WO2018055503A1 - Combination comprising a pd-1 antagonist and an fgfr4 inhibitor - Google Patents

Combination comprising a pd-1 antagonist and an fgfr4 inhibitor Download PDF

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WO2018055503A1
WO2018055503A1 PCT/IB2017/055632 IB2017055632W WO2018055503A1 WO 2018055503 A1 WO2018055503 A1 WO 2018055503A1 IB 2017055632 W IB2017055632 W IB 2017055632W WO 2018055503 A1 WO2018055503 A1 WO 2018055503A1
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seq
amino acid
acid sequence
methyl
carboxamide
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PCT/IB2017/055632
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French (fr)
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Sanela Bilic
John Cameron
Gary Gu
Danny HOWARD, Jr.
Luigi MANENTI
Haige SHEN
Diana Graus Porta
Andreas Weiss
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Novartis Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a pharmaceutical combination comprising an anti-
  • PD1 antibody molecule and an FGFR4 inhibitor in particular N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, to the use of said pharmaceutical combination in the treatment of cancer, including liver cancer, and to methods of treatment of cancer, including liver cancer, comprising administering said pharmaceutical combination.
  • an FGFR4 inhibitor in particular N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide
  • T cells T cells to mediate an immune response against an antigen requires two distinct signaling interactions (Viglietta, V. et al. (2007) Neurotherapeutics 4:666-675;
  • an antigen that has been arrayed on the surface of antigen-presenting cells is presented to an antigen-specific naive CD4 + T cell.
  • Such presentation delivers a signal via the T cell receptor (TCR) that directs the T cell to initiate an immune response specific to the presented antigen.
  • TCR T cell receptor
  • various co-stimulatory and inhibitory signals mediated through interactions between the APC and distinct T cell surface molecules trigger the activation and proliferation of the T cells and ultimately their inhibition.
  • the immune system is tightly controlled by a network of costimulatory and co- inhibitory ligands and receptors. These molecules provide the second signal for T cell activation and provide a balanced network of positive and negative signals to maximize immune responses against infection, while limiting immunity to self (Wang, L. et al. (Epub Mar. 7, 201 1) J. Exp. Med. 208(3):577-92; Lepenies, B. et al. (2008) Endocrine, Metabolic & Immune Disorders-Drug Targets 8:279-288).
  • costimulatory signals include the binding between the B7.1 (CD80) and B7.2 (CD86) ligands of the APC and the CD28 and CTLA-4 receptors of the CD4 + T-lymphocyte (Sharpe, A. H. et al. (2002) Nature Rev.
  • B7 Superfamily a group of related B7 molecules, also known as the "B7 Superfamily" (Coyle, A. J. et al. (2001) Nature Immunol. 2(3):203-209; Sharpe, A. H. et al. (2002) Nature Rev. Immunol. 2:1 16-126; Collins, M. et al. (2005) Genome Biol. 6:223.1 -223.7; Korman, A. J. et al. (2007) Adv. Immunol. 90:297-339).
  • B7 Superfamily Several members of the B7 Superfamily are known, including B7.1 (CD80), B7.2 (CD86), the inducible co-stimulator ligand (ICOS-L), the programmed death-1 ligand (PD-L1 ; B7-H1), the programmed death-2 ligand (PD-L2; B7-DC), B7-H3, B7-H4 and B7-H6 (Collins, M. et al. (2005) Genome Biol. 6:223.1 -223.7).
  • B7.1 CD80
  • B7.2 the inducible co-stimulator ligand
  • PD-L1 programmed death-1 ligand
  • PD-L2 programmed death-2 ligand
  • B7-DC B7-H3, B7-H4 and B7-H6
  • the Programmed Death 1 (PD-1) protein is an inhibitory member of the extended CD28/CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) J. Immunol. 170:71 1 -8).
  • Other members of the CD28 family include CD28, CTLA-4, ICOS and BTLA.
  • PD-1 is suggested to exist as a monomer, lacking the unpaired cysteine residue characteristic of other CD28 family members. PD-1 is expressed on activated B cells, T cells, and monocytes.
  • the PD-1 gene encodes a 55 kDa type I transmembrane protein (Agata et al. (1996) Int Immunol. 8:765-72). Although structurally similar to CTLA-4, PD-1 lacks the MYPPY motif (SEQ ID NO: 236) that is important for B7-1 and B7-2 binding.
  • Two ligands for PD-1 have been identified, PD-L1 (B7-H1) and PD-L2 (B7-DC), that have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43).
  • Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 , but do not bind to other CD28 family members.
  • PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9).
  • PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signals (Ishida, Y. et al. (1992) EMBO J. 1 1 :3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother. 56(5):739-745).
  • the interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, e.g., a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and/or immune evasion by cancerous cells (Dong et al. (2003) J. Mol. Med. 81 :281 -7; Blank et al. (2005) Cancer Immunol.
  • Therapies involving anti-PD-1 antibodies have been described for instance in WO2015/1 12900. Combination therapies comprising an immunomodulator and a second therapeutic agent for the treatment of cancer have been described in WO2016040892 and WO2016040880 .
  • Fibroblast Growth Factors constitute a family of over twenty structurally related polypeptides that are developmental ⁇ regulated and expressed in a wide variety of tissues. FGFs stimulate proliferation, cell migration and differentiation and play a major role in skeletal and limb development, wound healing, tissue repair, hematopoiesis, angiogenesis, and tumorigenesis (reviewed in Ornitz, Novartis Found Symp 232: 63-76; discussion 76-80, 272-82 (2001)).
  • FGFs The biological action of FGFs is mediated by specific cell surface receptors belonging to the Receptor Protein Tyrosine Kinase (RPTK) family of protein kinases. These proteins consist of an extracellular ligand binding domain, a single transmembrane domain and an intracellular tyrosine kinase domain which undergoes phosphorylation upon binding of FGF.
  • RPTK Receptor Protein Tyrosine Kinase
  • FGFR1 also called Fig, fms-like gene, fit- 2, bFGFR, N-bFGFR or Cekl
  • FGFR2 also called Bek-Bacterial Expressed Kinase, KGFR, Ksam, KsamI and Cek3
  • FGFR3 also called Cek2
  • FGFR4 also called Cek2
  • FGFRs All mature FGFRs share a common structure consisting of an amino terminal signal peptide, three extracellular immunoglobulin-like domains (Ig domain I, Ig domain II, Ig domain III), with an acidic region between Ig domains (the "acidic box” domain), a transmembrane domain, and intracellular kinase domains (Ullrich and Schlessinger, Cell 61 : 203,1990 ; Johnson and Williams (1992) Adv. Cancer Res. 60: 1 -41).
  • the distinct FGFR isoforms have different binding affinities for the different FGF ligands.
  • FGFR4 may play an important role in liver cancer in particular (PLoS One, 2012, volume 7, 36713).
  • Other studies have also implicated FGFR4 or its ligand FGF19 in other cancer types including breast, glioblastoma, prostate, rhabdomyosarcoma, gastric, ovarian, lung, colon (Int. J. Cancer 1993; 54:378-382;
  • Hepatocellular carcinoma is the seventh most common form of cancer worldwide and the third most common cause of cancer-related deaths.
  • Potentially curative therapies such as surgical resection, liver transplant or other local treatments, result in survival rates of between 50-70% at 5 years for patients with early stage HCC.
  • Sorafenib is the only approved drug in HCC with marginal improvement in overall survival (OS) and poor tolerability.
  • therapies for cancer including therapies for HCC, which have improved safety profiles, improved toxicity profiles, improved efficacy compared to a standard of care, are well tolerated by patients, offer advantages to patients in terms of overall response rate, overall survival, time to progression, and/or even cure the disease.
  • New therapies for cancer also could benefit such patients if they also provide tumor growth inhibition, decreased tumor volume, inhibition of cell proliferation, immunomodulatory activity, reduction in relapse time, apoptosis of cancer cells, increase in the time delay for the onset of exponential growth of tumor, reduction of required dosage, reduction of side effects, and/or reduction in resistance to treatment.
  • a pharmaceutical combination comprising a PD-1 antagonist and a second therapeutic agent may therefore be useful in treating a cancer, e.g. HCC.
  • the invention is expected to provide a solution at least to some of the needs outlined above by providing a pharmaceutical combination as defined herein.
  • a selective FGFR4 inhibitor may potentiate the effect of an immune checkpoint blocker such as an anti-PD-1 inhibitor.
  • the combined inhibition of the FGFR4 signal with the selective FGFR4 inhibitor described herein, in particular, N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, or a pharmaceutically acceptable salt thereof, and the inhibition of PD-1 with an anti-PD-1 antibody molecule described herein, in particular the exemplary antibody molecule described herein, may be of particular benefit to subjects suffering from a cancer such as HCC, including advanced HCC.
  • the combination of the invention may also have an acceptable and manageable overall toxicity profile.
  • the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form.
  • the invention relates to a pharmaceutical combination as defined herein for use as a medicament.
  • the invention relates to a pharmaceutical combination as defined herein for use in the treatment of cancer, in particular in the treatment of liver cancer.
  • the invention relates to a pharmaceutical combination comprising an FGFR4 inhibitor and anti-PD-1 antibody molecule for the treatment of liver cancer.
  • the invention in another aspect relates to a method of treating cancer, in particular liver cancer, in a subject in need thereof, comprising administering to said subject simultaneously or sequentially N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule.
  • the invention in another aspect, relates to a method of treating liver cancer in a subject suffering from liver cancer comprising administering to said subject simultaneously or sequentially an FGFR4 inhibitor and an anti-PD-1 antibody molecule.
  • the invention relates to a therapeutic regimen comprising the simultaneous or sequential administration of N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2- yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule.
  • the therapeutic regimen is for the treatment of cancer, in particular liver cancer.
  • the invention relates to a kit comprising a pharmaceutical combination as defined herein with instructions for simultaneous or sequential administration of N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin- 1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in
  • the present invention relates to a pharmaceutical combination comprising an anti- PD-1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form.
  • a "pharmaceutical combination” refers to a non-fixed combination.
  • non-fixed combination means that the active ingredients, e.g. an anti- PD- 1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form, are both administered to a patient or a subject in need thereof as separate entities either simultaneously or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two active ingredients in the body of the patient or a subject in need thereof.
  • active ingredients e.g. an anti- PD- 1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiper
  • the term “treat”, “treating” or “treatment” in connection to a disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • “treat”, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • “treat”, “treating” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.
  • a therapeutically effective amount when referring to the active ingredients present in the combination of the invention refers to the amounts of the active ingredients that, when administered to a subject either simultaneously or sequentially, will elicit a biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression.
  • a therapeutically effective amount when referring to the active ingredients present in the combination of the invention refers to the amounts of the active ingredients that, when administered to a subject either simultaneously or sequentially are effective to provide at least one of the following benefits:
  • improved safety profiles improved safety profiles, improved toxicity profiles, improved efficacy, well tolerated by patients or subjects suffering from cancer, improved overall response rate, improved overall survival, improved time to progression, tumor growth inhibition, decreased tumor volume, inhibition of cell proliferation, immunomodulatory activity, reduction in relapse time, apoptosis of cancer cells, increase in the time delay for the onset of exponential growth of tumor, reduction in resistance to treatment, synergistic effect leading to reduction of required dosage and/or reduction of side effects leading to an enhancement of the long-term clinical effectiveness of the combination.
  • the term “combination of the invention” or “combination of the present invention” refers to a combination comprising an anti-PD-1 antibody molecule and an FGFR4 inhibitor, in particular N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide, in free form or in pharmaceutically acceptable salt form, as defined herein.
  • an FGFR4 inhibitor in particular N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide, in free
  • the preferred FGFR4 inhibitor in the combination of the invention is N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, or a pharmaceutically acceptable salt thereof (e.g. the citrate salt).
  • This compound is also known as FGF401 and is currently undergoing clinical trials involving HCC patients (see NCT02325739).
  • active ingredient refers independently to each combination partner, namely the anti- PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form (e.g. the citrate salt form).
  • liver cancer also refers to hepatocellular carcinoma (HCC).
  • FGFR4 refers to fibroblast growth factor receptor 4, also known as CD334, JTK2, or TKF.
  • FGFR4 inhibitor is a compound which inhibits FGFR4.
  • the FGFR4 inhibitor is preferably a selective FGFR4 inhibitor.
  • the combinations and methods of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 85%, 90%, 95% identical or higher to the sequence specified.
  • the term "substantially identical" is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity.
  • amino acid sequences that contain a common structural domain having at least about 85%, 90%. 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.
  • nucleotide sequence in the context of nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity.
  • the term "functional variant” refers to polypeptides that have a substantially identical amino acid sequence to the naturally-occurring sequence, or are encoded by a substantially identical nucleotide sequence, and are capable of having one or more activities of the naturally-occurring sequence.
  • the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:1 1 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389- 3402.
  • the default parameters of the respective programs e.g. , XBLAST and NBLAST
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used.
  • hybridization conditions referred to herein are as follows: 1 ) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1 % SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45LC, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1 % SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified
  • anti-PD-1 antibody molecule used in the present invention may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.
  • amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids.
  • exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • amino acid includes both the D- or L- optical isomers and peptidomimetics.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • polymers of amino acids of any length may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • the polypeptide can be isolated from natural sources, can be a produced by recombinant techniques from a eukaryotic or prokaryotic host, or can be a product of synthetic procedures.
  • nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • the polynucleotide may be either single-stranded or double-stranded, and if single-stranded may be the coding strand or non-coding (antisense) strand.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • the nucleic acid may be a recombinant
  • polynucleotide or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a nonnatural arrangement.
  • isolated refers to material that is removed from its original or native environment (e.g. , the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated by human intervention from some or all of the co-existing materials in the natural system, is isolated.
  • polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature.
  • the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule and an FGFR4 inhibitor for use in the treatment of liver cancer.
  • the FGFR4 inhibitor used in the combination of the present invention is preferably a compound that inhibits FGFR4 selectively compared to FGFR1 , FGFR2 and FGFR3.
  • FGFR4 inhibitors may be small molecules or large molecules. Therapies involving FGFR4 blocking antibodies have been described for instance in the following patent applications
  • the FGFR4 inhibitor used in the combination of the present invention is any of the FGFR4 inhibitors described in any of patent applications
  • the FGFR4 inhibitor used in the combination of the present invention is any of the FGFR4 inhibitors described in WO2014/144737, WO2014/145909, WO2014/01 1900, WO2015/057963, WO2015/057938, WO2015/030021 , WO2015/107171 , WO2015/059668, WO2016/064960, WO2016/134320, WO2016/134314, WO2016/134294, in free form or in pharmaceutically acceptable salt form.
  • the FGFR4 inhibitor used in the combination of the present invention is a compound as defined in WO2015/059668, in free form or in pharmaceutically acceptable salt form.
  • the FGFR4 inhibitor used in the combination of the present invention is a compound of formula (I) in free form or in pharmaceutically acceptable salt form as described in patent application WO2015/059668. Therefore, in one embodiment, the FGFR4 inhibitor used in the combination of the present invention is a compound of formula (I) in free form or in pharmaceutically acceptable salt form
  • V is selected from CH 2 , O, CH(OH);
  • W is selected from CH 2 , CH 2 CH 2 , bond ;
  • X is C(R X ) or N;
  • Y is C(R Y ) or N;
  • Z is CH or N
  • R x is selected from hydrogen, halogen, haloC Csalkyl, cyano, C C 6 alkyl, hydroxyC C 6 alkyl;
  • R Y is selected from hydrogen, halogen, C Csalkyl, C ⁇ Cealkoxy, hydroxyC Csalkoxy, NR Y1 R Y2 , cyano, C CsalkoxyC Csalkoxy, C Csalkoxy-haloC Csalkoxy, di(C
  • R x and R Y together with the ring to which they are attached form a bicyclic aromatic ring system optionally further comprising one or two heteroatoms selected from N, O, or S, which ring system is optionally substituted with C Csalkyl;
  • R Y1 is hydrogen
  • R Y2 is selected from Ci-C 6 alkyl; hydroxyCrCealkyl; haloC Cealkyl optionally substituted with hydroxy; C 1 -C 4 alkoxyC 1 -C 6 alkyl; haloC CsalkoxyC Cealkyl; (CH 2 ) 0 -i-R Y4 ; di(C Csalky aminoC Cealkyl substituted with hydroxy; bicycloC 5 -C 8 alkyl optionally substituted with hydroxyCrCsalkyl; phenyl substituted with S(0) 2 -CH(CH 3 )2; C 2 -C 3 alkylsulfonic acid; or
  • R Y1 and R Y2 together with the N atom to which they are attached form a saturated or unsaturated non-aromatic 6-membered heterocyclic ring which may contain an O atom, which ring may be substituted once or twice by R Y5 ;
  • R Y3 is selected from quinuclidinyl, a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, or a 5- or 6-membered aromatic heterocyclic ring, which saturated or aromatic heterocyclic ring is optionally substituted with C Csalkyl and/or oxo;
  • R Y4 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O, or S, which ring is optionally substituted with C Csalkyl;
  • R Y5 is independently selected from C Csalkyl, hydroxy, d C CsalkyfyaminoC Csalkyl, or
  • R Y5 attached at the same carbon atom form together with the carbon atom to which they are attached a 5-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is substituted once or more than once with Ci-C 3 alkyl; R and R together with the carbon atom to which they are attached form a 6- membered saturated or unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N, O or S;
  • R 1 is selected from hydrogen; halogen; Crdalkyl; haloCrdalkyl; hydroxyC C 3 alkyl; C 3 -C 6 cycloalkyl; CH 2 NR 2 R 3 ; CH(CH 3 )NR 2 R 3 ; d-CsalkoxyC dalkyl; CH 2 C0 2 H; C(0)H; d-dalkoxy; a 5- or 6-membered saturated heterocyclic or aromatic heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted once or more than once with a group independently selected from d-dalkyl, haloC C 3 alkyl, oxetanyl or oxo;
  • R 2 is selected from d-dalkyl, di(d-dalkyl)aminod-dalkyl;
  • R 3 is selected from d-dalkyl, C(0)C C 3 alkyl, C(0)-CH 2 -OH, C(0)-CH 2 -0-CH 3 , C(O)-
  • R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, N- oxide, O or S, which ring may be substituted once or more than once with R 4 ;
  • R 4 is independently selected from d-dalkyl, di(d-dalkyl)amino, C(0)CH 3 , hydroxy; or
  • R 5 is selected from hydrogen or d-dalkyl.
  • d-dalkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and which is attached to the rest of the molecule by a single bond.
  • C 1 .C 4 alkyl is to be construed accordingly.
  • d-dalkyl is to be construed accordingly.
  • d-dalkyl examples include, but are not limited to, methyl, ethyl, n-propyl, 1 -methylethyl (/so-propyl), n-butyl, n-pentyl and 1 ,1 -dimethylethyl (f-butyl).
  • hydroxyd-dalkyl refers to a radical of formula -R a -OH, wherein R a is C ⁇ alkyl as defined above.
  • examples of hydroxyd-dalkyl include, but are not limited to, hydroxy-methyl, 2-hydroxy-ethyl, 2-hydroxy-propyl, 3-hydroxy-propyl and 5-hydroxy- pentyl.
  • C 3 -C 6 cycloalkyl refers to saturated monocyclic hydrocarbon groups of 3-6 carbon atoms. Examples of C 3 -C 6 cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • C Cealkoxy refers to a radical of the formula -OR a where R a is a C ⁇ Cealkyl radical as generally defined above.
  • C Csalkoxy is to be construed accordingly. Examples of C C 6 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentoxy, and hexoxy.
  • C 1 -C 4 alkoxyC 1 -C 6 alkyl refers to a radical of the formula - R b -0-R a where R a is a C 1 -C 4 alkyl radical and R b is a C C 6 alkyl radical as defined above.
  • R a is a C 1 -C 4 alkyl radical
  • R b is a C C 6 alkyl radical as defined above.
  • C CsalkoxyC Cealkyl is to be construed accordingly.
  • the oxygen atom may be bonded to any carbon atom in either alkyl radical.
  • C 1 -C 4 alkoxyC 1 -C 6 alkyl examples include, but are not limited to, methoxy-methyl, methoxy-ethyl, ethoxy-ethyl, 1 -ethoxy-propyl and 2-methoxy-butyl.
  • Halogen or halo refers to bromo, chloro, fluoro or iodo.
  • halogenCrCealkyl or “haloCrCealkyl” refers to C Cealkyl radical, as defined above, substituted by one or more halo radicals, as defined above.
  • halogenC Cealkyl examples include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1 -fluoromethyl-2-fluoroethyl, 3-bromo-2- fluoropropyl and 1 -bromomethyl-2-bromoethyl.
  • haloC Csalkoxy refers to C C 3 alkoxy as defined above, substituted by one or more halo radicals, as defined above.
  • haloC C 3 alkoxy include, but are not limited to, trifluoromethoxy, difluoromethoxy, trifluoroethoxy.
  • hydroxyC Csalkoxy refers to a C ⁇ Csalkoxy radical as defined above, wherein one of the hydrogen atoms of the ⁇ .Csalkoxy radical is replaced by OH.
  • hydroxy ⁇ .Csalkoxy include, but are not limited to, hydroxymethoxy, hydroxyethoxy.
  • C CsalkoxyC Csalkoxy refers to a C ⁇ Csalkoxy radical as defined above, wherein one of the hydrogen atoms of the C ⁇ alkoxy radical is replaced by - O-C Csalkyl.
  • Examples of C CsalkoxyC Csalkoxy include, but are not limited to, methoxymethoxy, ethoxymethoxy.
  • C Csalkoxy-haloC Csalkoxy refers to a haloC Csalkoxy radical as defined above, wherein one of the hydrogen atoms of the haloC Csalkoxy radical is replaced by -0-C C 3 alkyl.
  • Examples of C Csalkoxy-haloC Csalkoxy include, but are not limited to, methoxytrifluoropropyloxy.
  • diCC Csalky aminoC Cealkyl refers to a radical of the formula -R a i-N(R a2 )-R a2 where R a1 is a C ⁇ C ⁇ ky! radical as defined above and each R a2 is a C Csalkyl radical, which may be the same or different, as defined above.
  • the nitrogen atom may be bonded to any carbon atom in any alkyl radical.
  • the "diC CsalkylaminoC Cealkyl” may be substituted with hydroxy.
  • d CrCsalky aminoCrCealkoxy refers to a radical of the formula -R a i-N(R a2 )-Ra2 where R a1 is a C Cealkoxy radical as defined above and each R a2 is a C Csalkyl radical, which may be the same or different, as defined above.
  • 6-membered saturated heterocyclic ring comprising one heteroatom selected from N, O or S includes piperidyl, tetrahydropyranyl and
  • 6-membered unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N, O or S includes, but is not limited to, tetrahydropyridinyl, dihydropyranyl, dihydrothiopyranyl.
  • a 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S includes as examples, but is not limited to, azetidinyl, oxetanyl, pyrrolidyl, tetrahydrofuryl, tetrahydrothienyl, piperidyl, piperazinyl, tetrahydropyranyl, morpholinyl.
  • 5-membered saturated heterocyclic ring includes as example, but is not limited to, pyrrolidine.
  • a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, O or S includes as examples, but is not limited to, pyrrolidine, oxazolidine, piperazine, morpholine, thiomorpholine rings.
  • a "4-, 5- or 6-membered non-aromatic heterocyclic ring comprising at least one heteroatom selected from N, O or S” includes 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S as defined herein. It also includes 4-, 5-, or 6-membered unsaturated heterocyclic ring comprising at least one heteroatom selected from N, O or S.
  • bicyclic aromatic ring system optionally further comprising one or two heteroatoms selected from N, O or S
  • heteroatoms selected from N, O or S includes, but is not limited to
  • bicycloC 5 -C 8 alkyl refers to bicyclic hydrocarbon groups comprising 5 to 8 carbon atoms including, but not limited to, bicyclo[2.1 .1 ]hexyl, bicyclo[1 .1 .1 ]pentyl, bicyclo[2.2.1 ]heptanyl, bicyclo[2.2.2]octyl.
  • R Y , R x and R Y together, R Y2 , R Y3 , R Y4 includes unsubstituted or substituted once or twice.
  • substituted as used, for example in the description of R Y2 , two R Y5 , includes substituted once or twice, preferably once.
  • substituted R 4 includes 2, 3, 4, 5, or 6 times. Preferably, it includes 2 or 3 times.
  • the FGFR4 inhibitor used in the combination of the present invention is a compound in free form or in pharmaceutically acceptable salt form as described in the examples of patent application WO2015/059668.
  • the FGFR4 inhibitor used in the combination of the present invention is a compound in free form or in pharmaceutically acceptable salt form selected from the group consisting of:
  • Patent application WO2015/059668 describes how to make the compounds of formula (I) and the specific compounds described above.
  • the preferred FGFR4 inhibitor used in the combination of the present invention is N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in free form or in pharmaceutically acceptable salt form.
  • a preferred salt of the preferred FGFR4 inhibitor is the citrate salt .
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide is described specifically in example 83 of patent application WO2015/059668, which WO2015/059668 is hereby incorporated by reference in its entirety, and has the following structure:
  • the FGFR4 inhibitor used in the combination of the present invention is N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form.
  • the FGFR4 inhibitor used in the combination of the present invention is N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form.
  • the present invention relates to a pharmaceutical combination comprising an anti- PD-1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form.
  • the anti-PD-1 antibody molecule used in the combination of the present invention is one described in WO2015/1 12900, entitled “Antibody Molecules to PD-1 and Uses Thereof," which is hereby incorporated by reference in its entirety.
  • the anti-PD-1 antibody molecule comprises at least one antigen-binding region, e.g., a variable region or an antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
  • the anti-PD-1 antibody molecule comprises at least one, two, three or four variable regions from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
  • an antibody described herein e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequence
  • the anti-PD-1 antibody molecule comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
  • an antibody described herein e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
  • the anti-PD-1 antibody molecule comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
  • an antibody described herein e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
  • the anti-PD-1 antibody molecule includes a heavy chain constant region for an lgG4, e.g., a human lgG4.
  • the human lgG4 includes a substitution at position 228 according to EU numbering (e.g., a Ser to Pro substitution).
  • the anti-PD-1 antibody molecule includes a heavy chain constant region for an lgG1 , e.g., a human lgG1 .
  • the human lgG1 includes a substitution at position 297 according to EU numbering (e.g., an Asn to Ala substitution).
  • the human lgG1 includes a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both (e.g., an Asp to Ala substitution at position 265 and/or a Pro to Ala substitution at position 329).
