 Purepharm Inc. Topical glycopyrrolate product for the reduction of sweating
US7025982B2 (en) 2002-09-19 2006-04-11 Medtronic, Inc. Medical assembly suitable for long-term implantation and method for fabricating the same
EP1585768A2 (en) 2003-01-23 2005-10-19 Genentech, Inc. Methods for producing humanized antibodies and improving yield of antibodies or antigen binding fragments in cell culture
KR20060052681A (ko) 2003-05-23 2006-05-19 와이어쓰 Ｇｉｔｒ 리간드, ｇｉｔｒ 리간드-연관 분자, 항체 및그의 용도
RS20160203A1 (sr) 2003-05-30 2016-10-31 Genentech Inc Tretman sa anti-vegf antitelima
EP1660126A1 (en) 2003-07-11 2006-05-31 Schering Corporation Agonists or antagonists of the clucocorticoid-induced tumour necrosis factor receptor (gitr) or its ligand for the treatment of immune disorders, infections and cancer
US7758859B2 (en) 2003-08-01 2010-07-20 Genentech, Inc. Anti-VEGF antibodies
US20050106667A1 (en) 2003-08-01 2005-05-19 Genentech, Inc Binding polypeptides with restricted diversity sequences
US7182135B2 (en) 2003-11-14 2007-02-27 Halliburton Energy Services, Inc. Plug systems and methods for using plugs in subterranean formations
EP1692318A4 (en) 2003-12-02 2008-04-02 Genzyme Corp COMPOSITIONS AND METHODS FOR DIAGNOSIS AND TREATMENT OF LUNG CANCER
WO2005097832A2 (en) 2004-03-31 2005-10-20 Genentech, Inc. Humanized anti-tgf-beta antibodies
GB0409799D0 (en) 2004-04-30 2004-06-09 Isis Innovation Method of generating improved immune response
US20060002932A1 (en) 2004-06-04 2006-01-05 Duke University Methods and compositions for enhancement of immunity by in vivo depletion of immunosuppressive cell activity
US20060009360A1 (en) 2004-06-25 2006-01-12 Robert Pifer New adjuvant composition
WO2006010057A2 (en) * 2004-07-08 2006-01-26 Amgen Inc. Therapeutic peptides
US8367805B2 (en) 2004-11-12 2013-02-05 Xencor, Inc. Fc variants with altered binding to FcRn
BRPI0517837A (pt) 2004-11-12 2008-10-21 Xencor Inc variantes fc com ligação alterada a fcrn
CA2602375C (en) 2005-03-23 2018-07-24 Genmab A/S Antibodies against cd38 for treatment of multiple myeloma
AU2006230099B2 (en) 2005-03-25 2012-04-19 Gitr, Inc. GITR binding molecules and uses therefor
PL1874818T3 (pl) 2005-04-22 2011-09-30 Lilly Co Eli Przeciwciała swoiste wobec TGF-beta 1
WO2006117910A1 (ja) 2005-04-28 2006-11-09 Mochida Pharmaceutical Co., Ltd. 抗血小板膜糖蛋白質ⅵモノクローナル抗体
ITBS20050066A1 (it) 2005-06-01 2006-12-02 Sys Tec S R L Metodo e macchina per allineare lastre di stampa flessografica su cilindri di stampa
WO2006138627A2 (en) 2005-06-16 2006-12-28 Inhibitex, Inc. Monoclonal antibodies recognizing a coagulase-negative staphylococcal protein
AU2006330542B2 (en) 2005-12-23 2012-05-24 Eli Lilly And Company TGF-beta binding compositions
US20110212086A1 (en) 2006-01-19 2011-09-01 Genzyme Corporation GITR Antibodies For The Treatment of Cancer