  • the human lgG1 includes a substitution at position 234 according to EU numbering, a substitution at position 235 according to EU numbering, or both (e.g., a Leu to Ala substitution at position 234 and/or a Leu to Ala substitution at position 235).
  • the heavy chain constant region comprises an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.
  • the anti-PD-1 antibody molecule includes a kappa light chain constant region, e.g., a human kappa light chain constant region.
  • the light chain constant region comprises an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.
  • the anti-PD-1 antibody molecule includes a heavy chain constant region for an lgG4, e.g., a human lgG4, and a kappa light chain constant region, e.g., a human kappa light chain constant region, e.g., a heavy and light chain constant region comprising an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.
  • the human lgG4 includes a substitution at position 228 according to EU numbering (e.g., a Ser to Pro substitution).
  • the anti-PD-1 antibody molecule includes a heavy chain constant region for an lgG1 , e.g., a human lgG1 , and a kappa light chain constant region, e.g., a human kappa light chain constant region, e.g., a heavy and light chain constant region comprising an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.
  • the human lgG1 includes a substitution at position 297 according to EU numbering (e.g., an Asn to Ala substitution).
  • the human lgG1 includes a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both (e.g., an Asp to Ala substitution at position 265 and/or a Pro to Ala substitution at position 329).
  • the human lgG1 includes a substitution at position 234 according to EU numbering, a substitution at position 235 according to EU numbering, or both (e.g., a Leu to Ala substitution at position 234 and/or a Leu to Ala substitution at position 235).
  • the anti-PD-1 antibody molecule includes a heavy chain variable domain and a constant region, a light chain variable domain and a constant region, or both, comprising the amino acid sequence of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
  • the anti-PD-1 antibody molecule optionally, comprises a leader sequence from a heavy chain, a light chain, or both, as shown in Table 4; or a sequence substantially identical thereto.
  • the anti-PD-1 antibody molecule includes at least one, two, or three complementarity determining regions (CDRs) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone- B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
  • CDRs complementarity determining regions
  • the anti-PD-1 antibody molecule includes at least one, two, or three CDRs (or collectively all of the CDRs) from a heavy chain variable region comprising an amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 .
  • one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 .
  • the anti-PD-1 antibody molecule includes at least one, two, or three CDRs from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequence.
  • an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E
  • Table 1 or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequence.
  • the anti-PD-1 antibody molecule includes at least one, two, or three CDRs (or collectively all of the CDRs) from a light chain variable region comprising an amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 .
  • one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 .
  • the anti-PD-1 antibody molecule includes a substitution in a light chain CDR, e.g., one or more substitutions in a CDR1 , CDR2 and/or CDR3 of the light chain.
  • the anti-PD-1 antibody molecule includes a substitution in the light chain CDR3 at position 102 of the light variable region, e.g., a substitution of a cysteine to tyrosine, or a cysteine to serine residue, at position 102 of the light variable region according to Table 1 (e.g., SEQ ID NO: 54 or 70 for a modified sequence).
  • the anti-PD-1 antibody molecule includes at least one, two, three, four, five or six CDRs (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 .
  • one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 .
  • the anti-PD-1 antibody molecule includes all six CDRs from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 , or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).
  • the anti-PD-1 antibody molecule may include any CDR described herein.
  • the anti-PD-1 antibody molecule includes a substitution in a light chain CDR, e.g., one or more substitutions in a CDR1 , CDR2 and/or CDR3 of the light chain.
  • the anti- PD-1 antibody molecule includes a substitution in the light chain CDR3 at position 102 of the light variable region, e.g., a substitution of a cysteine to tyrosine, or a cysteine to serine residue, at position 102 of the light variable region according to Table 1 .
  • the anti-PD-1 antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat et al. definition as set out in Table 1) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat e
  • the anti-PD-1 antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat et al.
  • an antibody described herein e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Table 1 .
  • an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.
  • the anti-PD-1 antibody molecule includes at least one, two, three, four, five, or six CDRs according to Kabat et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Kabat et al.
  • an antibody described herein e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six CDRs according to Kabat et al. shown in Table 1 .
  • substitutions, deletions, or insertions e.g., conservative substitutions
  • the anti-PD-1 antibody molecule includes all six CDRs according to Kabat et al. (e.g., all six CDRs according to the Kabat et al. definition as set out in Table 1 ) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to Kabat et al. shown in Table 1 .
  • the anti-PD-1 antibody molecule includes at least one, two, or three Chothia hypervariable loops (e.g., at least one, two, or three hypervariable loops according to the Chothia et al. definition as set out in Table 1 ) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone- B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or at least the amino acids from those hypervariable loops that contact PD-1 ; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three hypervariable loops according to Chothia et al. shown in Table 1 .
  • Chothia hypervariable loops e.g., at least one,
  • the anti-PD-1 antibody molecule includes at least one, two, or three Chothia hypervariable loops (e.g., at least one, two, or three hypervariable loops according to the Chothia et al. definition as set out in Table 1 ) of a light chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or at least the amino acids from those hypervariable loops that contact PD-1 ; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three hypervariable loops according to Chothia et al. shown in Table 1 .
  • Chothia hypervariable loops e.g., at least one,
  • the anti-PD-1 antibody molecule includes at least one, two, three, four, five, or six hypervariable loops (e.g., at least one, two, three, four, five, or six hypervariable loops according to the Chothia et al.
  • an antibody described herein e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or at least the amino acids from those hypervariable loops that contact PD-1 ; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five or six hypervariable loops according to Chothia et al. shown in Table 1 .
  • an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or at least the amino acids from those hypervariable loops that contact PD-1 ; or which have at least one amino acid alteration, but not more
  • the anti-PD-1 antibody molecule includes all six hypervariable loops (e.g., all six hypervariable loops according to the Chothia et al. definition as set out in Table 1) of an antibody described herein, e.g., an antibody chosen from any of BAP049- Clone-B or BAP049-Clone-E, or closely related hypervariable loops, e.g., hypervariable loops which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions); or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions); or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative
  • the anti-PD-1 antibody molecule may include any one or more amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: amino acids having the following properties: (N-PD-1 antibody molecule, amino acids, amino acids, amino acids, amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the following amino acids having the
  • the anti-PD-1 antibody molecule includes at least one, two, or three hypervariable loops that have the same canonical structures as the corresponding hypervariable loop of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E, e.g., the same canonical structures as at least loop 1 and/or loop 2 of the heavy and/or light chain variable domains of an antibody described herein. See, e.g., Chothia et al. , (1992) J. Mol. Biol. 227:799-817; Tomlinson et a/. , (1992) J. Mol. Biol. 227:776-798 for descriptions of hypervariable loop canonical structures. These structures can be determined by inspection of the tables described in these references.
  • the anti-PD-1 antibody molecule includes a combination of
  • the anti-PD-1 antibody molecule includes at least one, two or three CDRs or hypervariable loops from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone- E, according to the Kabat et al. and Chothia et al. definition (e.g., at least one, two, or three CDRs or hypervariable loops according to the Kabat et al. and Chothia et al.
  • the anti-PD-1 antibody molecule can include VH CDR1 according to Kabat et al. or VH hypervariable loop 1 according to Chothia et al. , or a combination thereof, e.g., as shown in Table 1 .
  • the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO: 224), or an amino acid sequence substantially identical thereto (e.g., having at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions)).
  • the anti-PD-1 antibody molecule can further include, e.g., VH CDRs 2-3 according to Kabat et al. and VL CDRs 1 -3 according to Kabat et al., e.g., as shown in Table 1 .
  • framework regions are defined based on a combination of CDRs defined according to Kabat et al. and hypervariable loops defined according to Chothia et al.
  • the anti-PD-1 antibody molecule can include VH FW1 defined based on VH hypervariable loop 1 according to
  • the anti-PD-1 antibody molecule can further include, e.g., VH FWs 3-4 defined based on VH CDRs 2-3 according to Kabat et al. and VL FWs 1 -4 defined based on
  • VL CDRs 1 -3 according to Kabat et al.
  • the anti-PD-1 antibody molecule includes at least one, two or three CDRs from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E, according to the Kabat et al. and Chothia et al. definition (e.g., at least one, two, or three CDRs according to the
  • the anti-PD-1 antibody molecule includes
  • VH heavy chain variable region
  • VL light chain variable region
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ
  • VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33; or
  • VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ
  • VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32.
  • the anti-PD-1 antibody molecule includes:
  • VH heavy chain variable region
  • SEQ ID NO: 4 a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
  • VL light chain variable region
  • VH comprising a HCDR1 amino acid sequence chosen from SEQ ID NO: 1 ;
  • HCDR2 amino acid sequence of SEQ ID NO: 2 and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
  • VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32.
  • the anti-PD-1 antibody molecule comprises (i) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence chosen from SEQ ID NO: 1 , SEQ ID NO: 4, or SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and (ii) a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 or SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33.
  • the anti-PD-1 antibody molecule comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72.
  • the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-B or BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049- Clone-B or BAP049-Clone-E as described in Table 1 .
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 .
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-1 antibody molecule is selected from Nivolumab (Opdivo), Pembrolizumab (Keytruda) and Pidilizumab.
  • a preferred anti-PD-1 antibody molecule of the present combination is a molecule designated PDR001 , also referred to herein as Antibody Molecule A.
  • PDR001 is described in USSN 14/604,415, entitled “Antibody Molecules to PD-1 and Uses Thereof," and WO/2015/1 12900, Based on preliminary data, PDR001 is well tolerated, with a safety profile similar to those of other marketed anti-PD-1 antibodies. The combination of the present invention is therefore also expected to be well tolerated.
  • the combination of the invention comprises an anti-PD-1 antibody molecule designated PDR001 and comprising a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in
  • a preferred pharmaceutical combination is a pharmaceutical combination comprising an anti-PD-1 antibody molecule designated PDR001 and FGF401 , or a pharmaceutically acceptable salt thereof.
  • the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule designated PDR001 and comprising a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form.
  • VH heavy chain variable region
  • VL light chain variable region
  • the combinations of the invention are believed to result in one or more of: an increase in antigen presentation, an increase in effector cell function (e.g., one or more of T cell proliferation, IFN- ⁇ secretion or cytolytic function), inhibition of regulatory T cell function, an effect on the activity of multiple cell types, such as regulatory T cell, effector T cells and NK cells), an increase in tumor infiltrating lymphocytes, an increase in T-cell receptor mediated proliferation, and a decrease in immune evasion by cancerous cells.
  • the use of anti-PD-1 antibody molecule in the combination of the invention inhibits, reduces or neutralizes one or more activities of PD-1 , resulting in blockade or reduction of an immune checkpoint.
  • the combination of the invention can be used to treat disorders where enhancing an immune response in a subject is desired.
  • a method of modulating an immune response in a subject comprises administering to the subject in need thereof a combination of the invention (e.g., a combination comprising a therapeutically effective amount of an anti-PD-1 antibody molecule with a therapeutically effective amount of N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in
  • a combination of the invention e.g., a combination comprising a therapeutically effective amount of an anti-PD-1 antibody molecule with a therapeutically effective amount of N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1
  • the anti-PD-1 antibody molecule enhances, stimulates or increases the immune response in the subject.
  • the subject or patient is a human (e.g., a patient having a disorder described herein).
  • the subject is in need of enhancing an immune response.
  • the subject has a disorder described herein, e.g., a cancer, in particular liver cancer.
  • the subject is, or is at risk of being, immunocompromised.
  • the subject is undergoing or has undergone a chemotherapeutic treatment and/or radiation therapy.
  • the subject is, or is at risk of being, immunocompromised as a result of an infection.
  • administering or administration is to a subject in need thereof, and that such subject includes each subject stated in the foregoing paragraph; for example, a subject having cancer.
  • the embodiments of the invention include one or more methods of treating cancer comprising administering to a subject in need thereof a pharmaceutical combination comprising an anti-PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form.
  • a method of treating e.g., one or more of reducing, inhibiting, or delaying progression
  • the method comprises administering to the subject a combination of the invention (e.g., a combination comprising a therapeutically effective amount of an anti-PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form).
  • a combination of the invention e.g., a combination comprising a therapeutically effective amount of an anti-PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin
  • a method of treating cancer in a subject comprising administering to said subject simultaneously or sequentially N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • HCDR2 amino acid sequence of SEQ ID NO: 2 and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a
  • HCDR2 amino acid sequence of SEQ ID NO: 5 and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
  • VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224 a
  • HCDR2 amino acid sequence of SEQ ID NO: 2 and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • a method of treating cancer in a subject comprising administering to said subject simultaneously or sequentially N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ
  • VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
  • VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ
  • VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • SEQ ID NO: 70 or (f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72.
  • the cancer treated with the combination includes but is not limited to lung cancer, melanoma, renal cancer, liver cancer, breast cancer, colorectal cancer, thyroid cancer, cholangiocarcinoma, glioblastoma, prostate cancer,
  • rhabdomyosarcoma gastric cancer, or ovarian cancer.
  • the cancer is liver cancer.
  • the treatment is a first-line or a second-line treatment. In one embodiment, the treatment is a second-line treatment. In another embodiment, the treatment is a first-line treatment.
  • second-line treatment refers to a treatment that is given when an initial treatment (first-line treatment) does not work or no longer works.
  • the pharmaceutical combination may be particularly useful in treating hepatocellular carcinoma (HCC), e.g. advanced HCC.
  • HCC hepatocellular carcinoma
  • the pharmaceutical combination may be useful for subjects suffering from HCC, including advanced HCC, who have received prior treatment with sorafenib and progressed during or after discontinuation of sorafenib treatment.
  • Subjects suffering from HCC, including advanced HCC, who are intolerant to sorafenib treatment may also benefit from the administration of the pharmaceutical combination.
  • the hepatocellular carcinoma (HCC) to be treated by a pharmaceutical combination of the invention may be optionally characterized by positive FGFR4 and KLB expression.
  • the HCC to be treated may be characterized by positive FGFR4 and FGF19 expression or by positive FGFR4 and KLB expression.
  • the HCC to be treated is characterized by positive FGF19 expression.
  • the biomarkers FGFR4, KLB and FGF19 can be assayed using any method known in the art such as reverse Transcriptase PCR (RT-PCR), see, e.g. WO/2017/054483, published on 7th April 2016.
  • RT-PCR reverse Transcriptase PCR
  • the combination of the invention provides at least one of the following benefits:
  • improved safety profiles improved toxicity profiles, improved efficacy, well tolerated by subjects suffering from cancer, improved overall response rate, improved overall survival, improved time to progression, tumor growth inhibition, decreased tumor volume, inhibition of cell proliferation, immunomodulatory activity, reduction in relapse time, apoptosis of cancer cells, increase in the time delay for the onset of exponential growth of tumor, reduction in resistance to treatment, synergistic effect leading to reduction of required dosage and/or reduction of side effects leading to an enhancement of the long-term clinical effectiveness of the combination.
  • each active ingredient of the combination of the invention may be administered by different routes.
  • the anti-PD-1 antibody molecule is typically administered to the subject systemically (e.g. subcutaneously, intravenously, intramuscularly or
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl- 2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable form is preferably administered to the subject orally.
  • the anti-PD-1 antibody molecule used in the pharmaceutical combination of the invention is formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. , antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22 nd Ed. Pharmaceutical Press, 2013, pp. 1049-1070). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • the carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
  • the anti-PD-1 antibody molecule used in the present invention may, thus, be in a variety of forms. These include, for example, liquid or semi-solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions e.g., acetylcholine
  • the preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions.
  • the preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the anti-PD-1 antibody molecule disclosed herein is administered by intravenous infusion or injection.
  • the anti-PD-1 antibody molecule disclosed herein is administered by intramuscular or subcutaneous injection.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • compositions typically should be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution,
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the anti-PD-1 antibody molecule) in the required amount in an appropriate solvent with one or a combination of further carriers, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other carriers.
  • the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • An anti-PD-1 antibody molecule described herein can be formulated into a formulation (e.g., a dosage form) for administration (e.g., intravenous administration) to a subject.
  • a formulation e.g., a dosage form
  • administration e.g., intravenous administration
  • the formulation is a drug substance formulation.
  • the formulation is a lyophilized formulation, e.g., lyophilized from a drug substance formulation.
  • the formulation is a reconstituted formulation, e.g., reconstituted from a lyophilized formulation.
  • the formulation is a liquid formulation.
  • the formulation (e.g., drug substance formulation) comprises the anti-PD-1 antibody molecule and a buffering agent.
  • the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 10 to 50 mg/mL, e.g., 15 to 50 mg/mL, 20 to 45 mg/mL, 25 to 40 mg/mL, 30 to 35 mg/mL, 25 to 35 mg/mL, 30 to 40 mg/mL, e.g., 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 33.3 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, or 50 mg/mL.
  • the anti-PD-1 antibody molecule is present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL.
  • the formulation (e.g., drug substance formulation) comprises a buffering agent comprising histidine (e.g., a histidine buffer).
  • the buffering agent e.g., histine buffer
  • the buffering agent is present at a concentration of 1 mM to 20 mM, e.g., 2 mM to 15 mM, 3 mM to 10 mM, 4 mM to 9 mM, 5 mM to 8 mM, or 6 mM to 7 mM, e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 6.7 mM, 7 mM, 8 mM, 9 mM, 10 mM, 1 1 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM.
  • the buffering agent (e.g., histine buffer) is present at a concentration of 6 mM to 7 mM, e.g., 6.7 mM.
  • the buffering agent e.g., a histidine buffer
  • the buffering agent has a pH of 4 to 7, e.g., 5 to 6, e.g., 5, 5.5 or 6.
  • the buffering agent e.g., histidine buffer
  • the buffering agent comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5).
  • the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL; and a buffering agent that comprises histidine at a
  • concentration of 6 mM to 7 mM e.g., 6.7 mM
  • pH of 5 to 6 e.g., 5.5
  • the formulation (e.g., drug substance formulation) further comprises a carbohydrate.
  • the carbohydrate is sucrose.
  • the carbohydrate (e.g., sucrose) is present at a concentration of 50 mM to 150 mM, e.g., 25 mM to 150 mM, 50 mM to 100 mM, 60 mM to 90 mM, 70 mM to 80 mM, or 70 mM to 75 mM, e.g., 25 mM, 50 mM, 60 mM, 70 mM, 73.3 mM, 80 mM, 90 mM, 100 mM, or 150 mM.
  • the formulation comprises a carbohydrate or sucrose present at a concentration of 70 mM to 75 mM, e.g., 73.3 mM.
  • the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL; a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a carbohydrate or sucrose present at a concentration of 70 mM to 75 mM, e.g., 73.3 mM.
  • an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL
  • a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5)
  • a carbohydrate or sucrose present at a concentration of 70 mM to 75
  • the formulation (e.g., drug substance formulation) further comprises a surfactant.
  • the surfactant is polysorbate 20.
  • the surfactant or polysorbate 20) is present at a concentration of 0.005 % to 0.025% (w/w), e.g., 0.0075% to 0.02% or 0.01 % to 0.015% (w/w), e.g., 0.005%, 0.0075%, 0.01 %, 0.013%, 0.015%, or 0.02% (w/w).
  • the formulation comprises a surfactant or polysorbate 20 present at a concentration of 0.01 % to 0.015%, e.g., 0.013% (w/w).
  • the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL; a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a surfactant or polysorbate 20 present at a concentration of 0.01 % to 0.015%, e.g., 0.013% (w/w).
  • an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL
  • a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5)
  • a surfactant or polysorbate 20 present at a concentration of 0.01
  • the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL; a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); a carbohydrate or sucrose present at a concentration of 70 mM to 75 mM, e.g., 73.3 mM; and a surfactant or polysorbate 20 present at a concentration of 0.01 % to 0.015%, e.g., 0.013% (w/w).
  • a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5)
  • a carbohydrate or sucrose present at a concentration of 70 mM
  • the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 33.3 mg/mL; a buffering agent that comprises histidine at a concentration of 6.7 mM and has a pH of 5.5; sucrose present at a concentration of 73.3 mM; and polysorbate 20 present at a concentration of 0.013% (w/w).
  • the formulation is a lyophilized formulation.
  • the lyophilized formulation is lyophilized from a drug substance formulation described herein. For example, 2 to 5 mL, e.g., 3 to 4 mL, e.g., 3.6 mL, of the drug substance formulation described herein can be filled per container (e.g., vial) and lyophilized.
  • the lypophilized formulation is reconstituted with 0.5 mL to 2 mL, e.g., 1 mL, of water or buffer for injection. In certain embodiments, the lypophilized formulation is reconstituted with 1 mL of water for injection, e.g., at a clinical site.
  • the formulation (e.g., reconstituted formulation) comprises the PD-1 inhibitor (e.g., the anti-PD-1 antibody molecule) and a buffering agent.
  • the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 20 mg/mL to 200 mg/mL, e.g., 50 mg/mL to 150 mg/mL, 80 mg/mL to 120 mg/mL, or 90 mg/mL to 1 10 mg/mL, e.g., 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 1 10 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, or 200 mg/mL.
  • the PD-1 inhibitor e.g., the anti-PD-1 antibody molecule
  • the PD-1 inhibitor is present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL.
  • the formulation (e.g., reconstituted formulation) comprises a buffering agent comprising histidine (e.g., a histidine buffer).
  • the buffering agent e.g., histine buffer
  • the buffering agent is present at a concentration of 5 mM to 100 mM, e.g., 10 mM to 50 mM, 15 mM to 25 mM, e.g., 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.
  • the buffering agent (e.g., histine buffer) is present at a concentration of 15 mM to 25 mM, e.g., 20 mM.
  • the buffering agent e.g., a histidine buffer
  • the buffering agent has a pH of 4 to 7, e.g., 5 to 6, e.g., 5, 5.5 or 6.
  • the buffering agent e.g., histidine buffer
  • the buffering agent comprises histidine at a concentration of 15 mM to 25 mM (e.g., 20 mM) and has a pH of 5 to 6 (e.g., 5.5).
  • the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5).
  • the formulation (e.g., reconstituted formulation) further comprises a carbohydrate.
  • the carbohydrate is sucrose.
  • the carbohydrate (e.g., sucrose) is present at a concentration of 100 mM to 500 mM, e.g., 150 mM to 400 mM, 175 mM to 300 mM, or 200 mM to 250 mM, e.g., 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM.
  • the formulation comprises a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
  • the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
  • a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
  • the formulation (e.g., reconstituted formulation) further comprises a surfactant.
  • the surfactant is polysorbate 20.
  • the surfactant or polysorbate 20 is present at a concentration of 0.01 % to 0.1 % (w/w), e.g., 0.02% to 0.08%, 0.025% to 0.06% or 0.03 % to 0.05% (w/w), e.g., 0.01 %, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1 % (w/w).
  • the formulation comprises a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
  • the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
  • a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5)
  • a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
  • the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM; and a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
  • a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); a carbohydrate or sucrose present at a concentration of 200 mM to 250
  • the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6.7 mM and has a pH of 5.5; sucrose present at a concentration of 220 mM; and polysorbate 20 present at a concentration of 0.04% (w/w).
  • the formulation is reconstituted such that an extractable volume of at least 1 mL (e.g., at least 1 .5 mL, 2 mL, 2.5 mL, or 3 mL) of the reconstituted formulation can be withdrawn from the container (e.g., vial) containing the reconstituted formulation.
  • the formulation is reconstituted and/or extracted from the container (e.g., vial) at a clinical site.
  • the formulation e.g., reconstituted formulation
  • the formulation is injected to an infusion bag, e.g., within 1 hour (e.g., within 45 minutes, 30 minutes, or 15 minutes) before the infusion starts to the patient or the subject in need thereof.
  • the formulation is a liquid formulation.
  • the liquid formulation is prepared by diluting a drug substance formulation comprising the anti-PD-1 antibody molecule described herein.
  • a drug substance formulation can be diluted, e.g., 10 to 30 mg/mL (e.g., 25 mg/mL), e.g., with a solution comprising one or more excipients (e.g., concentrated excipients).
  • the solution comprises one, two, or all of histidine, sucrose, or polysorbate 20.
  • the solution comprises the same excipient(s) as the drug substance formulation.
  • the formulation (e.g., liquid formulation) comprises an anti- PD-1 antibody molecule present at a concentration of 5 mg/mL to 50 mg/mL, e.g., 10 mg/mL to 40 mg/mL, 15 mg/mL to 35 mg/mL, or 20 mg/mL to 30 mg/mL, e.g., 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, or 50 mg/mL.
  • the PD-1 inhibitor e.g., the anti-PD-1 antibody molecule
  • the formulation (e.g., liquid formulation) comprises a buffering agent comprising histidine (e.g., a histidine buffer).
  • the buffering agent e.g., histine buffer
  • the buffering agent is present at a concentration of 5 mM to 100 mM, e.g., 10 mM to 50 mM, 15 mM to 25 mM, e.g., 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.
  • the buffering agent e.g., histine buffer
  • the buffering agent is present at a concentration of 15 mM to 25 mM, e.g., 20 mM.
  • other buffering agent e.g., histine buffer
  • the buffering agent e.g., a histidine buffer
  • the buffering agent has a pH of 4 to 7, e.g., 5 to 6, e.g., 5, 5.5 or 6.
  • the buffering agent e.g., histidine buffer
  • the buffering agent comprises histidine at a concentration of 15 mM to 25 mM (e.g., 20 mM) and has a pH of 5 to 6 (e.g., 5.5).
  • the formulation (e.g., liquid formulation) comprises an anti- PD-1 antibody molecule present at a concentration of 20 to 30 mg/mL, e.g., 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5).
  • the formulation (e.g., liquid formulation) further comprises a carbohydrate.
  • the carbohydrate is sucrose.
  • the carbohydrate (e.g., sucrose) is present at a concentration of 100 mM to 500 mM, e.g., 150 mM to 400 mM, 175 mM to 300 mM, or 200 mM to 250 mM, e.g., 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM.
  • the formulation comprises a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
  • the formulation (e.g., liquid formulation) comprises an anti- PD-1 antibody molecule present at a concentration of 20 to 30 mg/mL, e.g., 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
  • a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
  • the formulation (e.g., liquid formulation) further comprises a surfactant.
  • the surfactant is polysorbate 20.
  • the surfactant or polysorbate 20 is present at a concentration of 0.01 % to 0.1 % (w/w), e.g., 0.02% to 0.08%, 0.025% to 0.06% or 0.03 % to 0.05% (w/w), e.g., 0.01 %, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1 % (w/w).
  • the formulation comprises a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
  • PD-1 antibody molecule present at a concentration of 20 to 30 mg/mL, e.g., 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
  • the formulation (e.g., liquid d formulation) comprises an anti-
  • PD-1 antibody molecule present at a concentration of 20 to 30 mg/mL, e.g., 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM; and a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
  • a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM;
  • the formulation (e.g., liquid formulation) comprises an anti- PD-1 antibody molecule present at a concentration of 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6.7 mM and has a pH of 5.5; sucrose present at a concentration of 220 mM; and polysorbate 20 present at a concentration of 0.04% (w/w).
  • 1 mL to 10 mL e.g., 2 mL to 8 mL, 3 mL to 7 mL, or 4 mL to 5 mL, e.g., 3 mL, 4 mL, 4.3 mL, 4.5 mL, 5 mL, or 6 mL
  • the liquid formulation is filled into a container (e.g., vial) such that an extractable volume of at least 2 mL (e.g., at least 3 mL, at least 4 mL, or at least 5 mL) of the liquid formulation can be withdrawn per container (e.g., vial).
  • the liquid formulation is diluted from the drug substance formulation and/or extracted from the container (e.g., vial) at a clinical site.
  • the formulation e.g., liquid formulation
  • the formulation is injected to an infusion bag, e.g., within 1 hour (e.g., within 45 minutes, 30 minutes, or 15 minutes) before the infusion starts to the patient or the subject in need thereof.
  • the container used for any of the formulations described herein can include, e.g., a vial, and optionally, a stopper, a cap, or both.
  • the vial is a glass vial, e.g., a 6R white glass vial.
  • the stopper is a rubber stopper, e.g., a grey rubber stopper.
  • the cap is a flip-off cap, e.g., an aluminum flip- off cap.
  • the container comprises a 6R white glass vial, a grey rubber stopper, and an aluminum flip-off cap.
  • the container (e.g., vial) is for a single-use container.
  • 50 mg to 150 mg e.g., 80 mg to 120 mg, 90 mg to 1 10 mg, 100 mg to 120 mg, 100 mg to 1 10 mg, 1 10 mg to 120 mg, or 1 10 mg to 130 mg, of the PD-1 inhibitor (e.g., the anti-PD-1 antibody molecule), is present in the container (e.g., vial).
  • exemplary buffering agents that can be used in the formulation described herein include, but are not limited to, an arginine buffer, a citrate buffer, or a phosphate buffer.
  • Other exemplary carbohydrates that can be used in the formulation described herein include, but are not limited to, trehalose, mannitol, sorbitol, or a combination thereof.
  • the formulation described herein may also contain a tonixity agent, e.g., sodium chloride, and/or a stabilizing agent, e.g., an amino acid (e.g., glycine, arginine, methionine, or a combination thereof).
  • a stabilizing agent e.g., an amino acid (e.g., glycine, arginine, methionine, or a combination thereof).
  • an amino acid e.g., glycine, arginine, methionine, or a combination thereof.
  • the antibody molecules can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. For example, the antibody molecules can be administered by intravenous infusion at a rate of more than 20 mg/min, e.g.
  • the antibody molecules can be administered by intravenous infusion at a rate of less than 10mg/min; preferably less than or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m 2 , preferably about 5 to 50 mg/m 2 , about 7 to 25 mg/m 2 and more preferably, about 10 mg/m 2 .
  • the route and/or mode of administration will vary depending upon the desired results.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form is preferably formulated for oral administration.
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders).
  • compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc.
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form is in the form of tablets or gelatin capsules comprising N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form together with one or more of:
  • diluents e.g. , lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine
  • lubricants e.g. , silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol
  • binders e.g. , magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and
  • Tablets may be either film coated or enteric coated according to methods known in the art.
  • compositions for oral administration include an effective amount of N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • compositions comprising N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide or a pharmaceutically acceptable salt thereof intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets may contain N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2- yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in citric acid salt form in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
  • Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example, peanut oil, liquid paraffin or olive oil.
  • the pharmaceutical combination of the invention comprises N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form in the form of a tablet.
  • the pharmaceutical combination of the invention comprises N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form in the form of a capsule comprising N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form only.
  • the pharmaceutical combination of the invention comprises N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form in the form of a capsule comprising N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form and a glidant.
  • Glidants are typically used to promote powder flow by reducing interparticle friction and cohesion.
  • Examples include colloidal silicon dioxide (fumed silica), talc and magnesium carbonate.
  • the pharmaceutical composition comprising N-(5-cyano- 4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide or a pharmaceutically acceptable salt thereof includes a glidant, and preferably, the glidant is colloidal silicon dioxide (sold under the trade name aerosil). Dosages of the active ingredients and therapeutic regimens of the combination of the invention can be determined by a skilled artisan.
  • the anti-PD-1 antibody molecule is administered to the subject in need thereof by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg.
  • the dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.
  • the anti-PD-1 antibody molecule is administered at a dose from about 10 to 20 mg/kg every other week.
  • the anti-PD-1 antibody molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose (e.g., a flat dose) of about 200 mg to 500 mg, e.g., about 250 mg to 450 mg, about 300 mg to 400 mg, about 250 mg to 350 mg, about 350 mg to 450 mg.
  • the dosing schedule e.g., flat dosing schedule
  • the anti-PD-1 antibody molecule e.g., the exemplary antibody molecule, is administered at a dose from about 300 mg to 400 mg once every three weeks or once every four weeks.
  • the anti-PD-1 antibody molecule is administered at a dose of 300mg once every three weeks. In an embodiment of the invention, the anti-PD-1 antibody molecule is administered at a dose of 400mg once every four weeks.
  • the anti-PD-1 antibody molecule is administered at a dose from about 300 mg once every four weeks.
  • the anti-PD-1 antibody molecule is administered at a dose from about 400 mg once every three weeks.
  • the anti-PD-1 antibody molecule is administered via i.v. infusion over 30 minutes (up to 2 hours, if clinically indicated) once every 3 weeks.
  • the anti-PD-1 antibody molecule is administered via i.v. infusion over 30 minutes (up to 2 hours, if clinically indicated) once every 4 weeks.
  • a method of treating e.g., one or more of reducing, inhibiting, or delaying progression
  • the method comprises administering, in combination with an FGFR4 inhibitor (e.g.
  • an anti- PD-1 antibody molecule e.g., the preferred anti-PD-1 antibody molecule described herein, at a dose of about 300 mg to 400 mg once every three weeks or once every four weeks.
  • the preferred anti-PD-1 antibody molecule is administered at a dose of about 300 mg once every three weeks.
  • the preferred anti-PD-1 antibody molecule is administered at a dose of about 400 mg once every four weeks.
  • the invention features a method of reducing an activity (e.g., growth, survival, or viability, or all), of a hyperproliferative (e.g., a cancer) cell.
  • the method includes contacting the cell with an anti-PD-1 antibody molecule, e.g., an anti-PD-1 antibody molecule described herein.
  • the method can be performed in a subject, e.g., as part of a therapeutic regimen in combination with an FGFR4 inhibitor, e.g., at a dose of about 300 mg to 400 mg of an anti-PD-1 antibody molecule once every three weeks or once every four weeks.
  • the dose or dosage of the FGFR4 inhibitor as described herein refers to the amount of the free base.
  • the dosage quoted is 80 mg
  • a pharmaceutically acceptable salt of the FGFR4 inhibitor such as the citrate salt thereof
  • the dose of 80mg corresponds to the amount of free base of the FGFR4 inhibitor, namely N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
  • the FGFR4 inhibitor of the combination of the present invention i.e. N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide may be administered at a total daily dose of about 25-200 mg, or about 50-150 mg (e.g., once per day).
  • the total daily dose of N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide (e.g. in citric acid salt form) which is administered may be 50 mg, 80 mg, 100 mg, 120 mg or 150 mg.
  • N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide may be administered at a dose of 80 mg daily or 120 mg daily.
  • the dose of the FGFR4 inhibitor may be administered once daily, or twice daily, or three times daily, or four times daily; preferably once or twice daily, more preferably once daily.
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide e.g. in citric acid salt form, may in particular be administered at a dose of 50 mg once daily, 80 mg once daily, 100 mg once daily, 120 mg once daily or 150 mg once daily.
  • N-(5-cyano- 4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide may be administered at a dose of 80 mg daily or 120 mg daily.
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide e.g. in citric acid salt form, is administered at a dose of 80mg daily, e.g. once daily.
  • the dose of 80mg corresponds to the amount of free base of N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
  • 6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide is administered at a dose of 120 mg daily, e.g. once daily.
  • the dose of 120 mg corresponds to the amount of free base of N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
  • N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form is administered orally.
  • PDR001 Antibody Molecule A
  • PDR001 Antibody Molecule A
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide may be administered at a daily dose of 50 mg, 80 mg, 100 mg, 120 mg or 200 mg.
  • the FGFR4 inhibitor may be administered once or twice daily.
  • PDR001 Antibody Molecule A
  • PDR001 Antibody Molecule A
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2- yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide may be administered at a daily dose of 50 mg, 80 mg, 100 mg, 120 mg or 200 mg.
  • the FGFR4 inhibitor may be administered once or twice daily.
  • the administration of the anti-PD-1 antibody molecule designated PDR001 comprising a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 is at least 1 hour after the administration of N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, e.g.
  • the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in free form or in pharmaceutically acceptable salt form, preferably in citric acid salt form, wherein the anti-PD-1 antibody molecule comprises:
  • VH heavy chain variable region
  • SEQ ID NO: 4 a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
  • VL light chain variable region
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
  • VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in free form or in pharmaceutically acceptable salt form, preferably in citric acid salt form, wherein the anti-PD-1 antibody molecule comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • LCDR1 amino acid sequence of SEQ ID NO: 13 a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
  • VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)- 7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide is administered at a dose of 80mg to 120 mg daily and the anti-PD-1 antibody molecule is administered at a dose of 300mg once every three weeks.
  • the invention provides a kit comprising a pharmaceutical combination comprising an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO:
  • VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO:
  • VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
  • VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
  • the invention provides a kit comprising a pharmaceutical combination comprising an anti-PD-1 antibody molecule designated PDR001 comprising a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form, with instructions for simultaneous or sequential administration of PDR001 and N-(5-cyano
  • the kit comprises two separate pharmaceutical compositions, one of which contains N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form and one of which contains the anti-PD-1 antibody molecule.
  • the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet.
  • An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like.
  • the kit of the invention may be used for administering different dosage forms, for administering the separate compositions at different dosage intervals.
  • the kit of the invention typically comprises directions for administration.
  • both active ingredients be manufactured and/or formulated by the same or different manufacturers.
  • the active ingredients of the combination of the invention may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the combination of the invention); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the subjects in need thereof themselves, e.g. during sequential administration of the active ingredients.
  • Example 1 Characterization of Humanized Anti-PD-1 Antibody
  • Murine anti-PD-1 monoclonal antibody BAP049 was humanized. The sequences and test samples of sixteen humanized BAP049 clones with unique variable region sequences were obtained. These clones were further analyzed for their biological functions (e.g. , antigen binding and ligand blocking), structural features, and transcient expression in CHO cells. Binding affinity and specificity
  • Humanization of BAP049 was performed using a combinatorial library of human germline variable region frameworks (FWs).
  • the technology entails transferring the murine CDRs in frame to a library of human variable regions (VRs) that had been constructed by randomly combining human germ line FW1 , FW2 and FW3 sequences. Only one FW4 sequence was used, which is WGQGTTVTVSS (SEQ ID NO: 67) for the heavy chain (HC) (Kabat human HC subgroup I) and FGQGTKVEIK (SEQ ID NO: 208) for the light chain (LC) (Kabat human ⁇ subgroup I).
  • the library of VR sequences was fused to human constant region (CR) sequences, human lgG4(S228P) of HC and human ⁇ CR of LC, and the resulting library of whole IgG was expressed in CHO cells for screening. Screening was performed with tissue culture supernatants measuring binding avidity on antigen-expressing cells in a whole cell ELISA format or on FACS.
  • CR constant region
  • the humanization process was performed in a stepwise manner starting with the construction and expression of the appropriate chimeric mAb (murine VR, lgG4(S228P), human ⁇ ), which can serve as a comparator for the screening of the humanized clones.
  • the constant region amino acid sequences for human lgG4(S228P) heavy chain and human kappa light chain are shown in Table 3.
  • the library of humanized LC was paired with the chimeric HC (murine VR, lgG4(S228P)) and the resulting "half-humanized" mAbs were screened for binding activity by ELISA.
  • the huLC of clones with adequate binding activity > binding of chimeric mAb
  • the library of humanized HC was paired with the chimeric LC (murine VR, human ⁇ ) and screened for binding activity by ELISA.
  • the huHC of clones with appropriate binding activity > binding of chimeric mAb
  • variable regions of the selected huLC and huHC were then sequenced to identify the huLC and huHC with unique sequences (some clones from the initial selection process may share the same LC or HC).
  • the unique huLC and huHC were then randomly combined to form a small library of humanized mAbs (humAbs), which was expressed in CHO cells and screened on antigen- expressing cells in an ELISA and FACS format. Clones with binding activities that were equal or better than the binding of the chimeric comparator mAb are the final product of the humanization process.
  • chimeric antibody Three variants of the chimeric antibody were prepared that either had a Cys, Tyr or Ser residue at position 102 of the LC sequence.
  • the three chimeric antibodies i.e.,
  • BAP049-chi (Cys), BAP049-chi (Tyr), and BAP049-chi (Ser) (also known as BAP049-chi, BAP049-chi-Y, and BAP049-chi-S, respectively), were expressed in CHO cells and tested for their ability to compete with labeled murine antibody for binding to PD-1 expressing Jurkat cells. The three variants were indistinguishable in the competition experiment. The results show that the three chimeric mAbs (Cys, Tyr, Ser) compete equally well with the binding of the labeled murine mAb BAP049. The slight difference between the chimeric mAb curves and the murine mAb curve is probably due to the different methods used for determining mAb concentrations. The concentration of the murine mAb was determined by OD280 measurement, whereas the chimeric mAb concentrations in supernatants were determined with an ELISA using an lgG4 standard. The germline residue Tyr was selected for humanized antibodies.
  • the process of humanization yielded sixteen clones with binding affinities comparable to that of the chimeric antibody.
  • the VR sequences were provided along with a sample of the mAb.
  • the samples had been prepared by transient transfections of CHO cells and were concentrated tissue culture supernatants.
  • the antibody concentrations in the solutions had been determined by an lgG4-specific ELISA.
  • the sixteen unique clones are combinations of four unique HC sequences and nine unique LC sequences.
  • the HC sequences are combinations of one of two different VHFW1 , one of three different VHFW2, and one of two different VHFW3 sequences.
  • the LC sequences are combinations of one of five different VLFW1 , one of three different VLFW2, and one of four different VLFW3 sequences.
  • the amino acid and nucleotide sequences of the heavy and light chain variable domains for the humanized BAP049 clones B and E are shown in Table 1 .
  • the amino acid and nucleotide sequences of the heavy and light chain CDRs of the humanized BAP049 clones are also shown in Table 1 .
  • the binding activity and specificity was measured in a competition binding assay using a constant concentration of Alexa 488-labeled murine mAb, serial dilutions of the test mAbs, and PD-1 -expressing 300.19 cells. Incubations with the mAb mixtures having different concentration ratios of test mAb to labeled mAb was at 4 °C for 30 min. Bound labeled murine mAb was then quantified using a FACS machine. The experiment was performed twice. Within the accuracy of the experiment, all humanized clones show similar activity for competing with binding of labeled murine mAb. The activity is also comparable to the activity of the parent murine mAb and chimeric mAb. MAbs were ranked relative to each other.
  • Selected clones including clones B and E were further tested for their ability to block the binding of PD-L1 and PD-L2 to PD-1 and for enhancing T cell activity in vitro assays with human PBMC.
  • Murine anti-PD-1 mAb blocks the binding of the natural ligands PD-L1 and PD-L2 to
  • the blocking capacity of the mAbs was evaluated in a competition binding assay using a constant concentration of PD-L1 -hulgG1 Fc fusion protein or PD-L2-hulgG1 Fc fusion protein, serial dilutions of the mAbs to be tested, and PD-1 -expressing 300.19 cells.
  • Single gene vectors were constructed using Lonza's GS Xceed vectors (lgG4proAk for heavy chain and Kappa for light chain). The SGVs were amplified and transiently co-transfected into CHOK1 SV GS-KO cells for expression at a volume of 2.8 L.
  • the sequences of the light and heavy chain variable domain encoding regions were synthesised by GeneArt AG. Light chain variable domain encoding regions were sub-cloned into pXC-Kappa and heavy chain variable domain encoding regions into pXC-lgG4pro ⁇ vectors respectively using the N-terminal restriction site Hind III and the C-terminal restriction sites BsiWI (light chain) and Apal (heavy chain). Positive clones were screened by PCR amplification (primers 1053: GCTGACAGACTAACAGACTGTTCC (SEQ ID NO: 226) and 1072: CAAATGTGGTATGGCTGA (SEQ ID NO: 227)) and verified by restriction digest
  • CHOK1 SV GS-KO cells were cultured in CD-CHO media (Invitrogen, 10743-029) supplemented with 6 mM glutamine (Invitrogen, 25030-123). Cells were incubated in a shaking incubator at 36.5 °C, 5% C0 2 , 85% humidity, 140 rpm. Cells were routinely sub- cultured every 3-4 days, seeding at 2 x 10 5 cells/ml and were propagated in order to have sufficient cells available for transfection. Cells were discarded by passage 20.
  • Transient transfections were performed using CHOK1 SV GS-KO cells which had been in culture a minimum two weeks. Cells were sub-cultured 24 h prior to transfection and cell viability was >99% at the time of transfection.
  • transfections were carried out via electroporation using a Gene Pulse MXCell (Bio-Rad), a plate based system for electroporation.
  • viable cells were resuspended in pre-warmed media to 2.86 x 10 7 cells/ml.
  • 80 ⁇ g DNA (1 :1 ratio of heavy and light chain SGVs) and 700 ⁇ cell suspension were aliquotted into each cuvette/well.
  • Cells were electroporated at 300 V, 1300 ⁇ .
  • Transfected cells were transferred to pre-warmed media in Erienmeyer flasks and the cuvette/wells rinsed twice with pre-warmed media which was also transferred to the flasks.
  • Transfected cell cultures were incubated in a shaking incubator at 36.5 °C, 5% C0 2 , 85% humidity, 140 rpm for 6 days. Cell viability and viable cell concentrations were measured at the time of harvest using a Cedex HiRes automated cell counter (Roche). Protein A Affinity Chromatography
  • Cell culture supernatant was harvested and clarified by centrifugation at 2000 rpm for 10 min, then filtered through a 0.22 ⁇ PES membrane filter. Clarified supernatant was purified using a pre-packed 5 ml HiTrap MabSelect SuRE column (GE Healthcare, 1 1 -0034- 94) on an AKTA purifier (10 ml/min). The column was equilibrated with 50 mM sodium phosphate, 125 mM sodium chloride, pH 7.0 (equilibration buffer) for 5 column volumes (CVs).
  • the column was washed with 2 CVs of equilibration buffer followed by 3 CVs of 50 mM sodium phosphate, 1 M sodium chloride pH 7.0 and a repeat wash of 2 CVs of equilibration buffer.
  • the Product was then eluted with 10 mM sodium formate, pH 3.5 over 5 CVs. Protein containing, eluted fractions were immediately pH adjusted to pH 7.2 and filtered through a 0.2 ⁇ filter.
  • Samples of Protein A purified antibodies were analyzed in duplicate by SE-HPLC on an Agilent 1200 series HPLC system, using a Zorbax GF-250 4 ⁇ 9.4 mm ID x 250 mm column (Agilent). Aliquots of sample at a concentration of 1 mg/ml were filtered through a 0.2 ⁇ filter prior to injection. 80 ⁇ aliquots were injected respectively and run at 1 ml/min for 15 minutes. Soluble aggregate levels were analysed using Chemstation (Agilent) software.
  • Chromatography profiles with retention time showing the percentage of the overall detected peak areas were obtained for the tested antibodies and a control lgG4 antibody.
  • the products show a single protein peak at approximately 8.65 to 8.72 min comparable to the human lgG4 antibody control (about 8.64 min) and consistent with a monomeric antibody.
  • Reduced samples were prepared for analysis by mixing with NuPage 4x LDS sample buffer (Invitrogen, NP0007) and NuPage 10x sample reducing agent (Invitrogen, NP0009), and incubated at 70 °C, 10 min. For non-reduced samples, the reducing agent and heat incubation were omitted. Samples were electrophoresed on 1 .5 mm NuPage 4-12% Bis-Tris Novex pre-cast gels (Invitrogen, NP0335PK2) with NuPage MES SDS running buffer under denaturing conditions.
  • control lgG4 antibody and one tested clone display an additional fainter band corresponding to a heavy plus light chain half- antibody at approximately 70 kDa under non-reducing conditions. This is expected for the control antibody.
  • Two bands were observed under reducing conditions consistent with the size of heavy (close to the position of the 49 kDa marker) and light chains (close to the position of the 28 kDa marker) and comparable with the bands found for the control lgG4 antibody.
  • Non-reduced samples of Protein A purified antibody were electrophoresed as described below.
  • the tested clones show charge isoforms between pH 7.4 and 8.0 markers.
  • the detected charge isoforms are slightly more basic than the theoretically calculated pis for these antibodies which were predicted to be between 6.99 and 7.56.
  • the general shift to more basic charge isoforms suggests the presence of post-translational modifications such as glycosylation on the molecules.
  • Clone C and Clone E show comparable charge isoforms, which is also consistent with the theorectically calculated pi being the same for both (6.99).
  • the control lgG4 antibody behaved as expected.
  • the binding of the same humanized anti-PD-1 antibody on human PD-1 -expressing 300.19 cells was measured using FACS analysis.
  • the result shows that the anti-PD-1 antibody (human lgG4) binds with high affinity to human PD-1 compared to a human lgG4 isotype control.
  • the exemplary humanized anti-PD-1 antibody was found to exhibit high affinity to cynomolgus PD-1 protein and cynomolgus PD-1 -expressing 300.19 cells. As measured by Biacore method, the anti-PD-1 antibody binds to cynomolgus PD-1 with a K D of 0.093 ⁇ 0.015 nM. The binding affinity to cynomolgus PD-1 is comparable to its binding affinity to human PD-1 .
  • the ability of the exemplary humanized anti-PD-1 antibody to block the interactions between PD-1 and both of its known ligands, PD-L1 and PD-L2 was examined.
  • the results show that the anti-PD-1 antibody blocked the binding of PD-L1 and PD-L2 on human PD-1 - expressing 300.19 cells compared to human lgG4 isotype control and no antibody control.
  • the anti-PD-1 antibody blocked PD-L1 binding on the 300.19 cells with an IC50 of 0.94 ⁇ 0.15 nM.
  • the same antibody blocked PD-L2 binding on the 300.19 cells with an IC50 of 1 .3 ⁇ 0.25 nM.
  • SEB Staphylococcal enterotoxin B
  • Example 2 A phase lb/11, multicenter, open-label study of oral FGF401 in combination with an anti-PD-1 antibody molecule ("Antibody Molecule A", detailed below) in adult patients with hepatocellular carcinoma (HCC).
  • the investigational drugs are FGF401 and Antibody Molecule A, an anti-PD-1 receptor recombinant humanized monoclonal antibody, designated PDR001 .
  • the exemplary antibody molecule, Antibody A, (BAP049-Clone-E) tested in this study is a humanized anti-programmed death-1 (PD-1) lgG4 monoclonal antibody (mAb) that blocks binding of programmed cell death ligand-1 (PD-L1) and programmed cell death ligand-2 (PD-L2) to PD-1 . It binds to PD-1 with high affinity and inhibits its biological activity.
  • PD-1 humanized anti-programmed death-1
  • mAb monoclonal antibody
  • amino acid sequences of this antibody molecule are described in Table 1 herein.
  • Antibody Molecule A is in the form of lyophilisate in vial for i.v. infusion.
  • the starting dose is 300 mg, and it is administered every 3 weeks (Q3W).
  • Antibody Molecule A is administered via i.v. infusion over 30 minutes (up to 2 hours, if clinically indicated) once every 3 weeks.
  • the next scheduled dose may be delayed by up to 7 days to recover from previous AEs (adverse events). If the next dose cannot be administered within the above mentioned 7-days delay, then the assessments should be shifted accordingly.
  • FGF401 FGF401
  • FGF401 corresponds to N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl- 6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
  • FGF401 may be used in citric acid salt form.
  • FGF401 may be given in the form of capsules.
  • the starting dose is 80mg of FGF401 (amount of free base) and it is administered once daily.
  • FGF401 may also be administered at a total daily dose of 50 mg, 80 mg, 100 mg, 120 mg or 150 mg.
  • Preferred dosages include 50 mg, 80 mg and 120 mg.
  • Phase I To estimate the MTD (maximum tolerated dose) and/or RP2D (recommended phase II dose) of FGF401 in combination with PDR001
  • Child-Pugh status must be calculated based on clinical findings and laboratory results during the Screening period.
  • hematopoietic colony-stimulating growth factors e.g. G-
  • erythroid stimulating agents ⁇ 2 weeks prior to start of study treatment. If erythroid stimulating agents were initiated more than 2 weeks prior to the first dose of study treatment and the patient is on a stable dose, they can be maintained.
  • TTP time to tumor progression
  • PFS progression free survival
  • OS overall survival
  • BOR best overall response
  • DCR disease control rate
  • Endpoints may also be determined by using Guidelines for immune-related Response Criteria (irRC). Table 1. Amino acid and nucleotide sequences for humanized antibody molecules.
  • the antibody molecules include BAP049-Clone-B and BAP049-Clone-E. The amino acid and nucleotide sequences of the heavy and light chain CDRs, the heavy and light chain variable regions, and the heavy and light chains are shown.
  • SEQ ID NO: 106 DNA VL AGAAGAACTTCCTGACCTGGTATCAG CAGAAGCCCGGTCAAGCCCCTAGACT
  • SEQ ID NO: 135 (Kabat) HCDR3 TGGACTACCGGCACAGGCGCCTAC

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Abstract

The present invention relates to the combination of an anti-PD-1 antibody molecule and an FGFR4 inhibitor and to the use of said combination in the treatment of cancer, in particular liver cancer.

Description

Combination comprising a PD-1 antagonist and an FGFR4 inhibitor Seguence listing
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 13, 2016, is named PAT057381_SL.txt and is 67,823 bytes in size.
Field of the invention
The present invention relates to a pharmaceutical combination comprising an anti-
PD1 antibody molecule and an FGFR4 inhibitor, in particular N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, to the use of said pharmaceutical combination in the treatment of cancer, including liver cancer, and to methods of treatment of cancer, including liver cancer, comprising administering said pharmaceutical combination.