EP2175884B8 (en) 2007-07-12 2017-02-22 GITR, Inc. Combination therapies employing gitr binding molecules
US8574577B2 (en) 2008-01-03 2013-11-05 The Scripps Research Institute VEGF antibodies comprising modular recognition domains
JO2913B1 (en) 2008-02-20 2015-09-15 امجين إنك, Antibodies directed towards angiopoietin-1 and angiopoietin-2 proteins and their uses
JP2011526794A (ja) 2008-07-02 2011-10-20 エマージェント プロダクト デベロップメント シアトル， エルエルシー ＴＧＦ−βアンタゴニスト多重標的結合性分子
WO2010019263A2 (en) * 2008-08-15 2010-02-18 Genzyme Corporation Soluble flt constructs for treating cancers
JPWO2010030002A1 (ja) 2008-09-12 2012-02-02 国立大学法人三重大学 外来性ｇｉｔｒリガンド発現細胞
US8268314B2 (en) * 2008-10-08 2012-09-18 Hoffmann-La Roche Inc. Bispecific anti-VEGF/anti-ANG-2 antibodies
WO2010115589A1 (en) 2009-04-07 2010-10-14 Roche Glycart Ag Trivalent, bispecific antibodies
CN102574924A (zh) 2009-09-03 2012-07-11 先灵公司 抗-gitr抗体
GB0919054D0 (en) 2009-10-30 2009-12-16 Isis Innovation Treatment of obesity
GB0919074D0 (en) 2009-10-30 2009-12-16 Cambridge Reactor Design Ltd Crystallisation process and apparatus
MX341796B (es) 2009-12-29 2016-09-02 Emergent Product Dev Seattle Proteinas de union heterodimericas y usos de las mismas.
US8993524B2 (en) * 2010-03-05 2015-03-31 The Johns Hopkins University Compositions and methods for targeted immunomodulatory antibodies and fusion proteins
US9527925B2 (en) 2011-04-01 2016-12-27 Boehringer Ingelheim International Gmbh Bispecific binding molecules binding to VEGF and ANG2
MX359833B (es) 2011-08-19 2018-10-12 Regeneron Pharma Anticuerpos anti-tie2 y usos de los mismos.
WO2013039954A1 (en) 2011-09-14 2013-03-21 Sanofi Anti-gitr antibodies
US8581886B2 (en) 2011-10-28 2013-11-12 Atmel Corporation Tuning algorithm for noise reduction in an active stylus
CA2858806A1 (en) 2011-12-23 2013-06-27 Innate Pharma Enzymatic conjugation of polypeptides
HUE044263T2 (hu) 2012-03-30 2019-10-28 Boehringer Ingelheim Int ANG2-kötõ molekulák
EP2861617A1 (en) 2012-06-15 2015-04-22 Pfizer Inc. Improved antagonist antibodies against gdf-8 and uses therefor
US20150368329A1 (en) * 2012-10-15 2015-12-24 Oncomed Pharmaceuticals, Inc. Methods of Treating Ocular Diseases
EP2922874A4 (en) 2012-11-21 2016-10-19 Wuhan Yzy Biopharma Co Ltd BISPECIFIC ANTIBODIES
US20140308285A1 (en) 2013-03-15 2014-10-16 Amgen Inc. Heterodimeric bispecific antibodies
WO2014203182A1 (en) * 2013-06-20 2014-12-24 Novartis Ag Use of a vegf antagonist in treating choroidal neovascularisation
EP3789036A1 (en) 2013-07-16 2021-03-10 F. Hoffmann-La Roche AG Methods of treating cancer using pd-1 axis binding antagonists and tigit inhibitors
TWI681969B (zh) 2014-01-23 2020-01-11 美商再生元醫藥公司 針對ｐｄ-１的人類抗體
JP6731346B2 (ja) * 2014-02-10 2020-07-29 メルク パテント ゲーエムベーハー 標的ＴＧＦβ阻害