Background
The ability of T cells to mediate an immune response against an antigen requires two distinct signaling interactions (Viglietta, V. et al. (2007) Neurotherapeutics 4:666-675;
Korman, A. J. et al. (2007) Adv. Immunol. 90:297-339). First, an antigen that has been arrayed on the surface of antigen-presenting cells (APC) is presented to an antigen-specific naive CD4+ T cell. Such presentation delivers a signal via the T cell receptor (TCR) that directs the T cell to initiate an immune response specific to the presented antigen. Second, various co-stimulatory and inhibitory signals mediated through interactions between the APC and distinct T cell surface molecules trigger the activation and proliferation of the T cells and ultimately their inhibition.
The immune system is tightly controlled by a network of costimulatory and co- inhibitory ligands and receptors. These molecules provide the second signal for T cell activation and provide a balanced network of positive and negative signals to maximize immune responses against infection, while limiting immunity to self (Wang, L. et al. (Epub Mar. 7, 201 1) J. Exp. Med. 208(3):577-92; Lepenies, B. et al. (2008) Endocrine, Metabolic & Immune Disorders-Drug Targets 8:279-288). Examples of costimulatory signals include the binding between the B7.1 (CD80) and B7.2 (CD86) ligands of the APC and the CD28 and CTLA-4 receptors of the CD4+ T-lymphocyte (Sharpe, A. H. et al. (2002) Nature Rev.
Immunol. 2:1 16-126; Lindley, P. S. et al. (2009) Immunol. Rev. 229:307-321 ). Binding of B7.1 or B7.2 to CD28 stimulates T cell activation, whereas binding of B7.1 or B7.2 to CTLA- 4 inhibits such activation (Dong, C. et al. (2003) Immunolog. Res. 28(1):39-48; Greenwald, R. J. et al. (2005) Ann. Rev. Immunol. 23:515-548). CD28 is constitutively expressed on the surface of T cells (Gross, J., et al. (1992) J. Immunol. 149:380-388), whereas CTLA-4 expression is rapidly up-regulated following T-cell activation (Linsley, P. et al. (1996) Immunity 4:535-543).
Other ligands of the CD28 receptor include a group of related B7 molecules, also known as the "B7 Superfamily" (Coyle, A. J. et al. (2001) Nature Immunol. 2(3):203-209; Sharpe, A. H. et al. (2002) Nature Rev. Immunol. 2:1 16-126; Collins, M. et al. (2005) Genome Biol. 6:223.1 -223.7; Korman, A. J. et al. (2007) Adv. Immunol. 90:297-339).
Several members of the B7 Superfamily are known, including B7.1 (CD80), B7.2 (CD86), the inducible co-stimulator ligand (ICOS-L), the programmed death-1 ligand (PD-L1 ; B7-H1), the programmed death-2 ligand (PD-L2; B7-DC), B7-H3, B7-H4 and B7-H6 (Collins, M. et al. (2005) Genome Biol. 6:223.1 -223.7).
The Programmed Death 1 (PD-1) protein is an inhibitory member of the extended CD28/CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) J. Immunol. 170:71 1 -8). Other members of the CD28 family include CD28, CTLA-4, ICOS and BTLA. PD-1 is suggested to exist as a monomer, lacking the unpaired cysteine residue characteristic of other CD28 family members. PD-1 is expressed on activated B cells, T cells, and monocytes.
The PD-1 gene encodes a 55 kDa type I transmembrane protein (Agata et al. (1996) Int Immunol. 8:765-72). Although structurally similar to CTLA-4, PD-1 lacks the MYPPY motif (SEQ ID NO: 236) that is important for B7-1 and B7-2 binding. Two ligands for PD-1 have been identified, PD-L1 (B7-H1) and PD-L2 (B7-DC), that have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 , but do not bind to other CD28 family members. PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9).
PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signals (Ishida, Y. et al. (1992) EMBO J. 1 1 :3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother. 56(5):739-745). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, e.g., a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and/or immune evasion by cancerous cells (Dong et al. (2003) J. Mol. Med. 81 :281 -7; Blank et al. (2005) Cancer Immunol.
Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well (Iwai et al. (2002) Proc. Natl Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257- 66). Therapies involving anti-PD-1 antibodies have been described for instance in WO2015/1 12900. Combination therapies comprising an immunomodulator and a second therapeutic agent for the treatment of cancer have been described in WO2016040892 and WO2016040880 .
Normal growth, as well as tissue repair and remodeling, require specific and delicate control of activating growth factors and their receptors. Fibroblast Growth Factors (FGFs) constitute a family of over twenty structurally related polypeptides that are developmental^ regulated and expressed in a wide variety of tissues. FGFs stimulate proliferation, cell migration and differentiation and play a major role in skeletal and limb development, wound healing, tissue repair, hematopoiesis, angiogenesis, and tumorigenesis (reviewed in Ornitz, Novartis Found Symp 232: 63-76; discussion 76-80, 272-82 (2001)).
The biological action of FGFs is mediated by specific cell surface receptors belonging to the Receptor Protein Tyrosine Kinase (RPTK) family of protein kinases. These proteins consist of an extracellular ligand binding domain, a single transmembrane domain and an intracellular tyrosine kinase domain which undergoes phosphorylation upon binding of FGF. Four FGFRs have been identified to date: FGFR1 (also called Fig, fms-like gene, fit- 2, bFGFR, N-bFGFR or Cekl), FGFR2 (also called Bek-Bacterial Expressed Kinase, KGFR, Ksam, KsamI and Cek3), FGFR3 (also called Cek2) and FGFR4. All mature FGFRs share a common structure consisting of an amino terminal signal peptide, three extracellular immunoglobulin-like domains (Ig domain I, Ig domain II, Ig domain III), with an acidic region between Ig domains (the "acidic box" domain), a transmembrane domain, and intracellular kinase domains (Ullrich and Schlessinger, Cell 61 : 203,1990 ; Johnson and Williams (1992) Adv. Cancer Res. 60: 1 -41). The distinct FGFR isoforms have different binding affinities for the different FGF ligands.
Alterations in FGFRs have been associated with a number of human cancers including myeloma, breast, stomach, colon, bladder, pancreatic and hepatocellular carcinomas. Recently, it was reported that FGFR4 may play an important role in liver cancer in particular (PLoS One, 2012, volume 7, 36713). Other studies have also implicated FGFR4 or its ligand FGF19 in other cancer types including breast, glioblastoma, prostate, rhabdomyosarcoma, gastric, ovarian, lung, colon (Int. J. Cancer 1993; 54:378-382;
Oncogene 2010; 29:1543-1552; Cancer Res 2010; 70:802-812; Cancer Res 201 1 ; 71 :4550- 4561 ; Clin Cancer Res 2004; 10:6169-6178; Cancer Res 2013; 73:2551 -2562; Clin Cancer Res 2012; 18:3780-3790; J. Clin. Invest. 2009; 1 19:3395-3407; Ann Surg Oncol
2010;17:3354-61 ; Cancer 201 1 ; 1 17:5304-13; Clin Cancer Res 2013; 19:809-820; PNAS 2013; 1 10:12426-12431 ; Oncogene 2008; 27:85-97). Hepatocellular carcinoma (HCC) is the seventh most common form of cancer worldwide and the third most common cause of cancer-related deaths. Potentially curative therapies, such as surgical resection, liver transplant or other local treatments, result in survival rates of between 50-70% at 5 years for patients with early stage HCC. However, despite advances in diagnostic techniques and increased surveillance, the majority of HCC cases present with advanced, inoperable tumors. Sorafenib is the only approved drug in HCC with marginal improvement in overall survival (OS) and poor tolerability.
There is thus a need to provide therapies for cancer, including therapies for HCC, which have improved safety profiles, improved toxicity profiles, improved efficacy compared to a standard of care, are well tolerated by patients, offer advantages to patients in terms of overall response rate, overall survival, time to progression, and/or even cure the disease. New therapies for cancer also could benefit such patients if they also provide tumor growth inhibition, decreased tumor volume, inhibition of cell proliferation, immunomodulatory activity, reduction in relapse time, apoptosis of cancer cells, increase in the time delay for the onset of exponential growth of tumor, reduction of required dosage, reduction of side effects, and/or reduction in resistance to treatment.
Given the importance of immune checkpoint pathways in regulating an immune response, the need also exists for developing novel combination therapies that modulate the activity of immunoinhibitory proteins, such as PD-1 , thus leading to activation of the immune system. Such agents can be used, e.g., for cancer immunotherapy and treatment of other conditions, such as chronic infection. Recent clinical studies have highlighted the potential of targeting the PD-L1 /PD-1 pathway in HCC. A pharmaceutical combination comprising a PD-1 antagonist and a second therapeutic agent may therefore be useful in treating a cancer, e.g. HCC.
Summary of the invention
The invention is expected to provide a solution at least to some of the needs outlined above by providing a pharmaceutical combination as defined herein.
A selective FGFR4 inhibitor may potentiate the effect of an immune checkpoint blocker such as an anti-PD-1 inhibitor. In particular, the combined inhibition of the FGFR4 signal with the selective FGFR4 inhibitor described herein, in particular, N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, or a pharmaceutically acceptable salt thereof, and the inhibition of PD-1 with an anti-PD-1 antibody molecule described herein, in particular the exemplary antibody molecule described herein, may be of particular benefit to subjects suffering from a cancer such as HCC, including advanced HCC. The combination of the invention may also have an acceptable and manageable overall toxicity profile. Thus, in a first aspect, the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form.
In another aspect, the invention relates to a pharmaceutical combination as defined herein for use as a medicament.
In a further aspect, the invention relates to a pharmaceutical combination as defined herein for use in the treatment of cancer, in particular in the treatment of liver cancer.
In another aspect, the invention relates to a pharmaceutical combination comprising an FGFR4 inhibitor and anti-PD-1 antibody molecule for the treatment of liver cancer.
In another aspect the invention relates to a method of treating cancer, in particular liver cancer, in a subject in need thereof, comprising administering to said subject simultaneously or sequentially N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule.
In another aspect, the invention relates to a method of treating liver cancer in a subject suffering from liver cancer comprising administering to said subject simultaneously or sequentially an FGFR4 inhibitor and an anti-PD-1 antibody molecule.
In a further aspect, the invention relates to a therapeutic regimen comprising the simultaneous or sequential administration of N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2- yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule. In a particular aspect, the therapeutic regimen is for the treatment of cancer, in particular liver cancer.
In another aspect, the invention relates to a kit comprising a pharmaceutical combination as defined herein with instructions for simultaneous or sequential administration of N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin- 1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in
pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule. Description
The present invention relates to a pharmaceutical combination comprising an anti- PD-1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form.
In the present description, a "pharmaceutical combination" refers to a non-fixed combination.
The term "non-fixed combination" means that the active ingredients, e.g. an anti- PD- 1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form, are both administered to a patient or a subject in need thereof as separate entities either simultaneously or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two active ingredients in the body of the patient or a subject in need thereof.
The skilled person will readily understand that when reference is made to a compound "in free form", the compound is present as the free acid or the free base, and not, e.g., as the salt.
As used herein, the term "treat", "treating" or "treatment" in connection to a disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In yet another embodiment, "treat", "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treat", "treating" or "treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder.
As used herein, the term "a therapeutically effective amount" when referring to the active ingredients present in the combination of the invention refers to the amounts of the active ingredients that, when administered to a subject either simultaneously or sequentially, will elicit a biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression.
In an embodiment, the term "a therapeutically effective amount" when referring to the active ingredients present in the combination of the invention refers to the amounts of the active ingredients that, when administered to a subject either simultaneously or sequentially are effective to provide at least one of the following benefits:
improved safety profiles, improved toxicity profiles, improved efficacy, well tolerated by patients or subjects suffering from cancer, improved overall response rate, improved overall survival, improved time to progression, tumor growth inhibition, decreased tumor volume, inhibition of cell proliferation, immunomodulatory activity, reduction in relapse time, apoptosis of cancer cells, increase in the time delay for the onset of exponential growth of tumor, reduction in resistance to treatment, synergistic effect leading to reduction of required dosage and/or reduction of side effects leading to an enhancement of the long-term clinical effectiveness of the combination.
As used herein, the term "combination of the invention" or "combination of the present invention" refers to a combination comprising an anti-PD-1 antibody molecule and an FGFR4 inhibitor, in particular N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide, in free form or in pharmaceutically acceptable salt form, as defined herein.
The preferred FGFR4 inhibitor in the combination of the invention is N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, or a pharmaceutically acceptable salt thereof (e.g. the citrate salt). This compound is also known as FGF401 and is currently undergoing clinical trials involving HCC patients (see NCT02325739).
As used herein, the term "active ingredient" refers independently to each combination partner, namely the anti- PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form (e.g. the citrate salt form).
As used herein "liver cancer" also refers to hepatocellular carcinoma (HCC).
As used herein, the term "FGFR4" refers to fibroblast growth factor receptor 4, also known as CD334, JTK2, or TKF.
As used herein, the term "FGFR4 inhibitor" is a compound which inhibits FGFR4. The FGFR4 inhibitor is preferably a selective FGFR4 inhibitor.
The term "about" generally means an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error may be within 10%, or within 5% of a given value or range of values.
The combinations and methods of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 85%, 90%, 95% identical or higher to the sequence specified. In the context of an amino acid sequence, the term "substantially identical" is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 85%, 90%. 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.
In the context of nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.
The term "functional variant" refers to polypeptides that have a substantially identical amino acid sequence to the naturally-occurring sequence, or are encoded by a substantially identical nucleotide sequence, and are capable of having one or more activities of the naturally-occurring sequence.
Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").
The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:1 1 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid (SEQ ID NO: 1) molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389- 3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. , XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1 ) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1 % SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45LC, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1 % SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.
It is understood that the anti-PD-1 antibody molecule used in the present invention may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.
The term "amino acid" is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
As used herein the term "amino acid" includes both the D- or L- optical isomers and peptidomimetics.
A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
The terms "polypeptide", "peptide" and "protein" (if single chain) are used
interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. The polypeptide can be isolated from natural sources, can be a produced by recombinant techniques from a eukaryotic or prokaryotic host, or can be a product of synthetic procedures. The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence," and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may be either single-stranded or double-stranded, and if single-stranded may be the coding strand or non-coding (antisense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The nucleic acid may be a recombinant
polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a nonnatural arrangement.
The term "isolated," as used herein, refers to material that is removed from its original or native environment (e.g. , the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated by human intervention from some or all of the co-existing materials in the natural system, is isolated. Such
polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature.
Various aspects of the invention are described in further detail below. Additional definitions are set out throughout the specification.
FGFR4 inhibitors
In an embodiment, the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule and an FGFR4 inhibitor for use in the treatment of liver cancer.
The FGFR4 inhibitor used in the combination of the present invention is preferably a compound that inhibits FGFR4 selectively compared to FGFR1 , FGFR2 and FGFR3. FGFR4 inhibitors may be small molecules or large molecules. Therapies involving FGFR4 blocking antibodies have been described for instance in the following patent applications
WO2009/009173, WO2007/136893, WO2012/138975, WO2010/026291 , WO2008/052798, WO2010/004204, WO2014/105849, WO2014/165287 and WO2016/023894.
WO2014/144737, WO2014/145909, WO2014/01 1900, WO2015/057963, WO2015/057938, WO2015/030021 , WO2015/107171 , WO2015/059668, WO2016/064960, WO2016/134320, WO2016/134314, WO2016/134294 also describe low molecular weight FGFR4 inhibitors. In an embodiment, the FGFR4 inhibitor used in the combination of the present invention is any of the FGFR4 inhibitors described in any of patent applications
WO2009/009173, WO2007/136893, WO2012/138975, WO2010/026291 , WO2008/052798, WO2010/004204, WO2014/105849, WO2014/165287, WO2016/023894, WO2014/144737, WO2014/145909, WO2014/01 1900, WO2015/057963, WO2015/057938, WO2015/030021 , WO2015/107171 , WO2015/059668, WO2016/064960, WO2016/134320, WO2016/134314, and WO2016/134294.
In an embodiment, the FGFR4 inhibitor used in the combination of the present invention is any of the FGFR4 inhibitors described in WO2014/144737, WO2014/145909, WO2014/01 1900, WO2015/057963, WO2015/057938, WO2015/030021 , WO2015/107171 , WO2015/059668, WO2016/064960, WO2016/134320, WO2016/134314, WO2016/134294, in free form or in pharmaceutically acceptable salt form.
In an embodiment, the FGFR4 inhibitor used in the combination of the present invention is a compound as defined in WO2015/059668, in free form or in pharmaceutically acceptable salt form.
In an embodiment, the FGFR4 inhibitor used in the combination of the present invention is a compound of formula (I) in free form or in pharmaceutically acceptable salt form as described in patent application WO2015/059668. Therefore, in one embodiment, the FGFR4 inhibitor used in the combination of the present invention is a compound of formula (I) in free form or in pharmaceutically acceptable salt form
Figure imgf000013_0001
(I)
wherein
V is selected from CH2, O, CH(OH);
W is selected from CH2, CH2CH2, bond; X is C(RX) or N;
Y is C(RY) or N;
Z is CH or N;
wherein when X is N, Y and Z are not N;
wherein when Y is N, X and Z are not N;
wherein when Z is N, X and Y are not N;
Rx is selected from hydrogen, halogen, haloC Csalkyl, cyano, C C6alkyl, hydroxyC C6alkyl;
RY is selected from hydrogen, halogen, C Csalkyl, C^Cealkoxy, hydroxyC Csalkoxy, NRY1RY2, cyano, C CsalkoxyC Csalkoxy, C Csalkoxy-haloC Csalkoxy, di(C
Csalky aminod-Cealkoxy, O-(CH2)0-i-RY3, CRY6RY7, S-Ci-C3alkyl, halod-Cealkoxy optionally substituted with hydroxy;
or
Rx and RY together with the ring to which they are attached form a bicyclic aromatic ring system optionally further comprising one or two heteroatoms selected from N, O, or S, which ring system is optionally substituted with C Csalkyl;
RY1 is hydrogen and
RY2 is selected from Ci-C6alkyl; hydroxyCrCealkyl; haloC Cealkyl optionally substituted with hydroxy; C1-C4alkoxyC1-C6alkyl; haloC CsalkoxyC Cealkyl; (CH2)0-i-RY4; di(C Csalky aminoC Cealkyl substituted with hydroxy; bicycloC5-C8alkyl optionally substituted with hydroxyCrCsalkyl; phenyl substituted with S(0)2-CH(CH3)2; C2-C3alkylsulfonic acid; or
RY1 and RY2 together with the N atom to which they are attached form a saturated or unsaturated non-aromatic 6-membered heterocyclic ring which may contain an O atom, which ring may be substituted once or twice by RY5;
RY3 is selected from quinuclidinyl, a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, or a 5- or 6-membered aromatic heterocyclic ring, which saturated or aromatic heterocyclic ring is optionally substituted with C Csalkyl and/or oxo;
RY4 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O, or S, which ring is optionally substituted with C Csalkyl; RY5 is independently selected from C Csalkyl, hydroxy, d C CsalkyfyaminoC Csalkyl, or
two RY5 attached at the same carbon atom form together with the carbon atom to which they are attached a 5-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is substituted once or more than once with Ci-C3alkyl; R and R together with the carbon atom to which they are attached form a 6- membered saturated or unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N, O or S;
R1 is selected from hydrogen; halogen; Crdalkyl; haloCrdalkyl; hydroxyC C3alkyl; C3-C6cycloalkyl; CH2NR2R3; CH(CH3)NR2R3; d-CsalkoxyC dalkyl; CH2C02H; C(0)H; d-dalkoxy; a 5- or 6-membered saturated heterocyclic or aromatic heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted once or more than once with a group independently selected from d-dalkyl, haloC C3alkyl, oxetanyl or oxo;
R2 is selected from d-dalkyl, di(d-dalkyl)aminod-dalkyl;
R3 is selected from d-dalkyl, C(0)C C3alkyl, C(0)-CH2-OH, C(0)-CH2-0-CH3, C(O)-
CH2-N(CH3)2, S(0)2CH3;
or
R2 and R3 together with the N atom to which they are attached form a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, N- oxide, O or S, which ring may be substituted once or more than once with R4;
R4 is independently selected from d-dalkyl, di(d-dalkyl)amino, C(0)CH3, hydroxy; or
two R4 attached at the same carbon atom form together with the carbon atom to which they are attached a 4-, 5- or 6-membered non-aromatic heterocyclic ring comprising at least one heteroatom selected from N, O or S;
or
two R4 attached at the same ring atom form an oxo group;
R5 is selected from hydrogen or d-dalkyl.
As used herein, the term "d-dalkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and which is attached to the rest of the molecule by a single bond. The term "C1.C 4alkyl" is to be construed accordingly. The term "d-dalkyl" is to be construed accordingly. Examples of d-dalkyl include, but are not limited to, methyl, ethyl, n-propyl, 1 -methylethyl (/so-propyl), n-butyl, n-pentyl and 1 ,1 -dimethylethyl (f-butyl).
As used herein, the term "hydroxyd-dalkyl" refers to a radical of formula -Ra-OH, wherein Ra is C^alkyl as defined above. Examples of hydroxyd-dalkyl include, but are not limited to, hydroxy-methyl, 2-hydroxy-ethyl, 2-hydroxy-propyl, 3-hydroxy-propyl and 5-hydroxy- pentyl. As used herein, the term "C3-C6cycloalkyl" refers to saturated monocyclic hydrocarbon groups of 3-6 carbon atoms. Examples of C3-C6cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
As used herein, the term "C Cealkoxy" refers to a radical of the formula -ORa where Ra is a C^Cealkyl radical as generally defined above. The term "C Csalkoxy" is to be construed accordingly. Examples of C C6alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentoxy, and hexoxy.
As used herein, the term "C1-C4alkoxyC1-C6alkyl" refers to a radical of the formula - Rb-0-Ra where Ra is a C1-C4alkyl radical and Rb is a C C6alkyl radical as defined above. The term "C CsalkoxyC Cealkyl" is to be construed accordingly. The oxygen atom may be bonded to any carbon atom in either alkyl radical. Examples of C1-C4alkoxyC1-C6alkyl include, but are not limited to, methoxy-methyl, methoxy-ethyl, ethoxy-ethyl, 1 -ethoxy-propyl and 2-methoxy-butyl.
"Halogen" or "halo" refers to bromo, chloro, fluoro or iodo.
As used herein, the term "halogenCrCealkyl" or "haloCrCealkyl" refers to C Cealkyl radical, as defined above, substituted by one or more halo radicals, as defined above.
Examples of halogenC Cealkyl include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1 -fluoromethyl-2-fluoroethyl, 3-bromo-2- fluoropropyl and 1 -bromomethyl-2-bromoethyl.
As used herein, the term "haloC Csalkoxy" refers to C C3alkoxy as defined above, substituted by one or more halo radicals, as defined above. Examples of haloC C3alkoxy include, but are not limited to, trifluoromethoxy, difluoromethoxy, trifluoroethoxy.
As used herein, the term "hydroxyC Csalkoxy" refers to a C^Csalkoxy radical as defined above, wherein one of the hydrogen atoms of the ^.Csalkoxy radical is replaced by OH. Examples of hydroxy^.Csalkoxy include, but are not limited to, hydroxymethoxy, hydroxyethoxy.
As used herein, the term "C CsalkoxyC Csalkoxy" refers to a C^Csalkoxy radical as defined above, wherein one of the hydrogen atoms of the C^alkoxy radical is replaced by - O-C Csalkyl. Examples of C CsalkoxyC Csalkoxy include, but are not limited to, methoxymethoxy, ethoxymethoxy.
As used herein, the term "C Csalkoxy-haloC Csalkoxy" refers to a haloC Csalkoxy radical as defined above, wherein one of the hydrogen atoms of the haloC Csalkoxy radical is replaced by -0-C C3alkyl. Examples of C Csalkoxy-haloC Csalkoxy include, but are not limited to, methoxytrifluoropropyloxy.
As used herein, the term "diCC Csalky aminoC Cealkyl" refers to a radical of the formula -Rai-N(Ra2)-Ra2 where Ra1 is a C^C^ky! radical as defined above and each Ra2 is a C Csalkyl radical, which may be the same or different, as defined above. The nitrogen atom may be bonded to any carbon atom in any alkyl radical. As described herein, the "diC CsalkylaminoC Cealkyl" may be substituted with hydroxy.
As used herein, the term "d CrCsalky aminoCrCealkoxy" refers to a radical of the formula -Rai-N(Ra2)-Ra2 where Ra1 is a C Cealkoxy radical as defined above and each Ra2 is a C Csalkyl radical, which may be the same or different, as defined above.
As used herein, the term "6-membered saturated heterocyclic ring comprising one heteroatom selected from N, O or S" includes piperidyl, tetrahydropyranyl and
tetrahydrothiopyranyl.
As used herein, the term "6-membered unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N, O or S" includes, but is not limited to, tetrahydropyridinyl, dihydropyranyl, dihydrothiopyranyl.
As used herein, the term "a 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S" includes as examples, but is not limited to, azetidinyl, oxetanyl, pyrrolidyl, tetrahydrofuryl, tetrahydrothienyl, piperidyl, piperazinyl, tetrahydropyranyl, morpholinyl.
As used herein, the term "5-membered saturated heterocyclic ring" includes as example, but is not limited to, pyrrolidine.
As used herein, the term "a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, O or S" in relation to the embodiments where R2 and R3 together with the N atom to which they are attached form said ring, includes as examples, but is not limited to, pyrrolidine, oxazolidine, piperazine, morpholine, thiomorpholine rings.
As used herein, the term a "4-, 5- or 6-membered non-aromatic heterocyclic ring comprising at least one heteroatom selected from N, O or S" includes 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S as defined herein. It also includes 4-, 5-, or 6-membered unsaturated heterocyclic ring comprising at least one heteroatom selected from N, O or S.
As used herein, the term "bicyclic aromatic ring system optionally further comprising one or two heteroatoms selected from N, O or S" includes, but is not limited to,
imidazopyridine and isothiazolopyridine.
As used herein, the term "bicycloC5-C8alkyl" refers to bicyclic hydrocarbon groups comprising 5 to 8 carbon atoms including, but not limited to, bicyclo[2.1 .1 ]hexyl, bicyclo[1 .1 .1 ]pentyl, bicyclo[2.2.1 ]heptanyl, bicyclo[2.2.2]octyl.
As used herein, the term "optionally substituted" as used in the description of RY, Rx and RY together, RY2, RY3, RY4 includes unsubstituted or substituted once or twice.
As used herein, the term "substituted" as used, for example in the description of RY2, two RY5, includes substituted once or twice, preferably once. As used herein, the term "more than once" when referring to substituent R4, includes 2, 3, 4, 5, or 6 times. Preferably, it includes 2 or 3 times.
In an embodiment, the FGFR4 inhibitor used in the combination of the present invention is a compound in free form or in pharmaceutically acceptable salt form as described in the examples of patent application WO2015/059668.
Therefore, in an embodiment, the FGFR4 inhibitor used in the combination of the present invention is a compound in free form or in pharmaceutically acceptable salt form selected from the group consisting of:
7-formyl-/V-(5-(trifluoromethyl)pyridin-2-yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
/V-(4,5-dichloropyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide; /V-(5-cyanopyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide; /V-(5-chloropyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide; 7-formyl-/V-(pyridin-2-yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide;
/V-(4,5-dimethylpyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide; 7-formyl-/V-(5-methylpyridin-2-yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide; /V-(5-cyanopyrimidin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
6- formyl-/V-(5-methylpyridin-2-yl)-2H-pyrido[3,2-b][1 ,4]oxazine-4(3H)-carboxamide; 6-chloro-/V-(5-cyanopyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
7- formyl-/V-(6-methoxypyrimidin-4-yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide; /V-(5-cyanopyrazin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide; /V-(5-cyano-4-methoxypyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
6-formyl-/V-(5-(trifluoromethyl)pyridin-2-yl)-2H-pyrido[3,2-b][1 ,4]oxazine-4(3H)- carboxamide;
6- fluoro-7-formyl-/V-(5-(trifluoromethyl)pyridin-2-yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
/V-(5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)-7-formyl-3,4-dihydro- 1 ,8-naphthyridine-1 (2H)-carboxamide;
/V-(4,5-dicyanopyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
7- formyl-6-(hydroxymethyl)-/V-(5-(trifluoromethyl)pyridin-2-yl)-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
/V-(5-cyano-4-ethoxypyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide; 7-formyl-6-methyl-A/-(5-(trifluoromethyl)pyridin-2-yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
/V-(5-cyanopyridin-2-yl)-7-formyl-6-methyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
7-formyl-/V-(5-(1 -hydroxypentyl)pyridin-2-yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
/V-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine- 1 (2H)-carboxamide;
/V-(4-chloro-5-cyanopyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
/V-(5-cyano-4-morpholinopyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
A/-(5-cyano-4-(4-hydroxy-4-methylpiperidin-1 -yl)pyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
/V-(5-cyanopyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
A/-(5-cyano-4-(2-methyl-2,8-diazaspiro[4.5]decan-8-yl)pyridin-2-yl)-7-formyl-3,4-dihydro- 1 ,8-naphthyridine-1 (2H)-carboxamide;
/V-(5-cyanopyridin-2-yl)-6-cyclopropyl-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
A/-(5-cyano-4-(3,6-dihydro-2H^yran-4-yl)pyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(tetrahydro-2H^yran-4-yl)pyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-chloro-4-((tetrahydrofuran-3-yl)oxy)pyrimidin-2-yl)-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
/V-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
A/-(5-cyano-4-((tetrahydrofuran-2-yl)methoxy)pyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(oxetan-2-ylmethoxy)pyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine- 1 (2H)-carboxamide;
A/-(5-cyano-4-((tetrahydro-2H^yran-2-yl)methoxy)pyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-cyanopyridin-2-yl)-6-(difluoromethyl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide; A/-(5-cyano-4-(2-(dimethylamino)ethoxy)pyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
/V-(5-cyano-4-((tetrahydrofuran-3-yl)oxy)pyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(4-hydroxy-4-methylpiperidin-1 -yl)pyridin-2-yl)-7-formyl-6-(hydroxymeth 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
7-acetyl-A/-(5-cyanopyridin-2-yl)-6-((dimethylamino)methyl)-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((tetrahydrofuran-2-yl)methoxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-
3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
/V-(5-cyano-4-((tetrahydro-2H-pyran-2-yl)methoxy)pyridin-2-yl)-7-formyl-6-
(hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(2-methyl-2,8-diazaspiro[4.5]decan-8-yl)pyridin-2-yl)-7-formyl-6-
(hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((1 -methylpyrrolidin-3-yl)oxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((1 -methylpiperidin-4-yl)oxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-^ dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((1 -methoxypropan-2-yl)oxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((tetrahydrofuran-3-yl)oxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((tetrahydrofuran-3-yl)oxy)pyridin-2-yl)-6-((dimethylamino)meth
3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
2-(8-((5-cyanopyridin-2-yl)carbamoyl)-2-formyl-5,6,7,8-tetrahydro-1 ,8-naphthyridin-3- yl)acetic acid;
A/-(5-cyano-4-((tetrahydro-2H-pyran-4-yl)oxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((1 -methylpyrrolidin-3-yl)oxy)pyridin-2-yl)-7-formyl-6-(hydroxymethy ^ dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(((tetrahydro-2H-pyran-3-yl)methyl)amino)pyridin-2-yl)-7-formyl-6- (hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((tetrahydrofuran-3-yl)amino)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide; A/-(5-cyano-4-((2-methoxypropyl)amino)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-chloro-4-((1 -methoxypropan-2-yl)oxy)pyrimidin-2-yl)-7-formyl-6-(hydroxymethyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(4-(4-chloro-2-hydroxybutoxy)-5-cyanopyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-6-(trifluoromethyl)-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
/V-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-6-cyclopropyl-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-6-(difluoromethyl)-7-formyl-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-6-((dimethylamino)methyl)-7-formyl-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((3-(hydroxymethyl)bicyclo[2.2.1 ]heptan-2-yl)amino)pyridin-2-yl)-7-formyl-
6- (hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((tetrahydro-2H^yran-4-yl)amino)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-6-(methoxymethyl)-3,4-dihydro- 1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyanopyridin-2-yl)-7-formyl-6-((A/-methylacetamido)methyl)-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-chloro-4-((tetrahydrofuran-3-yl)oxy)pyrimidin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((tetrahydrofuran-3-yl)oxy)pyrimidin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
7- formyl-6-(hydroxymethyl)-A/-(4-((tetrahydrofuran-3-yl)oxy)pyridin-2-yl)-3,4-dihydro-1 ,8- naphthyridine-1 (2H)-carboxamide;
A/-(5-chloro-4-((tetrahydrofuran-3-yl)oxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((1 -methylpiperidin-3-yl)methoxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((1 -methylpyrrolidin-2-yl)methoxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((1 -methylpiperidin-2-yl)methoxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide; A/-(5-fluoropyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
A/-(5-cyano-4-(2-(dimethylamino)ethoxy)pyridin-2-yl)-6-form-13C-yl-2H-pyrido[3,2- b][1 ,4]oxazine-4(3H)-carboxamide;
A/-(5-cyano-4-((1 -methylpiperidin-4-yl)meth^
3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
/V-(5-cyanopyridin-2-yl)-7-formyl-4-hydroxy-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
A/-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-(2-((dimethylamino)methyl)morpholino)pyridin-2-yl)-7-formyl-6-
(hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-(quinuclidin-3-yloxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4-dihydro-
1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin
1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
7-formyl-6-(hydroxymethyl)-N-(4-((2-methoxyethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-(4-((dimethylamino)methyl)-4-hydroxypiperidin-1 -yl)pyridin-2-yl)-7-form (hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-hydroxy-2-methylpropyl)amino)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((3-(dimethylamino)-2-hydroxy-2-methylpropyl)amino)pyridin-2-yl)-7-formy^ 6-(hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-fluoroethyl)amino)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4-dihydro- 1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4-dihydro- 1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-(isopropylamino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((3- oxomorpholino)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((3-hydroxy-2- oxopyrrolidin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide; N-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-ethylpyridin-2-yl)-6,7-diformyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide hydrochloride;
N-(5-cyano-4-((1 -methoxypropan-2-yl)oxy)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxooxazolidin-3-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((3-methyl-5- oxomorpholino)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
6- ((4-acetylpiperazin-1 -yl)methyl)-N-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-3,4- dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide;
A/-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(1 -(/V- methylacetamido)ethyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide;
A/-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-6-((2-hydroxy-A/-methylacetamido)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide;
/V-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-6-((/V-(2-
(dimethylamino)ethyl)acetamido)methyl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
/V-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-6-((/V-(2-
(dimethylamino)ethyl)methylsulfonamido)methyl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine- 1 (2/-/)-carboxamide;
A/-(5-cyano-4-isopropoxypyridin-2-yl)-6-((2-(dimethylamino)-A/-methylacetamido)meth
7- formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide;
A/-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((2-methoxy-/V- methylacetamido)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide;
A/-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((3- oxothiomorpholino)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide;
/V-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-6-((1 ,1 -dioxido-3- oxothiomorpholino)methyl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide; N-(5-cyano-4-(((4-methylmorpholin-2-yl)methyl)amino)pyridin-2-yl)-6-(difluoromethy formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((1 ,1 -trifluoro-3-methoxypropan-2-yl)oxy)pyridin-2-yl)-6-(difluoromethyl)- 7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-(trifluoromethoxy)ethyl)amino)pyridin-2-yl)-7-formyl-6-((N- methylacetamido)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide; 6- (2-oxa-5-azaspiro[3 ]octan-5-ylmethyl)-N-(5-cyano-4-((2-methoxyethyl)amino)pyridin- 2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(4-((2-(tert-butoxy)ethyl)amino)-5-cyanopyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-hydroxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-
1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-(2-hydroxyethoxy)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyanopyridin-2-yl)-2-formyl-7,8-dihydro-5H-pyrido[2,3-b]azepine-9(6H)- carboxamide;
N-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-2-formyl-7,8-dihydro-5H-pyrido[2,3- b]azepine-9(6H)-carboxamide;
N-(5-cyano-4-((1 -methoxypropan-2-yl)oxy)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
7-formyl-N-(1 -isopropyl-1 H-imidazo[4,5-c]pyridin-6-yl)-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
7- formyl-N-(1 -isopropyl-1 H-imidazo[4,5-c]pyridin-6-yl)-6-((2-oxopyrrolidin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
4-((8-((5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)carbamoyl)-2-formyl-5,6,7,8- tetrahydro-1 ,8-naphthyridin-3-yl)methyl)-1 -methyl-3-oxopiperazine 1 -oxide;
N-(5-cyano-4-((2-oxopiperidin-4-yl)methoxy)pyridin-2-yl)-7-formyl-6-((2-oxopyrrolidin-1 ^ yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-2-methyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-2-methyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-2-methyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((3-hydroxy-4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-isopropoxypyridin-2-yl)-6-formyl-2,3-dihydro-1 H-pyrrolo[2,3-b]pyridine-1 - carboxamide;
2- ((5-cyano-2-(7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-1 ,2,3,4-tetrahydro-1 ,8- naphthyridine-1 -carboxamido)pyridin-4-yl)amino)ethyl hydrogen sulfate;
N-(4-(bicyclo[1 .1 .1 ]pentan-1 -ylamino)-5-cyanopyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide; N-(5-cyano-4-(thiophen-2-ylmethoxy)pyridin-2-yl)-7-formyl-6-((N- methylacetamido)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-(isopropylthio)pyridin-2-yl)-7-formyl-6-((N-methylacetamido)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-6-((3,5-dimethylpiperazin-1 - yl)methyl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((3,3,4-trimethyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
6-amino-N-(5-cyanopyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(tetrahydro-2H-pyran-4-yl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-6-(1 ,3-dimethyl-1 H-pyrazol-4-yl)-7- formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(1 -methyl-1 H-pyrazol-4-yl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(2-methylthiazol-5-yl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(thiophen-2-yl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(1 H-imidazol-1 -yl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(pyridin-3-yl)-3,4-dihydro 1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(1 -methyl-1 H-pyrazol-5-yl)-
3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(3-methyl-1 H-1 ,2,4-triazol- 1 -yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(3-methyl-2-oxopyrrolidin- 1 -yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide;
A/-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(3-oxomorpholino)-3,4- dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide;
A/-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(2-oxooxazolidin-3-yl)-3,4- dihydro-1 ,8-naphthyridine-1 (2/-/)-carboxamide;
A/-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-6-(tetrahydrofuran-3-yl)-3,4-dihydro-1 ,8- naphthyridine-1 (2/-/)-carboxamide; N-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-6-(piperidin-4-yl)-3,4-dihydr^ ,8- naphthyridine-1 (2H)-carboxamide;
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(1 -(oxetan-3-yl)pi yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide; and
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-6-(1 -(2,2-difluoroethyl)piperidin-4-yl)- 7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
Patent application WO2015/059668 describes how to make the compounds of formula (I) and the specific compounds described above.
In an embodiment of the invention, the preferred FGFR4 inhibitor used in the combination of the present invention is N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in free form or in pharmaceutically acceptable salt form.
This compound is also known by the code name FGF401 . A preferred salt of the preferred FGFR4 inhibitor is the citrate salt .
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide is described specifically in example 83 of patent application WO2015/059668, which WO2015/059668 is hereby incorporated by reference in its entirety, and has the following structure:
Figure imgf000026_0001
In an embodiment of the invention, the FGFR4 inhibitor used in the combination of the present invention is N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form. In an embodiment of the invention, the FGFR4 inhibitor used in the combination of the present invention is N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form.
Antibody Molecules to PD-1
The present invention relates to a pharmaceutical combination comprising an anti- PD-1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form.
In one embodiment, the anti-PD-1 antibody molecule used in the combination of the present invention is one described in WO2015/1 12900, entitled "Antibody Molecules to PD-1 and Uses Thereof," which is hereby incorporated by reference in its entirety. In one embodiment, the anti-PD-1 antibody molecule comprises at least one antigen-binding region, e.g., a variable region or an antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
In yet another embodiment, the anti-PD-1 antibody molecule comprises at least one, two, three or four variable regions from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
In yet another embodiment, the anti-PD-1 antibody molecule comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
In yet another embodiment, the anti-PD-1 antibody molecule comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
In yet another embodiment, the anti-PD-1 antibody molecule includes a heavy chain constant region for an lgG4, e.g., a human lgG4. In one embodiment, the human lgG4 includes a substitution at position 228 according to EU numbering (e.g., a Ser to Pro substitution). In still another embodiment, the anti-PD-1 antibody molecule includes a heavy chain constant region for an lgG1 , e.g., a human lgG1 . In one embodiment, the human lgG1 includes a substitution at position 297 according to EU numbering (e.g., an Asn to Ala substitution). In one embodiment, the human lgG1 includes a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both (e.g., an Asp to Ala substitution at position 265 and/or a Pro to Ala substitution at position 329). In one embodiment, the human lgG1 includes a substitution at position 234 according to EU numbering, a substitution at position 235 according to EU numbering, or both (e.g., a Leu to Ala substitution at position 234 and/or a Leu to Ala substitution at position 235). In one embodiment, the heavy chain constant region comprises an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.
In yet another embodiment, the anti-PD-1 antibody molecule includes a kappa light chain constant region, e.g., a human kappa light chain constant region. In one embodiment, the light chain constant region comprises an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.
In another embodiment, the anti-PD-1 antibody molecule includes a heavy chain constant region for an lgG4, e.g., a human lgG4, and a kappa light chain constant region, e.g., a human kappa light chain constant region, e.g., a heavy and light chain constant region comprising an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto. In one embodiment, the human lgG4 includes a substitution at position 228 according to EU numbering (e.g., a Ser to Pro substitution). In yet another embodiment, the anti-PD-1 antibody molecule includes a heavy chain constant region for an lgG1 , e.g., a human lgG1 , and a kappa light chain constant region, e.g., a human kappa light chain constant region, e.g., a heavy and light chain constant region comprising an amino sequence set forth in Table 3, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto. In one embodiment, the human lgG1 includes a substitution at position 297 according to EU numbering (e.g., an Asn to Ala substitution). In one embodiment, the human lgG1 includes a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both (e.g., an Asp to Ala substitution at position 265 and/or a Pro to Ala substitution at position 329). In one embodiment, the human lgG1 includes a substitution at position 234 according to EU numbering, a substitution at position 235 according to EU numbering, or both (e.g., a Leu to Ala substitution at position 234 and/or a Leu to Ala substitution at position 235).
In another embodiment, the anti-PD-1 antibody molecule includes a heavy chain variable domain and a constant region, a light chain variable domain and a constant region, or both, comprising the amino acid sequence of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences. The anti-PD-1 antibody molecule, optionally, comprises a leader sequence from a heavy chain, a light chain, or both, as shown in Table 4; or a sequence substantially identical thereto.
In yet another embodiment, the anti-PD-1 antibody molecule includes at least one, two, or three complementarity determining regions (CDRs) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone- B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.
In yet another embodiment, the anti-PD-1 antibody molecule includes at least one, two, or three CDRs (or collectively all of the CDRs) from a heavy chain variable region comprising an amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 . In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 .
In yet another embodiment, the anti-PD-1 antibody molecule includes at least one, two, or three CDRs from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequence.
In yet another embodiment, the anti-PD-1 antibody molecule includes at least one, two, or three CDRs (or collectively all of the CDRs) from a light chain variable region comprising an amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 . In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 . In certain embodiments, the anti-PD-1 antibody molecule includes a substitution in a light chain CDR, e.g., one or more substitutions in a CDR1 , CDR2 and/or CDR3 of the light chain. In one embodiment, the anti-PD-1 antibody molecule includes a substitution in the light chain CDR3 at position 102 of the light variable region, e.g., a substitution of a cysteine to tyrosine, or a cysteine to serine residue, at position 102 of the light variable region according to Table 1 (e.g., SEQ ID NO: 54 or 70 for a modified sequence).
In another embodiment, the anti-PD-1 antibody molecule includes at least one, two, three, four, five or six CDRs (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 . In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 1 , or encoded by a nucleotide sequence shown in Table 1 .
In one embodiment, the anti-PD-1 antibody molecule includes all six CDRs from an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 , or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In one embodiment, the anti-PD-1 antibody molecule may include any CDR described herein. In certain embodiments, the anti-PD-1 antibody molecule includes a substitution in a light chain CDR, e.g., one or more substitutions in a CDR1 , CDR2 and/or CDR3 of the light chain. In one embodiment, the anti- PD-1 antibody molecule includes a substitution in the light chain CDR3 at position 102 of the light variable region, e.g., a substitution of a cysteine to tyrosine, or a cysteine to serine residue, at position 102 of the light variable region according to Table 1 .
In another embodiment, the anti-PD-1 antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat et al. definition as set out in Table 1) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Table 1 . In another embodiment, the anti-PD-1 antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat et al. definition as set out in Table 1) from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Table 1 .
In yet another embodiment, the anti-PD-1 antibody molecule includes at least one, two, three, four, five, or six CDRs according to Kabat et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Kabat et al. definition as set out in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six CDRs according to Kabat et al. shown in Table 1 .
In yet another embodiment, the anti-PD-1 antibody molecule includes all six CDRs according to Kabat et al. (e.g., all six CDRs according to the Kabat et al. definition as set out in Table 1 ) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to Kabat et al. shown in Table 1 . In one embodiment, the anti-PD-1 antibody molecule may include any CDR described herein.
In another embodiment, the anti-PD-1 antibody molecule includes at least one, two, or three Chothia hypervariable loops (e.g., at least one, two, or three hypervariable loops according to the Chothia et al. definition as set out in Table 1 ) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone- B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or at least the amino acids from those hypervariable loops that contact PD-1 ; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three hypervariable loops according to Chothia et al. shown in Table 1 .
In another embodiment, the anti-PD-1 antibody molecule includes at least one, two, or three Chothia hypervariable loops (e.g., at least one, two, or three hypervariable loops according to the Chothia et al. definition as set out in Table 1 ) of a light chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or at least the amino acids from those hypervariable loops that contact PD-1 ; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three hypervariable loops according to Chothia et al. shown in Table 1 .
In yet another embodiment, the anti-PD-1 antibody molecule includes at least one, two, three, four, five, or six hypervariable loops (e.g., at least one, two, three, four, five, or six hypervariable loops according to the Chothia et al. definition as set out in Table 1) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E; or as described in Table 1 , or encoded by the nucleotide sequence in Table 1 ; or at least the amino acids from those hypervariable loops that contact PD-1 ; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five or six hypervariable loops according to Chothia et al. shown in Table 1 .
In one embodiment, the anti-PD-1 antibody molecule includes all six hypervariable loops (e.g., all six hypervariable loops according to the Chothia et al. definition as set out in Table 1) of an antibody described herein, e.g., an antibody chosen from any of BAP049- Clone-B or BAP049-Clone-E, or closely related hypervariable loops, e.g., hypervariable loops which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions); or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative
substitutions) relative to all six hypervariable loops according to Chothia et al. shown in Table 1 . In one embodiment, the anti-PD-1 antibody molecule may include any
hypervariable loop described herein. In still another embodiment, the anti-PD-1 antibody molecule includes at least one, two, or three hypervariable loops that have the same canonical structures as the corresponding hypervariable loop of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E, e.g., the same canonical structures as at least loop 1 and/or loop 2 of the heavy and/or light chain variable domains of an antibody described herein. See, e.g., Chothia et al. , (1992) J. Mol. Biol. 227:799-817; Tomlinson et a/. , (1992) J. Mol. Biol. 227:776-798 for descriptions of hypervariable loop canonical structures. These structures can be determined by inspection of the tables described in these references.
In certain embodiments, the anti-PD-1 antibody molecule includes a combination of
CDRs or hypervariable loops defined according to the Kabat et al. and Chothia et al. as described herein in table 1 .
In one embodiment, the anti-PD-1 antibody molecule includes at least one, two or three CDRs or hypervariable loops from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone- E, according to the Kabat et al. and Chothia et al. definition (e.g., at least one, two, or three CDRs or hypervariable loops according to the Kabat et al. and Chothia et al. definition as set out in Table 1 ); or encoded by the nucleotide sequence in Table 1 ; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs or hypervariable loops according to Kabat and/or Chothia shown in Table 1 .
For example, the anti-PD-1 antibody molecule can include VH CDR1 according to Kabat et al. or VH hypervariable loop 1 according to Chothia et al. , or a combination thereof, e.g., as shown in Table 1 . In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO: 224), or an amino acid sequence substantially identical thereto (e.g., having at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions)). The anti-PD-1 antibody molecule can further include, e.g., VH CDRs 2-3 according to Kabat et al. and VL CDRs 1 -3 according to Kabat et al., e.g., as shown in Table 1 . Accordingly, in some embodiments, framework regions (FW) are defined based on a combination of CDRs defined according to Kabat et al. and hypervariable loops defined according to Chothia et al. For example, the anti-PD-1 antibody molecule can include VH FW1 defined based on VH hypervariable loop 1 according to
Chothia et al. and VH FW2 defined based on VH CDRs 1 -2 according to Kabat et al. , e.g., as shown in Table 1 . The anti-PD-1 antibody molecule can further include, e.g., VH FWs 3-4 defined based on VH CDRs 2-3 according to Kabat et al. and VL FWs 1 -4 defined based on
VL CDRs 1 -3 according to Kabat et al.
In one embodiment, the anti-PD-1 antibody molecule includes at least one, two or three CDRs from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any of BAP049-Clone-B or BAP049-Clone-E, according to the Kabat et al. and Chothia et al. definition (e.g., at least one, two, or three CDRs according to the
Kabat et al. and Chothia et al. definition as set out in Table 1 ).
In one embodiment, the anti-PD-1 antibody molecule includes
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ
ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33; or
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ
ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32.
In one embodiment, the anti-PD-1 antibody molecule includes:
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of
SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence chosen from SEQ ID NO: 1 ; a
HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33; or
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32.
In another embodiment, the anti-PD-1 antibody molecule comprises (i) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence chosen from SEQ ID NO: 1 , SEQ ID NO: 4, or SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and (ii) a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 or SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In another embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70.
In another embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72.
In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-B or BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049- Clone-B or BAP049-Clone-E as described in Table 1 .
In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 .
In one embodiment, the anti-PD-1 antibody molecule is selected from Nivolumab (Opdivo), Pembrolizumab (Keytruda) and Pidilizumab.
A preferred anti-PD-1 antibody molecule of the present combination is a molecule designated PDR001 , also referred to herein as Antibody Molecule A.
PDR001 is described in USSN 14/604,415, entitled "Antibody Molecules to PD-1 and Uses Thereof," and WO/2015/1 12900, Based on preliminary data, PDR001 is well tolerated, with a safety profile similar to those of other marketed anti-PD-1 antibodies. The combination of the present invention is therefore also expected to be well tolerated.
Thus, in one embodiment, the combination of the invention comprises an anti-PD-1 antibody molecule designated PDR001 and comprising a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in
pharmaceutically acceptable salt form.
A preferred pharmaceutical combination is a pharmaceutical combination comprising an anti-PD-1 antibody molecule designated PDR001 and FGF401 , or a pharmaceutically acceptable salt thereof.
Thus, in an embodiment, the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule designated PDR001 and comprising a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form. Uses of the Combination
The combinations of the invention are believed to result in one or more of: an increase in antigen presentation, an increase in effector cell function (e.g., one or more of T cell proliferation, IFN-γ secretion or cytolytic function), inhibition of regulatory T cell function, an effect on the activity of multiple cell types, such as regulatory T cell, effector T cells and NK cells), an increase in tumor infiltrating lymphocytes, an increase in T-cell receptor mediated proliferation, and a decrease in immune evasion by cancerous cells. In one embodiment, the use of anti-PD-1 antibody molecule in the combination of the invention inhibits, reduces or neutralizes one or more activities of PD-1 , resulting in blockade or reduction of an immune checkpoint. Thus, the combination of the invention can be used to treat disorders where enhancing an immune response in a subject is desired.
In ongoing clinical trials, administrating N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide, e.g. in citric acid salt form, to a patient with liver cancer resulted in confirmed tumor shrinkage of 70%. Thus, the combination of the invention may also be used to treat cancers such as liver cancer.
Accordingly, in another aspect, a method of modulating an immune response in a subject is provided. The method comprises administering to the subject in need thereof a combination of the invention (e.g., a combination comprising a therapeutically effective amount of an anti-PD-1 antibody molecule with a therapeutically effective amount of N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in
pharmaceutically acceptable salt form), such that the immune response in the subject is modulated. In one embodiment, the anti-PD-1 antibody molecule enhances, stimulates or increases the immune response in the subject.
As described herein, the subject or patient is a human (e.g., a patient having a disorder described herein). In one embodiment, the subject is in need of enhancing an immune response. In one embodiment, the subject has a disorder described herein, e.g., a cancer, in particular liver cancer. In certain embodiments, the subject is, or is at risk of being, immunocompromised. For example, the subject is undergoing or has undergone a chemotherapeutic treatment and/or radiation therapy. Alternatively, or in combination, the subject is, or is at risk of being, immunocompromised as a result of an infection.