US20170335281A1 (en) * 2014-03-15 2017-11-23 Novartis Ag Treatment of cancer using chimeric antigen receptor
KR102349370B1 (ko) 2014-05-26 2022-01-10 삼성전자주식회사 인간화 또는 친화도 성숙된 항 앤지오포이에틴-2 항체 및 그의 용도
TWI790593B (zh) 2014-08-19 2023-01-21 美商默沙東有限責任公司 抗tigit抗體
CN107073126A (zh) * 2014-11-06 2017-08-18 豪夫迈·罗氏有限公司 包含ox40结合激动剂和tigit抑制剂的组合疗法
EP3237448A1 (en) * 2014-12-23 2017-11-01 Bristol-Myers Squibb Company Antibodies to tigit
WO2016163842A1 (ko) 2015-04-10 2016-10-13 엘지전자 (주) 무선 통신 시스템에서 채널 상태 정보를 보고하기 위한 방법 및 이를 위한 장치
TWI715587B (zh) 2015-05-28 2021-01-11 美商安可美德藥物股份有限公司 Ｔｉｇｉｔ結合劑和彼之用途
TWI773646B (zh) * 2015-06-08 2022-08-11 美商宏觀基因股份有限公司 結合lag-3的分子和其使用方法
CN105111314B (zh) * 2015-08-13 2019-01-08 成都百世博生物技术有限公司 一种新型融合蛋白、药物组合物及其制备方法和用途
CA2996996A1 (en) * 2015-08-31 2017-03-09 National Research Council Of Canada Tgf-.beta.-receptor ectodomain fusion molecules and uses thereof
EP3433277A4 (en) 2016-03-23 2020-06-17 Mabspace Biosciences (Suzhou) Co., Ltd NEW ANTI-PD-L1 ANTIBODIES
CA3026151A1 (en) 2016-06-14 2017-12-21 Xencor, Inc. Bispecific checkpoint inhibitor antibodies
RU2769282C2 (ru) * 2016-06-20 2022-03-30 Кимаб Лимитед Анти-PD-L1 и IL-2 цитокины
WO2018128939A1 (en) 2017-01-05 2018-07-12 Gensun Biopharma Inc. Checkpoint regulator antagonists
WO2020006511A1 (en) * 2018-06-29 2020-01-02 Gensun Biopharma, Inc. Trispecific antagonists
US10851157B2 (en) * 2019-07-01 2020-12-01 Gensun Biopharma, Inc. Antagonists targeting the TGF-β pathway
TWI815184B (zh) * 2020-09-23 2023-09-11 大陸商海正生物製藥有限公司 Tgfbr2-ecd突變體及包含其的融合蛋白與應用
US20240279324A1 (en) * 2021-06-03 2024-08-22 Gensun Biopharma Inc. Multispecific antagonists

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060099150A1 (en) * 2000-10-02 2006-05-11 Houston L L Compositions and methods for the transport of biologically active agents across cellular barriers
WO2016187594A1 (en) * 2015-05-21 2016-11-24 Harpoon Therapeutics, Inc. Trispecific binding proteins and methods of use
US20170275353A1 (en) * 2016-03-24 2017-09-28 Gensun Biopharma Inc Trispecific inhibitors for cancer treatment

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022059800A1 (en) * 2020-09-15 2022-03-24 Santen Pharmaceutical Co., Ltd. Bispecific binding molecules against vegf and ang2
WO2022174451A1 (zh) * 2021-02-22 2022-08-25 浙江道尔生物科技有限公司 一种具有抗癌活性的多结构域融合蛋白
WO2022174781A1 (zh) * 2021-02-22 2022-08-25 浙江道尔生物科技有限公司 多结构域融合蛋白及其应用
CN116761814A (zh) * 2021-02-22 2023-09-15 浙江道尔生物科技有限公司 多结构域融合蛋白及其应用
JP2024507856A (ja) * 2021-02-22 2024-02-21 浙江道尓生物科技有限公司 マルチドメイン融合タンパク質及びその応用
CN116761814B (zh) * 2021-02-22 2024-08-27 浙江道尔生物科技有限公司 多结构域融合蛋白及其应用
JP7702493B2 (ja) 2021-02-22 2025-07-03 浙江道尓生物科技有限公司 マルチドメイン融合タンパク質及びその応用
AU2022221660B2 (en) * 2021-02-22 2025-12-04 Zhejiang Doer Biologics Co., Ltd. Multi-domain fusion protein and use thereof

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