Alternatively, or in combination with any and all embodiments of the invention comprising administering to a subject or administration, it is recognized that the
administering or administration is to a subject in need thereof, and that such subject includes each subject stated in the foregoing paragraph; for example, a subject having cancer. For example, the embodiments of the invention include one or more methods of treating cancer comprising administering to a subject in need thereof a pharmaceutical combination comprising an anti-PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form.
In one aspect, a method of treating (e.g., one or more of reducing, inhibiting, or delaying progression) a cancer or a tumor in a subject is provided. The method comprises administering to the subject a combination of the invention (e.g., a combination comprising a therapeutically effective amount of an anti-PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form).
Thus there is provided a method of treating cancer in a subject comprising administering to said subject simultaneously or sequentially N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule comprises:
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a
HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a
HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224 a
HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70; or
(f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72.
There is also provided a method of treating cancer in a subject comprising administering to said subject simultaneously or sequentially N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule comprises:
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ
ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ
ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of
SEQ ID NO: 70; or (f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72.
In certain embodiments, the cancer treated with the combination, includes but is not limited to lung cancer, melanoma, renal cancer, liver cancer, breast cancer, colorectal cancer, thyroid cancer, cholangiocarcinoma, glioblastoma, prostate cancer,
rhabdomyosarcoma, gastric cancer, or ovarian cancer.
In one preferred embodiment, the cancer is liver cancer.
In one embodiment, the treatment is a first-line or a second-line treatment. In one embodiment, the treatment is a second-line treatment. In another embodiment, the treatment is a first-line treatment.
As used herein, the term "second-line treatment" refers to a treatment that is given when an initial treatment (first-line treatment) does not work or no longer works. The pharmaceutical combination may be particularly useful in treating hepatocellular carcinoma (HCC), e.g. advanced HCC. The pharmaceutical combination may be useful for subjects suffering from HCC, including advanced HCC, who have received prior treatment with sorafenib and progressed during or after discontinuation of sorafenib treatment. Subjects suffering from HCC, including advanced HCC, who are intolerant to sorafenib treatment may also benefit from the administration of the pharmaceutical combination.
The hepatocellular carcinoma (HCC) to be treated by a pharmaceutical combination of the invention may be optionally characterized by positive FGFR4 and KLB expression. In one embodiment, the HCC to be treated may be characterized by positive FGFR4 and FGF19 expression or by positive FGFR4 and KLB expression. In one embodiment, the HCC to be treated is characterized by positive FGF19 expression. Thus there is provided a pharmaceutical combination described herein for use in the treatment of FGF19+ HCC, including for use in the treatment of FGF19+ advanced HCC.
The biomarkers FGFR4, KLB and FGF19 can be assayed using any method known in the art such as reverse Transcriptase PCR (RT-PCR), see, e.g. WO/2016/054483, published on 7th April 2016.
In an embodiment of the invention, the combination of the invention provides at least one of the following benefits:
improved safety profiles, improved toxicity profiles, improved efficacy, well tolerated by subjects suffering from cancer, improved overall response rate, improved overall survival, improved time to progression, tumor growth inhibition, decreased tumor volume, inhibition of cell proliferation, immunomodulatory activity, reduction in relapse time, apoptosis of cancer cells, increase in the time delay for the onset of exponential growth of tumor, reduction in resistance to treatment, synergistic effect leading to reduction of required dosage and/or reduction of side effects leading to an enhancement of the long-term clinical effectiveness of the combination.
Each active ingredient of the combination of the invention may be administered by different routes. For instance, the anti-PD-1 antibody molecule is typically administered to the subject systemically (e.g. subcutaneously, intravenously, intramuscularly or
intraperitoneally). N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl- 2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable form is preferably administered to the subject orally.
Thus, in another aspect, the anti-PD-1 antibody molecule used in the pharmaceutical combination of the invention is formulated together with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. , antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22nd Ed. Pharmaceutical Press, 2013, pp. 1049-1070). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
The anti-PD-1 antibody molecule used in the present invention may, thus, be in a variety of forms. These include, for example, liquid or semi-solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the anti-PD-1 antibody molecule disclosed herein is administered by intravenous infusion or injection. In another preferred embodiment, the anti-PD-1 antibody molecule disclosed herein is administered by intramuscular or subcutaneous injection. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Therapeutic compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure suitable to high antibody concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the anti-PD-1 antibody molecule) in the required amount in an appropriate solvent with one or a combination of further carriers, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other carriers. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
An anti-PD-1 antibody molecule described herein can be formulated into a formulation (e.g., a dosage form) for administration (e.g., intravenous administration) to a subject.
In certain embodiments, the formulation is a drug substance formulation. In other embodiments, the formulation is a lyophilized formulation, e.g., lyophilized from a drug substance formulation. In other embodiments, the formulation is a reconstituted formulation, e.g., reconstituted from a lyophilized formulation. In other embodiments, the formulation is a liquid formulation.
In some embodiments, the formulation (e.g., drug substance formulation) comprises the anti-PD-1 antibody molecule and a buffering agent.
In some embodiments, the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 10 to 50 mg/mL, e.g., 15 to 50 mg/mL, 20 to 45 mg/mL, 25 to 40 mg/mL, 30 to 35 mg/mL, 25 to 35 mg/mL, 30 to 40 mg/mL, e.g., 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 33.3 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, or 50 mg/mL. In certain embodiments, the anti-PD-1 antibody molecule is present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL.
In some embodiments, the formulation (e.g., drug substance formulation) comprises a buffering agent comprising histidine (e.g., a histidine buffer). In certain embodiments, the buffering agent (e.g., histine buffer) is present at a concentration of 1 mM to 20 mM, e.g., 2 mM to 15 mM, 3 mM to 10 mM, 4 mM to 9 mM, 5 mM to 8 mM, or 6 mM to 7 mM, e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 6.7 mM, 7 mM, 8 mM, 9 mM, 10 mM, 1 1 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM. In some embodiments, the buffering agent (e.g., histine buffer) is present at a concentration of 6 mM to 7 mM, e.g., 6.7 mM. In other embodiments, the buffering agent (e.g., a histidine buffer) has a pH of 4 to 7, e.g., 5 to 6, e.g., 5, 5.5 or 6. In some embodiments, the buffering agent (e.g., histidine buffer) has a pH of 5 to 6, e.g., 5.5. In certain embodiments, the buffering agent comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5). In some embodiments, the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL; and a buffering agent that comprises histidine at a
concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5).
In some embodiments, the formulation (e.g., drug substance formulation) further comprises a carbohydrate. In certain embodiments, the carbohydrate is sucrose. In some embodiments, the carbohydrate (e.g., sucrose) is present at a concentration of 50 mM to 150 mM, e.g., 25 mM to 150 mM, 50 mM to 100 mM, 60 mM to 90 mM, 70 mM to 80 mM, or 70 mM to 75 mM, e.g., 25 mM, 50 mM, 60 mM, 70 mM, 73.3 mM, 80 mM, 90 mM, 100 mM, or 150 mM. In some embodiments, the formulation comprises a carbohydrate or sucrose present at a concentration of 70 mM to 75 mM, e.g., 73.3 mM.
In some embodiments, the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL; a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a carbohydrate or sucrose present at a concentration of 70 mM to 75 mM, e.g., 73.3 mM.
In some embodiments, the formulation (e.g., drug substance formulation) further comprises a surfactant. In certain embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant or polysorbate 20) is present at a concentration of 0.005 % to 0.025% (w/w), e.g., 0.0075% to 0.02% or 0.01 % to 0.015% (w/w), e.g., 0.005%, 0.0075%, 0.01 %, 0.013%, 0.015%, or 0.02% (w/w). In some embodiments, the formulation comprises a surfactant or polysorbate 20 present at a concentration of 0.01 % to 0.015%, e.g., 0.013% (w/w).
In some embodiments, the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL; a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a surfactant or polysorbate 20 present at a concentration of 0.01 % to 0.015%, e.g., 0.013% (w/w).
In some embodiments, the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 30 to 35 mg/mL, e.g., 33.3 mg/mL; a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); a carbohydrate or sucrose present at a concentration of 70 mM to 75 mM, e.g., 73.3 mM; and a surfactant or polysorbate 20 present at a concentration of 0.01 % to 0.015%, e.g., 0.013% (w/w).
In some embodiments, the formulation (e.g., drug substance formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 33.3 mg/mL; a buffering agent that comprises histidine at a concentration of 6.7 mM and has a pH of 5.5; sucrose present at a concentration of 73.3 mM; and polysorbate 20 present at a concentration of 0.013% (w/w). In some embodiments, the formulation is a lyophilized formulation. In certain embodiments, the lyophilized formulation is lyophilized from a drug substance formulation described herein. For example, 2 to 5 mL, e.g., 3 to 4 mL, e.g., 3.6 mL, of the drug substance formulation described herein can be filled per container (e.g., vial) and lyophilized.
In some embodiments, the lypophilized formulation is reconstituted with 0.5 mL to 2 mL, e.g., 1 mL, of water or buffer for injection. In certain embodiments, the lypophilized formulation is reconstituted with 1 mL of water for injection, e.g., at a clinical site.
In some embodiments, the formulation (e.g., reconstituted formulation) comprises the PD-1 inhibitor (e.g., the anti-PD-1 antibody molecule) and a buffering agent.
In some embodiments, the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 20 mg/mL to 200 mg/mL, e.g., 50 mg/mL to 150 mg/mL, 80 mg/mL to 120 mg/mL, or 90 mg/mL to 1 10 mg/mL, e.g., 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 1 10 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, or 200 mg/mL. In certain embodiments, the PD-1 inhibitor (e.g., the anti-PD-1 antibody molecule) is present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL.
In some embodiments, the formulation (e.g., reconstituted formulation) comprises a buffering agent comprising histidine (e.g., a histidine buffer). In certain embodiments, the buffering agent (e.g., histine buffer) is present at a concentration of 5 mM to 100 mM, e.g., 10 mM to 50 mM, 15 mM to 25 mM, e.g., 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. In some embodiments, the buffering agent (e.g., histine buffer) is present at a concentration of 15 mM to 25 mM, e.g., 20 mM. In other embodiments, the buffering agent (e.g., a histidine buffer) has a pH of 4 to 7, e.g., 5 to 6, e.g., 5, 5.5 or 6. In some embodiments, the buffering agent (e.g., histidine buffer) has a pH of 5 to 6, e.g., 5.5. In certain embodiments, the buffering agent comprises histidine at a concentration of 15 mM to 25 mM (e.g., 20 mM) and has a pH of 5 to 6 (e.g., 5.5).
In some embodiments, the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5).
In some embodiments, the formulation (e.g., reconstituted formulation) further comprises a carbohydrate. In certain embodiments, the carbohydrate is sucrose. In some embodiments, the carbohydrate (e.g., sucrose) is present at a concentration of 100 mM to 500 mM, e.g., 150 mM to 400 mM, 175 mM to 300 mM, or 200 mM to 250 mM, e.g., 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM. In some embodiments, the formulation comprises a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
In some embodiments, the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
In some embodiments, the formulation (e.g., reconstituted formulation) further comprises a surfactant. In certain embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant or polysorbate 20 is present at a concentration of 0.01 % to 0.1 % (w/w), e.g., 0.02% to 0.08%, 0.025% to 0.06% or 0.03 % to 0.05% (w/w), e.g., 0.01 %, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1 % (w/w). In some embodiments, the formulation comprises a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
In some embodiments, the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w). In some embodiments, the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 80 to 120 mg/mL, e.g., 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM; and a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
In some embodiments, the formulation (e.g., reconstituted formulation) comprises an anti-PD-1 antibody molecule present at a concentration of 100 mg/mL; and a buffering agent that comprises histidine at a concentration of 6.7 mM and has a pH of 5.5; sucrose present at a concentration of 220 mM; and polysorbate 20 present at a concentration of 0.04% (w/w). In some embodiments, the formulation is reconstituted such that an extractable volume of at least 1 mL (e.g., at least 1 .5 mL, 2 mL, 2.5 mL, or 3 mL) of the reconstituted formulation can be withdrawn from the container (e.g., vial) containing the reconstituted formulation. In certain embodiments, the formulation is reconstituted and/or extracted from the container (e.g., vial) at a clinical site. In certain embodiments, the formulation (e.g., reconstituted formulation) is injected to an infusion bag, e.g., within 1 hour (e.g., within 45 minutes, 30 minutes, or 15 minutes) before the infusion starts to the patient or the subject in need thereof. In certain embodiments, the formulation is a liquid formulation. In some
embodiments, the liquid formulation is prepared by diluting a drug substance formulation comprising the anti-PD-1 antibody molecule described herein. For example, a drug substance formulation can be diluted, e.g., 10 to 30 mg/mL (e.g., 25 mg/mL), e.g., with a solution comprising one or more excipients (e.g., concentrated excipients). In some embodiments, the solution comprises one, two, or all of histidine, sucrose, or polysorbate 20. In certain embodiments, the solution comprises the same excipient(s) as the drug substance formulation.
In some embodiments, the formulation (e.g., liquid formulation) comprises an anti- PD-1 antibody molecule present at a concentration of 5 mg/mL to 50 mg/mL, e.g., 10 mg/mL to 40 mg/mL, 15 mg/mL to 35 mg/mL, or 20 mg/mL to 30 mg/mL, e.g., 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, or 50 mg/mL. In certain embodiments, the PD-1 inhibitor (e.g., the anti-PD-1 antibody molecule) is present at a concentration of 20 to 30 mg/mL, e.g., 25 mg/mL.
In some embodiments, the formulation (e.g., liquid formulation) comprises a buffering agent comprising histidine (e.g., a histidine buffer). In certain embodiments, the buffering agent (e.g., histine buffer) is present at a concentration of 5 mM to 100 mM, e.g., 10 mM to 50 mM, 15 mM to 25 mM, e.g., 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. In some embodiments, the buffering agent (e.g., histine buffer) is present at a concentration of 15 mM to 25 mM, e.g., 20 mM. In other
embodiments, the buffering agent (e.g., a histidine buffer) has a pH of 4 to 7, e.g., 5 to 6, e.g., 5, 5.5 or 6. In some embodiments, the buffering agent (e.g., histidine buffer) has a pH of 5 to 6, e.g., 5.5. In certain embodiments, the buffering agent comprises histidine at a concentration of 15 mM to 25 mM (e.g., 20 mM) and has a pH of 5 to 6 (e.g., 5.5).
In some embodiments, the formulation (e.g., liquid formulation) comprises an anti- PD-1 antibody molecule present at a concentration of 20 to 30 mg/mL, e.g., 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5).
In some embodiments, the formulation (e.g., liquid formulation) further comprises a carbohydrate. In certain embodiments, the carbohydrate is sucrose. In some embodiments, the carbohydrate (e.g., sucrose) is present at a concentration of 100 mM to 500 mM, e.g., 150 mM to 400 mM, 175 mM to 300 mM, or 200 mM to 250 mM, e.g., 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM. In some embodiments, the formulation comprises a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
In some embodiments, the formulation (e.g., liquid formulation) comprises an anti- PD-1 antibody molecule present at a concentration of 20 to 30 mg/mL, e.g., 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM.
In some embodiments, the formulation (e.g., liquid formulation) further comprises a surfactant. In certain embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant or polysorbate 20 is present at a concentration of 0.01 % to 0.1 % (w/w), e.g., 0.02% to 0.08%, 0.025% to 0.06% or 0.03 % to 0.05% (w/w), e.g., 0.01 %, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1 % (w/w). In some embodiments, the formulation comprises a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
In some embodiments, the formulation (e.g., liquid formulation) comprises an anti-
PD-1 antibody molecule present at a concentration of 20 to 30 mg/mL, e.g., 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); and a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
In some embodiments, the formulation (e.g., liquid d formulation) comprises an anti-
PD-1 antibody molecule present at a concentration of 20 to 30 mg/mL, e.g., 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6 mM to 7 mM (e.g., 6.7 mM) and has a pH of 5 to 6 (e.g., 5.5); a carbohydrate or sucrose present at a concentration of 200 mM to 250 mM, e.g., 220 mM; and a surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, e.g., 0.04% (w/w).
In some embodiments, the formulation (e.g., liquid formulation) comprises an anti- PD-1 antibody molecule present at a concentration of 25 mg/mL; and a buffering agent that comprises histidine at a concentration of 6.7 mM and has a pH of 5.5; sucrose present at a concentration of 220 mM; and polysorbate 20 present at a concentration of 0.04% (w/w).
In certain embodiments, 1 mL to 10 mL (e.g., 2 mL to 8 mL, 3 mL to 7 mL, or 4 mL to 5 mL, e.g., 3 mL, 4 mL, 4.3 mL, 4.5 mL, 5 mL, or 6 mL) of the liquid formulation is filled per container (e.g., vial). In other embodiments, the liquid formulation is filled into a container (e.g., vial) such that an extractable volume of at least 2 mL (e.g., at least 3 mL, at least 4 mL, or at least 5 mL) of the liquid formulation can be withdrawn per container (e.g., vial). In certain embodiments, the liquid formulation is diluted from the drug substance formulation and/or extracted from the container (e.g., vial) at a clinical site. In certain embodiments, the formulation (e.g., liquid formulation) is injected to an infusion bag, e.g., within 1 hour (e.g., within 45 minutes, 30 minutes, or 15 minutes) before the infusion starts to the patient or the subject in need thereof.
The container used for any of the formulations described herein can include, e.g., a vial, and optionally, a stopper, a cap, or both. In certain embodiments, the vial is a glass vial, e.g., a 6R white glass vial. In other embodiments, the stopper is a rubber stopper, e.g., a grey rubber stopper. In other embodiments, the cap is a flip-off cap, e.g., an aluminum flip- off cap. In some embodiments, the container comprises a 6R white glass vial, a grey rubber stopper, and an aluminum flip-off cap. In some embodiments, the container (e.g., vial) is for a single-use container. In certain embodiments, 50 mg to 150 mg, e.g., 80 mg to 120 mg, 90 mg to 1 10 mg, 100 mg to 120 mg, 100 mg to 1 10 mg, 1 10 mg to 120 mg, or 1 10 mg to 130 mg, of the PD-1 inhibitor (e.g., the anti-PD-1 antibody molecule), is present in the container (e.g., vial). Other exemplary buffering agents that can be used in the formulation described herein include, but are not limited to, an arginine buffer, a citrate buffer, or a phosphate buffer. Other exemplary carbohydrates that can be used in the formulation described herein include, but are not limited to, trehalose, mannitol, sorbitol, or a combination thereof. The formulation described herein may also contain a tonixity agent, e.g., sodium chloride, and/or a stabilizing agent, e.g., an amino acid (e.g., glycine, arginine, methionine, or a combination thereof). The antibody molecules can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. For example, the antibody molecules can be administered by intravenous infusion at a rate of more than 20 mg/min, e.g. , 20-40 mg/min, and typically greater than or equal to 40 mg/min to reach a dose of about 35 to 440 mg/m2, typically about 70 to 310 mg/m2, and more typically, about 1 10 to 130 mg/m2. In embodiments, the antibody molecules can be administered by intravenous infusion at a rate of less than 10mg/min; preferably less than or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m 2, preferably about 5 to 50 mg/m2, about 7 to 25 mg/m2 and more preferably, about 10 mg/m2. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form is preferably formulated for oral administration. In addition, N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders). The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc.
Typically, N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form is in the form of tablets or gelatin capsules comprising N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form together with one or more of:
a) diluents, e.g. , lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g. , silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also
c) binders, e.g. , magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and
e) absorbents, colorants, flavors and sweeteners.
Tablets may be either film coated or enteric coated according to methods known in the art.
Suitable compositions for oral administration include an effective amount of N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions comprising N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide or a pharmaceutically acceptable salt thereof intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2- yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in citric acid salt form in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
In an embodiment, the pharmaceutical combination of the invention comprises N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form in the form of a tablet. In another embodiment, the pharmaceutical combination of the invention comprises N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form in the form of a capsule comprising N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form only.
In another embodiment, the pharmaceutical combination of the invention comprises N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form in the form of a capsule comprising N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6- ((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form and a glidant.
Glidants are typically used to promote powder flow by reducing interparticle friction and cohesion. Examples include colloidal silicon dioxide (fumed silica), talc and magnesium carbonate.
In a preferred embodiment, the pharmaceutical composition comprising N-(5-cyano- 4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide or a pharmaceutically acceptable salt thereof includes a glidant, and preferably, the glidant is colloidal silicon dioxide (sold under the trade name aerosil). Dosages of the active ingredients and therapeutic regimens of the combination of the invention can be determined by a skilled artisan. In certain embodiments, the anti-PD-1 antibody molecule is administered to the subject in need thereof by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose from about 10 to 20 mg/kg every other week.
In some embodiments, the anti-PD-1 antibody molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose (e.g., a flat dose) of about 200 mg to 500 mg, e.g., about 250 mg to 450 mg, about 300 mg to 400 mg, about 250 mg to 350 mg, about 350 mg to 450 mg. The dosing schedule (e.g., flat dosing schedule) can vary from e.g., once a week to once every 2, 3, 4, 5, or 6 weeks. In one embodiment, the anti-PD-1 antibody molecule, e.g., the exemplary antibody molecule, is administered at a dose from about 300 mg to 400 mg once every three weeks or once every four weeks.
In an embodiment of the invention, the anti-PD-1 antibody molecule is administered at a dose of 300mg once every three weeks. In an embodiment of the invention, the anti-PD-1 antibody molecule is administered at a dose of 400mg once every four weeks.
In one embodiment, the anti-PD-1 antibody molecule is administered at a dose from about 300 mg once every four weeks.
In one embodiment, the anti-PD-1 antibody molecule is administered at a dose from about 400 mg once every three weeks.
In one embodiment, the anti-PD-1 antibody molecule is administered via i.v. infusion over 30 minutes (up to 2 hours, if clinically indicated) once every 3 weeks.
In one embodiment, the anti-PD-1 antibody molecule is administered via i.v. infusion over 30 minutes (up to 2 hours, if clinically indicated) once every 4 weeks.
In one embodiment, a method of treating (e.g., one or more of reducing, inhibiting, or delaying progression) a cancer or a tumor in a subject is provided. The method comprises administering, in combination with an FGFR4 inhibitor (e.g. N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form), to the subject an anti- PD-1 antibody molecule, e.g., the preferred anti-PD-1 antibody molecule described herein, at a dose of about 300 mg to 400 mg once every three weeks or once every four weeks. In certain embodiments, the preferred anti-PD-1 antibody molecule is administered at a dose of about 300 mg once every three weeks. In other embodiments, the preferred anti-PD-1 antibody molecule is administered at a dose of about 400 mg once every four weeks.
In another aspect, the invention features a method of reducing an activity (e.g., growth, survival, or viability, or all), of a hyperproliferative (e.g., a cancer) cell. The method includes contacting the cell with an anti-PD-1 antibody molecule, e.g., an anti-PD-1 antibody molecule described herein. The method can be performed in a subject, e.g., as part of a therapeutic regimen in combination with an FGFR4 inhibitor, e.g., at a dose of about 300 mg to 400 mg of an anti-PD-1 antibody molecule once every three weeks or once every four weeks.
The dose or dosage of the FGFR4 inhibitor as described herein refers to the amount of the free base. For example, if the dosage quoted is 80 mg, and a pharmaceutically acceptable salt of the FGFR4 inhibitor is used, such as the citrate salt thereof, the dose of 80mg corresponds to the amount of free base of the FGFR4 inhibitor, namely N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide. The FGFR4 inhibitor of the combination of the present invention, i.e. N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide may be administered at a total daily dose of about 25-200 mg, or about 50-150 mg (e.g., once per day). The total daily dose of N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide (e.g. in citric acid salt form) which is administered may be 50 mg, 80 mg, 100 mg, 120 mg or 150 mg. In particular N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide may be administered at a dose of 80 mg daily or 120 mg daily.
The dose of the FGFR4 inhibitor may be administered once daily, or twice daily, or three times daily, or four times daily; preferably once or twice daily, more preferably once daily.
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, e.g. in citric acid salt form, may in particular be administered at a dose of 50 mg once daily, 80 mg once daily, 100 mg once daily, 120 mg once daily or 150 mg once daily. In particular N-(5-cyano- 4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide may be administered at a dose of 80 mg daily or 120 mg daily.
In an embodiment, N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, e.g. in citric acid salt form, is administered at a dose of 80mg daily, e.g. once daily. The dose of 80mg corresponds to the amount of free base of N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
In another embodiment, N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-
6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, e.g. in citric acid salt form, is administered at a dose of 120 mg daily, e.g. once daily. The dose of 120 mg corresponds to the amount of free base of N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide. In a preferred embodiment, N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form is administered orally. PDR001 (Antibody Molecule A) may be administered at a dose of 300mg once every three weeks and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide may be administered at a daily dose of 50 mg, 80 mg, 100 mg, 120 mg or 200 mg. The FGFR4 inhibitor may be administered once or twice daily.
In another embodiment, PDR001 (Antibody Molecule A) may be administered at a dose of 400mg once every four weeks and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2- yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide may be administered at a daily dose of 50 mg, 80 mg, 100 mg, 120 mg or 200 mg. The FGFR4 inhibitor may be administered once or twice daily.
In an embodiment, N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, e.g. in citric acid salt form, is administered prior to the anti-PD-1 antibody molecule. In an embodiment, the administration of the anti-PD-1 antibody molecule designated PDR001 comprising a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 is at least 1 hour after the administration of N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, e.g. in citric acid salt form. In an embodiment, the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in free form or in pharmaceutically acceptable salt form, preferably in citric acid salt form, wherein the anti-PD-1 antibody molecule comprises:
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of
SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70; or
(f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72;
for use in a therapeutic regimen, wherein N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-
7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide is administered at a dose of 80mg to 120 mg daily and the anti-PD-1 antibody molecule is administered at a dose of 300mg once every three weeks.
In an embodiment, the invention relates to a pharmaceutical combination comprising an anti-PD-1 antibody molecule and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide in free form or in pharmaceutically acceptable salt form, preferably in citric acid salt form, wherein the anti-PD-1 antibody molecule comprises:
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a
LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32; (c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70; or
(f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72;
for use in a therapeutic regimen, wherein N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)- 7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide is administered at a dose of 80mg to 120 mg daily and the anti-PD-1 antibody molecule is administered at a dose of 300mg once every three weeks.
In one embodiment, the invention provides a kit comprising a pharmaceutical combination comprising an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule comprises:
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID
NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID
NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70; or
(f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72;
and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form, with instructions for simultaneous or sequential administration of the anti-PD-1 antibody molecule and N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide. In one embodiment, the invention provides a kit comprising a pharmaceutical combination comprising an anti-PD-1 antibody molecule designated PDR001 comprising a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-E as described in Table 1 and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form, with instructions for simultaneous or sequential administration of PDR001 and N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)- 7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)- carboxamide.
In one embodiment, the kit comprises two separate pharmaceutical compositions, one of which contains N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in citric acid salt form and one of which contains the anti-PD-1 antibody molecule. In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like.
The kit of the invention may be used for administering different dosage forms, for administering the separate compositions at different dosage intervals. To assist compliance, the kit of the invention typically comprises directions for administration.
In the combination of the invention, both active ingredients be manufactured and/or formulated by the same or different manufacturers. Moreover, the active ingredients of the combination of the invention may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the combination of the invention); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the subjects in need thereof themselves, e.g. during sequential administration of the active ingredients.
The following Examples illustrate the invention described above; they are not, however, intended to limit the scope of the invention in any way.
Examples
Example 1 Example 1: Characterization of Humanized Anti-PD-1 Antibody
Binding affinity and specificity
The Generation of humanized BAP049-Clone-B and BAP049-Clone E and characterization thereof is described in PCT application PCT/US2015/012754, which was published on 30th July 2015, as WO/2015/1 12900.
Murine anti-PD-1 monoclonal antibody BAP049 was humanized. The sequences and test samples of sixteen humanized BAP049 clones with unique variable region sequences were obtained. These clones were further analyzed for their biological functions (e.g. , antigen binding and ligand blocking), structural features, and transcient expression in CHO cells. Binding affinity and specificity
The binding of an exemplary humanized anti-PD-1 antibody on human PD-1 protein was measured using Biacore method. The results are: Ka = 2.78 X 105 M"1s"1 ; Kd = 2.13 x 10" 4 s_1 ; KD = 0.0827±0.005505 nM.
Humanization Technology and Process
Humanization of BAP049 was performed using a combinatorial library of human germline variable region frameworks (FWs). The technology entails transferring the murine CDRs in frame to a library of human variable regions (VRs) that had been constructed by randomly combining human germ line FW1 , FW2 and FW3 sequences. Only one FW4 sequence was used, which is WGQGTTVTVSS (SEQ ID NO: 67) for the heavy chain (HC) (Kabat human HC subgroup I) and FGQGTKVEIK (SEQ ID NO: 208) for the light chain (LC) (Kabat human κ subgroup I). The library of VR sequences was fused to human constant region (CR) sequences, human lgG4(S228P) of HC and human κ CR of LC, and the resulting library of whole IgG was expressed in CHO cells for screening. Screening was performed with tissue culture supernatants measuring binding avidity on antigen-expressing cells in a whole cell ELISA format or on FACS.
The humanization process was performed in a stepwise manner starting with the construction and expression of the appropriate chimeric mAb (murine VR, lgG4(S228P), human κ), which can serve as a comparator for the screening of the humanized clones. The constant region amino acid sequences for human lgG4(S228P) heavy chain and human kappa light chain are shown in Table 3.
Humanization of the VR of LC and HC were performed in two independent steps.
The library of humanized LC (huLC) was paired with the chimeric HC (murine VR, lgG4(S228P)) and the resulting "half-humanized" mAbs were screened for binding activity by ELISA. The huLC of clones with adequate binding activity (> binding of chimeric mAb) were selected. Analogously, the library of humanized HC (huHC) was paired with the chimeric LC (murine VR, human κ) and screened for binding activity by ELISA. The huHC of clones with appropriate binding activity (> binding of chimeric mAb) were selected.
The variable regions of the selected huLC and huHC were then sequenced to identify the huLC and huHC with unique sequences (some clones from the initial selection process may share the same LC or HC). The unique huLC and huHC were then randomly combined to form a small library of humanized mAbs (humAbs), which was expressed in CHO cells and screened on antigen- expressing cells in an ELISA and FACS format. Clones with binding activities that were equal or better than the binding of the chimeric comparator mAb are the final product of the humanization process.
Construction of Chimeric Antibody
Three variants of the chimeric antibody were prepared that either had a Cys, Tyr or Ser residue at position 102 of the LC sequence. The three chimeric antibodies, i.e.,
BAP049-chi (Cys), BAP049-chi (Tyr), and BAP049-chi (Ser) (also known as BAP049-chi, BAP049-chi-Y, and BAP049-chi-S, respectively), were expressed in CHO cells and tested for their ability to compete with labeled murine antibody for binding to PD-1 expressing Jurkat cells. The three variants were indistinguishable in the competition experiment. The results show that the three chimeric mAbs (Cys, Tyr, Ser) compete equally well with the binding of the labeled murine mAb BAP049. The slight difference between the chimeric mAb curves and the murine mAb curve is probably due to the different methods used for determining mAb concentrations. The concentration of the murine mAb was determined by OD280 measurement, whereas the chimeric mAb concentrations in supernatants were determined with an ELISA using an lgG4 standard. The germline residue Tyr was selected for humanized antibodies.
Humanized Antibody Clones
The process of humanization yielded sixteen clones with binding affinities comparable to that of the chimeric antibody. In addition to binding data, for each clone, the VR sequences were provided along with a sample of the mAb. The samples had been prepared by transient transfections of CHO cells and were concentrated tissue culture supernatants. The antibody concentrations in the solutions had been determined by an lgG4-specific ELISA.
The sixteen unique clones are combinations of four unique HC sequences and nine unique LC sequences. For the HC FW regions, the HC sequences are combinations of one of two different VHFW1 , one of three different VHFW2, and one of two different VHFW3 sequences. For the LC FW regions, the LC sequences are combinations of one of five different VLFW1 , one of three different VLFW2, and one of four different VLFW3 sequences. The amino acid and nucleotide sequences of the heavy and light chain variable domains for the humanized BAP049 clones B and E are shown in Table 1 . The amino acid and nucleotide sequences of the heavy and light chain CDRs of the humanized BAP049 clones are also shown in Table 1 .
Analysis of Humanized Clones
Analysis of binding activity and binding specificity
The binding activity and specificity was measured in a competition binding assay using a constant concentration of Alexa 488-labeled murine mAb, serial dilutions of the test mAbs, and PD-1 -expressing 300.19 cells. Incubations with the mAb mixtures having different concentration ratios of test mAb to labeled mAb was at 4 °C for 30 min. Bound labeled murine mAb was then quantified using a FACS machine. The experiment was performed twice. Within the accuracy of the experiment, all humanized clones show similar activity for competing with binding of labeled murine mAb. The activity is also comparable to the activity of the parent murine mAb and chimeric mAb. MAbs were ranked relative to each other. For example, it can be a weaker competitor if in both experiments the curve of a certain clone is to the right of the chimeric mAb curve or it can be a better competitor if the curve of a certain clone is to the left of the chimeric mAb curve. Selection of humanized clones
Selected clones including clones B and E were further tested for their ability to block the binding of PD-L1 and PD-L2 to PD-1 and for enhancing T cell activity in vitro assays with human PBMC.
Blocking ofligand binding
Murine anti-PD-1 mAb blocks the binding of the natural ligands PD-L1 and PD-L2 to
PD-1 expressed on cells at low concentrations. Whether the humanized clones had preserved the blocking capacity of the parent murine mAb was tested in comparative experiments with murine and chimeric antibodies.
The blocking capacity of the mAbs was evaluated in a competition binding assay using a constant concentration of PD-L1 -hulgG1 Fc fusion protein or PD-L2-hulgG1 Fc fusion protein, serial dilutions of the mAbs to be tested, and PD-1 -expressing 300.19 cells.
Incubation was at 4 °C for 30 min. Bound ligand fusion proteins were detected with PE- conjugated F(ab')2 fragment of goat anti-human IgG which doesn't recognize lgG4 mAbs
(Southern Biotech 2043-09), and flow cytometry. Wthin the accuracy of the experiments, the humanized clones, chimeric antibody and murine parent mAb demonstrated comparable blocking activity for both the PD-L1 and PD-L2 ligands. Expression of Humanized Anti-PD-1 Antibody, BAP049
Five humanized clones were selected for evaluation of expression in Chinese Hamster Ovary (CHO) cells.
Single gene vectors (SGVs) were constructed using Lonza's GS Xceed vectors (lgG4proAk for heavy chain and Kappa for light chain). The SGVs were amplified and transiently co-transfected into CHOK1 SV GS-KO cells for expression at a volume of 2.8 L.
Expression cultures were harvested Day 6 post-transfection and clarified by centrifugation and sterile filtration. The clarified cell culture supernatant was purified using one-step Protein A chromatography. Product quality analysis in the form of SE-HPLC, SDS- PAGE, IEF, and LAL was carried out using purified material at a concentration of 1 mg/ml inicluding an antibody as a control sample.
Vector Construction
The sequences of the light and heavy chain variable domain encoding regions were synthesised by GeneArt AG. Light chain variable domain encoding regions were sub-cloned into pXC-Kappa and heavy chain variable domain encoding regions into pXC-lgG4pro ΔΚ vectors respectively using the N-terminal restriction site Hind III and the C-terminal restriction sites BsiWI (light chain) and Apal (heavy chain). Positive clones were screened by PCR amplification (primers 1053: GCTGACAGACTAACAGACTGTTCC (SEQ ID NO: 226) and 1072: CAAATGTGGTATGGCTGA (SEQ ID NO: 227)) and verified by restriction digest
(using a double digest of EcoRI-HF and Hindlll-HF) and nucleotide sequencing of the gene of interest.
DNA Amplification
A single bacterial colony was picked into 15 ml Luria Bertani (LB) medium (LB Broth,
Sigma-Aldrich, L7275) containing 50 μg/ml ampicillin and incubated at 37 °C overnight with shaking at 220 rpm. The resulting starter culture was used to inoculate 1 L Luria Bertani (LB) medium containing 50 μg/ml ampicillin and incubated at 37 °C overnight with shaking at 220 rpm. Vector DNA was isolated using the QIAGEN Plasmid Plus Gigaprep system (QIAGEN, 12991 ). In all instances, DNA concentration was measured using a Nanodrop 1000 spectrophotometer (Thermo-Scientific) and adjusted to 1 mg/ml with EB buffer (10 mM Tris-CI, pH 8.5). DNA quality for the single gene vectors was assessed by measuring the absorbance ratio A260/A280. This was found to be between 1 .88 and 1 .90.
Culture of CHOKISV GS-KO Cells
CHOK1 SV GS-KO cells were cultured in CD-CHO media (Invitrogen, 10743-029) supplemented with 6 mM glutamine (Invitrogen, 25030-123). Cells were incubated in a shaking incubator at 36.5 °C, 5% C02, 85% humidity, 140 rpm. Cells were routinely sub- cultured every 3-4 days, seeding at 2 x 105 cells/ml and were propagated in order to have sufficient cells available for transfection. Cells were discarded by passage 20.
Transient Transfections of CHOK1SV GS-KO Cells
Transient transfections were performed using CHOK1 SV GS-KO cells which had been in culture a minimum two weeks. Cells were sub-cultured 24 h prior to transfection and cell viability was >99% at the time of transfection.
All transfections were carried out via electroporation using a Gene Pulse MXCell (Bio-Rad), a plate based system for electroporation. For each transfection, viable cells were resuspended in pre-warmed media to 2.86 x 107 cells/ml. 80 μg DNA (1 :1 ratio of heavy and light chain SGVs) and 700 μΙ cell suspension were aliquotted into each cuvette/well. Cells were electroporated at 300 V, 1300 μΡ. Transfected cells were transferred to pre-warmed media in Erienmeyer flasks and the cuvette/wells rinsed twice with pre-warmed media which was also transferred to the flasks. Transfected cell cultures were incubated in a shaking incubator at 36.5 °C, 5% C02, 85% humidity, 140 rpm for 6 days. Cell viability and viable cell concentrations were measured at the time of harvest using a Cedex HiRes automated cell counter (Roche). Protein A Affinity Chromatography
Cell culture supernatant was harvested and clarified by centrifugation at 2000 rpm for 10 min, then filtered through a 0.22 μηι PES membrane filter. Clarified supernatant was purified using a pre-packed 5 ml HiTrap MabSelect SuRE column (GE Healthcare, 1 1 -0034- 94) on an AKTA purifier (10 ml/min). The column was equilibrated with 50 mM sodium phosphate, 125 mM sodium chloride, pH 7.0 (equilibration buffer) for 5 column volumes (CVs). After sample loading, the column was washed with 2 CVs of equilibration buffer followed by 3 CVs of 50 mM sodium phosphate, 1 M sodium chloride pH 7.0 and a repeat wash of 2 CVs of equilibration buffer. The Product was then eluted with 10 mM sodium formate, pH 3.5 over 5 CVs. Protein containing, eluted fractions were immediately pH adjusted to pH 7.2 and filtered through a 0.2 μηι filter.
A single protein-containing peak was observed during the elution phase. This peak was shown to contain the mAb, when analyzed by SE-HPLC and SDS-PAGE. Recovered protein yield is shown in Table 5. The clones expressed transiently in a range from 32.4 to 43.0 mg/L. Table 5. Summary of yield, titre, monomer content and endotoxin levels
Figure imgf000064_0001
SE-HPLC Analysis
Samples of Protein A purified antibodies were analyzed in duplicate by SE-HPLC on an Agilent 1200 series HPLC system, using a Zorbax GF-250 4 μηι 9.4 mm ID x 250 mm column (Agilent). Aliquots of sample at a concentration of 1 mg/ml were filtered through a 0.2 μηι filter prior to injection. 80 μΙ aliquots were injected respectively and run at 1 ml/min for 15 minutes. Soluble aggregate levels were analysed using Chemstation (Agilent) software.
Chromatography profiles with retention time showing the percentage of the overall detected peak areas were obtained for the tested antibodies and a control lgG4 antibody. The products show a single protein peak at approximately 8.65 to 8.72 min comparable to the human lgG4 antibody control (about 8.64 min) and consistent with a monomeric antibody. Small amounts (up to about 4-5%) of higher molecular weight impurities, consistent with soluble aggregates, were detected at retention times between about 7.43 and 8.08 min.
SDS-PAGE Analysis
Reduced samples were prepared for analysis by mixing with NuPage 4x LDS sample buffer (Invitrogen, NP0007) and NuPage 10x sample reducing agent (Invitrogen, NP0009), and incubated at 70 °C, 10 min. For non-reduced samples, the reducing agent and heat incubation were omitted. Samples were electrophoresed on 1 .5 mm NuPage 4-12% Bis-Tris Novex pre-cast gels (Invitrogen, NP0335PK2) with NuPage MES SDS running buffer under denaturing conditions. 10 μΙ aliquots of SeeBlue Plus 2 pre-stained molecular weight standard (Invitrogen, LC5925) and a control lgG4 antibody at 1 mg/ml were included on the gel. 1 μΙ of each sample at 1 mg/ml were loaded onto the gel. Once electrophoresed, gels were stained with InstantBlue (TripleRed, ISB01 L) for 30 min at room temperature. Images of the stained gels were analysed on a BioSpectrum Imaging System (UVP). The analysis confirmed the presence of the antibody products and good levels of purity. Under non-reducing conditions, a predominant protein band close to 98 kDa was observed comparable with the control lgG4 antibody. The control lgG4 antibody and one tested clone display an additional fainter band corresponding to a heavy plus light chain half- antibody at approximately 70 kDa under non-reducing conditions. This is expected for the control antibody. Two bands were observed under reducing conditions consistent with the size of heavy (close to the position of the 49 kDa marker) and light chains (close to the position of the 28 kDa marker) and comparable with the bands found for the control lgG4 antibody.
Iso-electric Focussing (IEF) Analysis
Non-reduced samples of Protein A purified antibody were electrophoresed as described below.
5 μg of Protein A purified samples were electrophoresed on a 1 .0 mm Novex pH 3-10 gradient gel (Invitrogen, EC66552BOX) using manufacturers recommended running conditions. A 10 μΙ aliquot of IEF pH 3 - 10 markers (Invitrogen, 39212-01) was included on the gel. Once electrophoresed, gels were fixed with 10% TCA solution for 30 min and then stained with InstantBlue (TripleRed, ISB01 L) over night at room temperature. Images of the stained gels were analysed on a BioSpectrum Imaging System (UVP).
The tested clones show charge isoforms between pH 7.4 and 8.0 markers. The detected charge isoforms are slightly more basic than the theoretically calculated pis for these antibodies which were predicted to be between 6.99 and 7.56. The general shift to more basic charge isoforms suggests the presence of post-translational modifications such as glycosylation on the molecules. Clone C and Clone E show comparable charge isoforms, which is also consistent with the theorectically calculated pi being the same for both (6.99). The control lgG4 antibody behaved as expected.
Table 6. Charge isoforms as detected by Novex IEF analysis
Product pi of predominant Acidic charge Basic charge
charge isoform* isoforms* isoforms*
Clone A 7.6 2x; 7.5 to 7.55 7.7
Clone B 7.75 2x; 7.5 to 7.6 7.8
Clone C 7.5 2x; 7.4 to 7.5 7.55
Clone D 8.0 7.9 8.1
Clone E 7.5 2x; 7.4 to 7.5 7.55
*pl readings are estimated from the staining positions correlated against the IEF 3-10
Figure imgf000066_0001
Characterization of Humanized Anti-PD-1 Antibodies
Binding affinity and specificity
The binding of exemplary humanized anti-PD-1 antibodies including Clone B and Clone E as shown in Table 1 on human PD-1 protein was measured using Biacore method. The results are: Ka = 2.78 X 105 M ; Kd = 2.13 X 10"4 s_1 ; KD = 0.0827±0.005505 nM.
The binding of the same humanized anti-PD-1 antibody on human PD-1 -expressing 300.19 cells was measured using FACS analysis. The result shows that the anti-PD-1 antibody (human lgG4) binds with high affinity to human PD-1 compared to a human lgG4 isotype control.
The exemplary humanized anti-PD-1 antibody was found to exhibit high affinity to cynomolgus PD-1 protein and cynomolgus PD-1 -expressing 300.19 cells. As measured by Biacore method, the anti-PD-1 antibody binds to cynomolgus PD-1 with a KD of 0.093±0.015 nM. The binding affinity to cynomolgus PD-1 is comparable to its binding affinity to human PD-1 .
Additional binding analyses show that the exemplary humanized anti-PD-1 antibody is not cross- reactive with mouse PD-1 or cross-reactive with parental cell line.
Blocking of interactions between PD-1 and its ligands
The ability of the exemplary humanized anti-PD-1 antibody to block the interactions between PD-1 and both of its known ligands, PD-L1 and PD-L2 was examined. The results show that the anti-PD-1 antibody blocked the binding of PD-L1 and PD-L2 on human PD-1 - expressing 300.19 cells compared to human lgG4 isotype control and no antibody control. The anti-PD-1 antibody blocked PD-L1 binding on the 300.19 cells with an IC50 of 0.94±0.15 nM. The same antibody blocked PD-L2 binding on the 300.19 cells with an IC50 of 1 .3±0.25 nM.
Cellular activity
The ability of the exemplary humanized anti-PD-1 antibody to enhance the
Staphylococcal enterotoxin B (SEB)-stimulated expression of IL-2 was tested in human whole blood ex vivo assay. Diluted human whole blood was incubated with the anti-PD-1 antibody in the presence or absence of SEB at 37°C for 48 hours prior to IL-2
measurement. The result shows that the anti-PD-1 antibody increased SEB-stimulated IL-2 expression by 2.28±0.32 fold compared to a human lgG4 isotype control (25 μg/ml SEB; n=5 donors). Example 2: A phase lb/11, multicenter, open-label study of oral FGF401 in combination with an anti-PD-1 antibody molecule ("Antibody Molecule A", detailed below) in adult patients with hepatocellular carcinoma (HCC). For this study, the investigational drugs are FGF401 and Antibody Molecule A, an anti-PD-1 receptor recombinant humanized monoclonal antibody, designated PDR001 .
Antibody Molecule A:
The exemplary antibody molecule, Antibody A, (BAP049-Clone-E) tested in this study is a humanized anti-programmed death-1 (PD-1) lgG4 monoclonal antibody (mAb) that blocks binding of programmed cell death ligand-1 (PD-L1) and programmed cell death ligand-2 (PD-L2) to PD-1 . It binds to PD-1 with high affinity and inhibits its biological activity.
The amino acid sequences of this antibody molecule are described in Table 1 herein.
Results from pre-clinical toxicology studies have shown that it has a favorable safety profile. Its pharmacodynamic activity has also been demonstrated in vivo.
Antibody Molecule A is in the form of lyophilisate in vial for i.v. infusion. The starting dose is 300 mg, and it is administered every 3 weeks (Q3W).
Antibody Molecule A is administered via i.v. infusion over 30 minutes (up to 2 hours, if clinically indicated) once every 3 weeks. The next scheduled dose may be delayed by up to 7 days to recover from previous AEs (adverse events). If the next dose cannot be administered within the above mentioned 7-days delay, then the assessments should be shifted accordingly. FGF401
FGF401 corresponds to N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl- 6-((4-methyl-2-oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide. FGF401 may be used in citric acid salt form. FGF401 may be given in the form of capsules. The starting dose is 80mg of FGF401 (amount of free base) and it is administered once daily. FGF401 may also be administered at a total daily dose of 50 mg, 80 mg, 100 mg, 120 mg or 150 mg. Preferred dosages include 50 mg, 80 mg and 120 mg.
Objective of the trial:
Main objective:
Primary objective
• Phase I: To estimate the MTD (maximum tolerated dose) and/or RP2D (recommended phase II dose) of FGF401 in combination with PDR001
• Phase II: To investigate the anti-tumor activity of FGF401 in combination with PDR001 Secondary objective
• To characterize the safety and tolerability of FGF401 in combination with PDR001 in advanced HCC
• To further investigate the anti-tumor activity of FGF401 in combination with PDR001
PRINCIPAL INCLUSION CRITERIA
Key Inclusion Diagnosis of advanced HCC (hepatocellular carcinoma) criteria according to the AASLD (American Association for the
Study of Liver Diseases) Guidelines
HCC stage C according to the BCLC (Barcelona-Clinic
Liver Cancer) staging classification
Prior systemic treatment with sorafenib for advanced HCC with documented disease progression during or after discontinuation of sorafenib treatment, or intolerance to sorafenib treatment. Specifically, this can be defined as:
• Documented radiological confirmation (radiology scans or report) of disease progression during or after sorafenib treatment. [For France only: patients must have received at least 8 weeks of prior sorafenib treatment.]
• Intolerance to sorafenib (at any dose and/or duration) is defined as documented sorafenib-related Grade 3 or 4 adverse events that led to sorafenib discontinuation.
• Patients for whom sorafenib is contraindicated as national full prescribing information.
Current cirrhotic status of Child-Pugh class A (5-6 points), with no encephalopathy and/or ascites. Child-Pugh status must be calculated based on clinical findings and laboratory results during the Screening period.
Key Exclusion 1 . History of severe hypersensitivity to study treatment ingredients criteria or other monoclonal antibodies and/or their excipients or which in the opinion of the investigator may pose an increased risk of serious infusion reaction.
2. Impaired cardiac function or clinically significant cardiac
disease, including any of the following:
• Clinically significant and/or uncontrolled heart disease such as congestive heart failure requiring treatment (NYHA
Grade > 2), uncontrolled hypertension or clinically significant arrhythmia
• QTcF > 470 msec on screening ECG or congenital long QT syndrome
• Acute myocardial infarction or unstable angina pectoris < 3 months prior to study entry
3. HIV infection
4. Active HBV or HCV infection tested during screening. Patients whose disease is controlled under antiviral therapy should not be excluded. The adequately controlled hepatitis is defined as:
• patients with positive HBV-DNA and/or positive HbsAg results, must be on antiviral therapy (according to institutions guideline), as prophylaxis at least 1 week prior to start of study treatment
• having undetectable level of serum HCV RNA level prior to enrollment.
5. Active, known or suspected autoimmune disease. Patients with vitiligo, type I diabetes, residual hypothyroidism only requiring hormone replacement, psoriasis not requiring systemic treatment or conditions not expected to recur in the absence of an external trigger should not be excluded.
6. Prior anti-PD-1 /PD-L1 treatment
7. Systemic chronic steroid therapy or any immunosuppressive therapy (> 10mg/day prednisone or equivalent). Topical, inhaled, nasal and ophthalmic steroids are allowed.
8. Use of any live vaccines against infectious diseases within 4 weeks of initiation of study treatment.
9. Use of hematopoietic colony-stimulating growth factors (e.g. G-
CSF, GM-CSF, M-CSF), thrombopoietin mimetics or erythroid stimulating agents < 2 weeks prior to start of study treatment. If erythroid stimulating agents were initiated more than 2 weeks prior to the first dose of study treatment and the patient is on a stable dose, they can be maintained.
10. Patients with a history of drug-induced pneumonitis or current pneumonitis.
END POINTS:
Objective Endpoint
Primary
Phase I part Incidence rate and characteristics of DLT (dose limiting
To estimate the MTD and/or toxicities) during the first two cycles of FGF401 co¬
RP2D of FGF401 in administration with PDR001
combination with PDR001
Phase II part FGF401 in combination with PDR001
To investigate the anti-tumor ORR (overall response rate) based on local assessment activity of FGF401 in per RECIST v1 .1
combination with PDR001
Secondary (Phase I and II part)
To characterize the safety and Safety: Incidence and severity of AEs and SAEs, including tolerability of FGF401 in changes in laboratory values, vital signs and ECGs combination with PDR001 Tolerability: Dose interruptions and reductions
To further investigate the antiPhase I part
tumor activity of FGF401 in BOR (best overall response), ORR (overall response rate), combination with PDR001 DCR (disease control rate), TTP (time to tumor
progression) and OS (overall survival), based on local assessment per RECIST v1 .1
Phase II part
FGF401 in combination with PDR001
TTP (time to tumor progression), PFS (progression free survival), OS (overall survival), BOR (best overall response) and DCR (disease control rate)
Endpoints may also be determined by using Guidelines for immune-related Response Criteria (irRC). Table 1. Amino acid and nucleotide sequences for humanized antibody molecules. The antibody molecules include BAP049-Clone-B and BAP049-Clone-E. The amino acid and nucleotide sequences of the heavy and light chain CDRs, the heavy and light chain variable regions, and the heavy and light chains are shown.
BAP049-Clone-B HC
SEQ ID NO 1 (Kabat) HCDR1 TYWMH
SEQ ID NO 2 (Kabat) HCDR2 NIYPGTGGSNFDEKFKN
SEQ ID NO 3 (Kabat) HCDR3 WTTGTGAY
SEQ ID NO 4 (Chothia) HCDR1 GYTFTTY
SEQ ID NO 5 (Chothia) HCDR2 YPGTGG
SEQ ID NO 3 (Chothia) HCDR3 WTTGTGAY
EVQLVQSGAEVKKPGESLRISCKGSGY
TFTTYWMHWVRQATGQGLEWMGNIYP
GTGGSNFDEKFKNRVTITADKSTSTAY
MELSSLRSEDTAVYYCTRWTTGTGAY
SEQ ID NO: 38 VH WGQGTTVTVSS
GAGGTGCAGCTGGTGCAGTCAGGCG
CCGAAGTGAAGAAGCCCGGCGAGTC
ACTGAGAATTAGCTGTAAAGGTTCAG
GCTACACCTTCACTACCTACTGGATG
CACTGGGTCCGCCAGGCTACCGGTC
AAGGCCTCGAGTGGATGGGTAATATC
TACCCCGGCACCGGCGGCTCTAACTT
CGACGAGAAG I I I AAGAATAGAGTGA
CTATCACCGCCGATAAGTCTACTAGC
ACCGCCTATATGGAACTGTCTAGCCT
GAGATCAGAGGACACCGCCGTCTACT
ACTGCACTAGGTGGACTACCGGCACA
GGCGCCTACTGGGGTCAAGGCACTA
SEQ ID NO: 95 DNA VH CCGTGACCGTGTCTAGC
EVQLVQSGAEVKKPGESLRISCKGSGY
TFTTYWMHWVRQATGQGLEWMGNIYP
GTGGSNFDEKFKNRVTITADKSTSTAY
MELSSLRSEDTAVYYCTRWTTGTGAY
WGQGTTVTVSSASTKGPSVFPLAPCSR
SEQ ID NO: 91 HC STS ESTAALG C L VKD YF P E P VTVS WN S GALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEFLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPE
VQFNWYVDGVEVHNAKTKPREEQFNS
TYRWSVLTVLHQDWLNGKEYKCKVSN
KGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLY
SRLTVDKSRWQEGNVFSCSVMHEALH
NHYTQKSLSLSLG
GAGGTGCAGCTGGTGCAGTCAGGCG
CCGAAGTGAAGAAGCCCGGCGAGTC
ACTGAGAATTAGCTGTAAAGGTTCAG
GCTACACCTTCACTACCTACTGGATG
CACTGGGTCCGCCAGGCTACCGGTC
AAGGCCTCGAGTGGATGGGTAATATC
TACCCCGGCACCGGCGGCTCTAACTT
CGACGAGAAGTTTAAGAATAGAGTGA
CTATCACCGCCGATAAGTCTACTAGC
ACCGCCTATATGGAACTGTCTAGCCT
GAGATCAGAGGACACCGCCGTCTACT
ACTGCACTAGGTGGACTACCGGCACA
GGCGCCTACTGGGGTCAAGGCACTA
CCGTGACCGTGTCTAGCGCTAGCACT
AAGGGCCCGTCCGTGTTCCCCCTGG
CACCTTGTAGCCGGAGCACTAGCGAA
TCCACCGCTGCCCTCGGCTGCCTGGT
CAAGGATTACTTCCCGGAGCCCGTGA
CCGTGTCCTGGAACAGCGGAGCCCT
GACCTCCGGAGTGCACACCTTCCCCG
CTGTGCTGCAGAGCTCCGGGCTGTAC
TCGCTGTCGTCGGTGGTCACGGTGCC
TTCATCTAGCCTGGGTACCAAGACCT
ACACTTGCAACGTGGACCACAAGCCT
SEQ ID NO: 96 DNA HC TCCAACACTAAGGTGGACAAGCGCGT
Figure imgf000073_0001
WASTRESGVPSRFSGSGSGTDFTFTIS
SLQPEDIATYYCQNDYSYPYTFGQGTK VEIK
GAGATCGTCCTGACTCAGTCACCCGC
TACCCTGAGCCTGAGCCCTGGCGAG
CGGGCTACACTGAGCTGTAAATCTAG
TCAGTCACTGCTGGATAGCGGTAATC
AGAAGAACTTCCTGACCTGGTATCAG
CAGAAGCCCGGTAAAGCCCCTAAGCT
GCTGATCTACTGGGCCTCTACTAGAG
AATCAGGCGTGCCCTCTAGGTTTAGC
GGTAGCGGTAGTGGCACCGACTTCAC
CTTCACTATCTCTAGCCTGCAGCCCG
AGGATATCGCTACCTACTACTGTCAG
AACGACTATAGCTACCCCTACACCTTC
GGTCAAGGCACTAAGGTCGAGATTAA
SEQ ID NO: 97 DNA VL G
EIVLTQSPATLSLSPGERATLSCKSSQS
LLDSGNQKNFLTWYQQKPGKAPKLLIY
WASTRESGVPSRFSGSGSGTDFTFTIS
SLQPEDIATYYCQNDYSYPYTFGQGTK
VEIKRTVAAPSVFIFPPSDEQLKSGTAS
VVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNR
SEQ ID NO: 56 LC GEC
GAGATCGTCCTGACTCAGTCACCCGC
TACCCTGAGCCTGAGCCCTGGCGAG
CGGGCTACACTGAGCTGTAAATCTAG
TCAGTCACTGCTGGATAGCGGTAATC
AGAAGAACTTCCTGACCTGGTATCAG
CAGAAGCCCGGTAAAGCCCCTAAGCT
GCTGATCTACTGGGCCTCTACTAGAG
AATCAGGCGTGCCCTCTAGGTTTAGC
GGTAGCGGTAGTGGCACCGACTTCAC
SEQ ID NO: 98 DNA LC CTTCACTATCTCTAGCCTGCAGCCCG AGGATATCGCTACCTACTACTGTCAG
AACGACTATAGCTACCCCTACACCTTC
GGTCAAGGCACTAAGGTCGAGATTAA
GCGTACGGTGGCCGCTCCCAGCGTG
TTCATCTTCCCCCCCAGCGACGAGCA
GCTGAAGAGCGGCACCGCCAGCGTG
GTGTGCCTGCTGAACAACTTCTACCC
CCGGGAGGCCAAGGTGCAGTGGAAG
GTGGACAACGCCCTGCAGAGCGGCA
ACAGCCAGGAGAGCGTCACCGAGCA
GGACAGCAAGGACTCCACCTACAGCC
TGAGCAGCACCCTGACCCTGAGCAAG
GCCGACTACGAGAAGCATAAGGTGTA
CGCCTGCGAGGTGACCCACCAGGGC
CTGTCCAGCCCCGTGACCAAGAGCTT
CAACAGGGGCGAGTGC
GAAGTGCAGCTGGTGCAGTCTGGCG
CCGAAGTGAAGAAGCCTGGCGAGTC
CCTGCGGATCTCCTGCAAGGGCTCTG
GCTACACCTTCACCACCTACTGGATG
CACTGGGTGCGACAGGCTACCGGCC
AGGGCCTGGAATGGATGGGCAACATC
TATCCTGGCACCGGCGGCTCCAACTT
CGACGAGAAGTTCAAGAACAGAGTGA
CCATCACCGCCGACAAGTCCACCTCC
ACCGCCTACATGGAACTGTCCTCCCT
GAGATCCGAGGACACCGCCGTGTACT
ACTGCACCCGGTGGACAACCGGCAC
AGGCGCTTATTGGGGCCAGGGCACC
ACAGTGACCGTGTCCTCTGCTTCTAC
CAAGGGGCCCAGCGTGTTCCCCCTG
GCCCCCTGCTCCAGAAGCACCAGCG
AGAGCACAGCCGCCCTGGGCTGCCT
GGTGAAGGACTACTTCCCCGAGCCCG
TGACCGTGTCCTGGAACAGCGGAGC
SEQ ID NO: 92 DNA HC CCTGACCAGCGGCGTGCACACCTTCC CCGCCGTGCTGCAGAGCAGCGGCCT
GTACAGCCTGAGCAGCGTGGTGACC
GTGCCCAGCAGCAGCCTGGGCACCA
AGACCTACACCTGTAACGTGGACCAC
AAGCCCAGCAACACCAAGGTGGACAA
GAGGGTGGAGAGCAAGTACGGCCCA
CCCTGCCCCCCCTGCCCAGCCCCCG
AGTTCCTGGGCGGACCCAGCGTGTTC
CTGTTCCCCCCCAAGCCCAAGGACAC
CCTGATGATCAGCAGAACCCCCGAGG
TGACCTGTGTGGTGGTGGACGTGTCC
CAGGAGGACCCCGAGGTCCAGTTCAA
CTGGTACGTGGACGGCGTGGAGGTG
CACAACGCCAAGACCAAGCCCAGAGA
GGAGCAGTTTAACAGCACCTACCGGG
TGGTGTCCGTGCTGACCGTGCTGCAC
CAGGACTGGCTGAACGGCAAAGAGTA
CAAGTGTAAGGTCTCCAACAAGGGCC
TGCCAAGCAGCATCGAAAAGACCATC
AGCAAGGCCAAGGGCCAGCCTAGAG
AGCCCCAGGTCTACACCCTGCCACCC
AGCCAAGAGGAGATGACCAAGAACCA
GGTGTCCCTGACCTGTCTGGTGAAGG
GCTTCTACCCAAGCGACATCGCCGTG
GAGTGGGAGAGCAACGGCCAGCCCG
AGAACAACTACAAGACCACCCCCCCA
GTGCTGGACAGCGACGGCAGCTTCTT
CCTGTACAGCAGGCTGACCGTGGACA
AGTCCAGATGGCAGGAGGGCAACGT
CTTTAGCTGCTCCGTGATGCACGAGG
CCCTGCACAACCACTACACCCAGAAG
AGCCTGAGCCTGTCCCTGGGC
BAP049-Clone-E HC
SEQ ID NO: 1 (Kabat) HCDR1 TYWMH
SEQ ID NO: 2 (Kabat) HCDR2 NIYPGTGGSNFDEKFKN
SEQ ID NO: 3 (Kabat) HCDR3 WTTGTGAY SEQ ID NO: 4 (Chothia) HCDR1 GYTFTTY
SEQ ID NO: 5 (Chothia) HCDR2 YPGTGG
SEQ ID NO: 3 (Chothia) HCDR3 WTTGTGAY
EVQLVQSGAEVKKPGESLRISCKGSGY
TFTTYWMHWVRQATGQGLEWMGNIYP
GTGGSNFDEKFKNRVTITADKSTSTAY
MELSSLRSEDTAVYYCTRWTTGTGAY
SEQ ID NO: 38 VH WGQGTTVTVSS
GAGGTGCAGCTGGTGCAGTCAGGCG
CCGAAGTGAAGAAGCCCGGCGAGTC
ACTGAGAATTAGCTGTAAAGGTTCAG
GCTACACCTTCACTACCTACTGGATG
CACTGGGTCCGCCAGGCTACCGGTC
AAGGCCTCGAGTGGATGGGTAATATC
TACCCCGGCACCGGCGGCTCTAACTT
CGACGAGAAGTTTAAGAATAGAGTGA
CTATCACCGCCGATAAGTCTACTAGC
ACCGCCTATATGGAACTGTCTAGCCT
GAGATCAGAGGACACCGCCGTCTACT
ACTGCACTAGGTGGACTACCGGCACA
GGCGCCTACTGGGGTCAAGGCACTA
SEQ ID NO: 95 DNA VH CCGTGACCGTGTCTAGC
EVQLVQSGAEVKKPGESLRISCKGSGY
TFTTYWMHWVRQATGQGLEWMGNIYP
GTGGSNFDEKFKNRVTITADKSTSTAY
MELSSLRSEDTAVYYCTRWTTGTGAY
WGQGTTVTVSSASTKGPSVFPLAPCSR
STSESTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEFLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPE
VQFNWYVDGVEVHNAKTKPREEQFNS
TYRWSVLTVLHQDWLNGKEYKCKVSN
KGLPSSIEKTISKAKGQPREPQVYTLPP
SEQ ID NO: 91 HC SQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLY
SRLTVDKSRWQEGNVFSCSVMHEALH
NHYTQKSLSLSLG
GAGGTGCAGCTGGTGCAGTCAGGCG
CCGAAGTGAAGAAGCCCGGCGAGTC
ACTGAGAATTAGCTGTAAAGGTTCAG
GCTACACCTTCACTACCTACTGGATG
CACTGGGTCCGCCAGGCTACCGGTC
AAGGCCTCGAGTGGATGGGTAATATC
TACCCCGGCACCGGCGGCTCTAACTT
CGACGAGAAG 1 1 1 AAGAATAGAGTGA
CTATCACCGCCGATAAGTCTACTAGC
ACCGCCTATATGGAACTGTCTAGCCT
GAGATCAGAGGACACCGCCGTCTACT
ACTGCACTAGGTGGACTACCGGCACA
GGCGCCTACTGGGGTCAAGGCACTA
CCGTGACCGTGTCTAGCGCTAGCACT
AAGGGCCCGTCCGTGTTCCCCCTGG
CACCTTGTAGCCGGAGCACTAGCGAA
TCCACCGCTGCCCTCGGCTGCCTGGT
CAAGGATTACTTCCCGGAGCCCGTGA
CCGTGTCCTGGAACAGCGGAGCCCT
GACCTCCGGAGTGCACACCTTCCCCG
CTGTGCTGCAGAGCTCCGGGCTGTAC
TCGCTGTCGTCGGTGGTCACGGTGCC
TTCATCTAGCCTGGGTACCAAGACCT
ACACTTGCAACGTGGACCACAAGCCT
TCCAACACTAAGGTGGACAAGCGCGT
CGAATCGAAGTACGGCCCACCGTGCC
CGCCTTGTCCCGCGCCGGAGTTCCTC
GGCGGTCCCTCGGTC 1 1 1 CTGTTCCC
ACCGAAGCCCAAGGACAC 1 1 1 GATGA
1 1 1 CCCGCACCCCTGAAGTGACATGC
GTGGTCGTGGACGTGTCACAGGAAGA
TCCGGAGGTGCAGTTCAATTGGTACG
SEQ ID NO: 96 DNA HC TGGATGGCGTCGAGGTGCACAACGC CAAAACCAAGCCGAGGGAGGAGCAG
TTCAACTCCACTTACCGCGTCGTGTC
CGTGCTGACGGTGCTGCATCAGGACT
GGCTGAACGGGAAGGAGTACAAGTG
CAAAGTGTCCAACAAGGGACTTCCTA
GCTCAATCGAAAAGACCATCTCGAAA
GCCAAGGGACAGCCCCGGGAACCCC
AAGTGTATACCCTGCCACCGAGCCAG
GAAGAAATGACTAAGAACCAAGTCTC
ATTGACTTGCCTTGTGAAGGGCTTCTA
CCCATCGGATATCGCCGTGGAATGGG
AGTCCAACGGCCAGCCGGAAAACAAC
TACAAGACCACCCCTCCGGTGCTGGA
CTCAGACGGATCCTTCTTCCTCTACTC
GCGGCTGACCGTGGATAAGAGCAGAT
GGCAGGAGGGAAATGTGTTCAGCTGT
TCTGTG ATG C ATG AAG CC CTG C AC AA
CCACTACACTCAGAAGTCCCTGTCCC
TCTCCCTGGGA
BAP049-Clone-E LC
SE a ID NO: 10 (Kabat) LCDR1 KSSQSLLDSGNQKNFLT
SEQ ID NO 11 (Kabat) LCDR2 WASTRES
SEQ ID NO 32 (Kabat) LCDR3 QNDYSYPYT
SEQ ID NO Ϊ3 (Chothia) LCDR1 "SQSTLDSGNQKNF
SEQ ID NO 14 (Chothia) LCDR2 WAS
SEQ ID NO 33 OTOthia) LCDR3 DYSYPY
EIVLTQSPATLSLSPGERATLSCKSSQS LLDSGNQKNFLTWYQQKPGQAPRLLIY WASTRESGVPSRFSGSGSGTDFTFTIS SLEAEDAATYYCQNDYSYPYTFGQGTK
SEQ ID NO: 70 VL VEIK
GAGATCGTCCTGACTCAGTCACCCGC
TACCCTGAGCCTGAGCCCTGGCGAG
CGGGCTACACTGAGCTGTAAATCTAG
TCAGTCACTGCTGGATAGCGGTAATC
SEQ ID NO: 106 DNA VL AGAAGAACTTCCTGACCTGGTATCAG CAGAAGCCCGGTCAAGCCCCTAGACT
GCTGATCTACTGGGCCTCTACTAGAG
AATCAGGCGTGCCCTCTAGGTTTAGC
GGTAGCGGTAGTGGCACCGACTTCAC
CTTCACTATCTCTAGCCTGGAAGCCG
AGGACGCCGCTACCTACTACTGTCAG
AACGACTATAGCTACCCCTACACCTTC
GGTCAAGGCACTAAGGTCGAGATTAA
G
EIVLTQSPATLSLSPGERATLSCKSSQS
LLDSGNQKNFLTWYQQKPGQAPRLLIY
WASTRESGVPSRFSGSGSGTDFTFTIS
SLEAEDAATYYCQNDYSYPYTFGQGTK
VEIKRTVAAPSVFIFPPSDEQLKSGTAS
VVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNR
SEQ ID NO: 72 LC GEC
GAGATCGTCCTGACTCAGTCACCCGC
TACCCTGAGCCTGAGCCCTGGCGAG
CGGGCTACACTGAGCTGTAAATCTAG
TCAGTCACTGCTGGATAGCGGTAATC
AGAAGAACTTCCTGACCTGGTATCAG
CAGAAGCCCGGTCAAGCCCCTAGACT
GCTGATCTACTGGGCCTCTACTAGAG
AATCAGGCGTGCCCTCTAGGTTTAGC
GGTAGCGGTAGTGGCACCGACTTCAC
CTTCACTATCTCTAGCCTGGAAGCCG
AGGACGCCGCTACCTACTACTGTCAG
AACGACTATAGCTACCCCTACACCTTC
GGTCAAGGCACTAAGGTCGAGATTAA
GCGTACGGTGGCCGCTCCCAGCGTG
TTCATCTTCCCCCCCAGCGACGAGCA
GCTGAAGAGCGGCACCGCCAGCGTG
GTGTGCCTGCTGAACAACTTCTACCC
SEQ ID NO: 107 DNA LC CCGGGAGGCCAAGGTGCAGTGGAAG GTGGACAACGCCCTGCAGAGCGGCA
ACAGCCAGGAGAGCGTCACCGAGCA
GGACAGCAAGGACTCCACCTACAGCC
TGAGCAGCACCCTGACCCTGAGCAAG
GCCGACTACGAGAAGCATAAGGTGTA
CGCCTGCGAGGTGACCCACCAGGGC
CTGTCCAGCCCCGTGACCAAGAGCTT
CAACAGGGGCGAGTGC
BAP049-Clone-B HC
SEQ ID NO: 133 (Kabat) HCDR1 ACCTACTGGATGCAC
AATATCTACCCCGGCACCGGCGGCTC
SEQ ID NO 134 (Kabat) HCDR2 TAACTTCGACGAGAAG I I I AAGAAT
SEQ ID NO 135 (Kabat) HCDR3 TGGACTACCGGCACAGGCGCCTAC
SEQ ID NO 136 (Chothia) HCDR1 GGCTACACCTTCACTACCTAC
SEQ ID NO 137 (Chothia) HCDR2 TACCCCGGCACCGGCGGC
SEQ ID NO 135 (Chothia) HCDR3 TGGACTACCGGCACAGGCGCCTAC
BAP049-Clone-B LC
AAATCTAGTCAGTCACTGCTGGATAG
SEQ ID NO: 138 (Kabat) LCDR1 CGGTAATCAGAAGAACTTCCTGACC
SEQ ID NO: 139 (Kabat) LCDR2 TGGGCCTCTACTAGAGAATCA
CAGAACGACTATAGCTACCCCTACAC
SEQ ID NO: 140 (Kabat) LCDR3 C
AGTCAGTCACTGCTGGATAGCGGTAA
SEQ ID NO: 141 (Chothia) LCDR1 TCAGAAGAACTTC
SEQ ID NO: 142 (Chothia) LCDR2 TGGGCCTCT
SEQ ID NO: 143 (Chothia) LCDR3 GACTATAGCTACCCCTAC
BAP049-Clone-E HC
SEQ ID NO: 133 (Kabat) HCDR1 ACCTACTGGATGCAC
AATATCTACCCCGGCACCGGCGGCTC
SEQ ID NO: 134 (Kabat) HCDR2 TAACTTCGACGAGAAG I I I AAGAAT
SEQ ID NO: 135 (Kabat) HCDR3 TGGACTACCGGCACAGGCGCCTAC
SEQ ID NO: 136 (Chothia) HCDR1 GGCTACACCTTCACTACCTAC
SEQ ID NO: 137 (Chothia) HCDR2 TACCCCGGCACCGGCGGC
SEQ ID NO: 135 (Chothia) HCDR3 TGGACTACCGGCACAGGCGCCTAC
BAP049-Clone-E LC AAATCTAGTCAGTCACTGCTGGATAG
SEQ ID NO: 138 (Kabat) LCDR1 CGGTAATCAGAAGAACTTCCTGACC
SEQ ID NO: 139 (Kabat) LCDR2 TGGGCCTCTACTAGAGAATCA
CAGAACGACTATAGCTACCCCTACAC
SEQ ID NO: 140 (Kabat) LCDR3 C
AGTCAGTCACTGCTGGATAGCGGTAA
SEQ ID NO: 141 (Chothia) LCDR1 TCAGAAGAACTTC
SEQ ID NO: 142 (Chothia) LCDR2 TGGGCCTCT
SEQ ID NO: 143 (Chothia) LCDR3 GACTATAGCTACCCCTAC
Table 2. Amino acid and nucleotide sequences of the heavy and light chain framework regions for humanized mAbs BAP049-Clone-B and BAP049-Clone-E
Amino Acid Sequence Nucleotide Sequence
VHFW1 EVQLVQSGAEVKKPGESLRISCK GAAGTGCAGCTGGTGCAGTCTGGAGCAGA
(type a) GS (SEQ ID NO: 147) GGTGAAAAAGCCCGGGGAGTCTCTGAGGA
TCTCCTGTAAGGGTTCT (SEQ ID NO: 148)
GAAGTGCAGCTGGTGCAGTCTGGCGCCGA AGTGAAGAAGCCTGGCGAGTCCCTGCGGA TCTCCTGCAAGGGCTCT (SEQ ID NO: 149)
GAGGTGCAGCTGGTGCAGTCAGGCGCCGA AGTGAAGAAGCCCGGCGAGTCACTGAGAA TTAG CTGTAAAG GTTC A (SEQ ID NO: 150)
VHFW1 QVQLVQSGAEVKKPGASVKVSC CAGGTTCAGCTGGTGCAGTCTGGAGCTGA
(type b) KAS (SEQ ID NO: 151) GGTGAAGAAGCCTGGGGCCTCAGTGAAGG
TCTCCTGCAAGGCTTCT (SEQ ID NO: 152)
VHFW2 WVRQATGQGLEWMG TGGGTGCGACAGGCCACTGGACAAGGGCT
(type a) (SEQ ID NO: 153) TGAGTGGATGGGT (SEQ ID NO: 154)
TGGGTGCGACAGGCTACCGGCCAGGGCCT GGAATGGATGGGC (SEQ ID NO: 155)
TGGGTCCGCCAGGCTACCGGTCAAGGCCT CGAGTGGATGGGT (SEQ ID NO: 156)
VHFW2 WIRQSPSRGLEWLG TGGATCAGGCAGTCCCCATCGAGAGGCCTT
(type b) (SEQ ID NO: 157) GAGTGGCTGGGT (SEQ ID NO: 158)
TGGATCCGGCAGTCCCCCTCTAGGGGCCT GGAATGGCTGGGC (SEQ ID NO: 159)
VHFW2 WVRQAPGQGLEWMG TGGGTGCGACAGGCCCCTGGACAAGGGCT
(type c) (SEQ ID NO: 160) TGAGTGGATGGGT (SEQ ID NO: 161)
VHFW3 RVTITADKSTSTAYMELSSLRSE AGAGTCACGATTACCGCGGACAAATCCACG
(type a) DTAVYYCTR (SEQ ID NO: 162) AGCACAGCCTACATGGAGCTGAGCAGCCT
GAGATCTGAGGACACGGCCGTGTATTACTG TACAAGA (SEQ ID NO: 163)
AGAGTGACCATCACCGCCGACAAGTCCACC TCCACCGCCTACATGGAACTGTCCTCCCTG AGATCCGAGGACACCGCCGTGTACTACTGC ACCCGG (SEQ ID NO: 164)
AGAGTGACTATCACCGCCGATAAGTCTACT AGCACCGCCTATATGGAACTGTCTAGCCTG AGATCAGAGGACACCGCCGTCTACTACTGC ACTAGG (SEQ ID NO: 165)
VHFW3 RFTISRDNSKNTLYLQMNSLRAE AGATTCACCATCTCCAGAGACAATTCCAAG
(type b) DTAVYYCTR (SEQ ID NO: 166) AACACGCTGTATCTTCAAATGAACAGCCTG
AGAGCCGAGGACACGGCCGTGTATTACTGT ACAAGA (SEQ ID NO: 167)
AGGTTCACCATCTCCCGGGACAACTCCAAG AACACCCTGTACCTGCAGATGAACTCCCTG CGGGCCGAGGACACCGCCGTGTACTACTG TACCAGA (SEQ ID NO: 168)
VHFW4 WGQGTTVTVSS TGGGGCCAGGGCACCACCGTGACCGTGTC
(SEQ ID NO: 169) CTCC (SEQ ID NO: 170) TGGGGCCAGGGCACCACAGTGACCGTGTC
CTCT (SEQ ID NO: 171)
TGGGGTCAAGGCACTACCGTGACCGTGTCT AGC (SEQ ID NO: 172)
TGGGGCCAGGGCACAACAGTGACCGTGTC CTCC (SEQ ID NO: 173)
VLFW1 EIVLTQSPDFQSVTPKEKVTITC GAAATTGTGCTGACTCAGTCTCCAGACTTT
(type a) (SEQ ID NO: 174) CAGTCTGTGACTCCAAAGGAGAAAGTCACC
ATCACCTGC (SEQ ID NO: 175)
GAGATCGTGCTGACCCAGTCCCCCGACTTC CAGTCCGTGACCCCCAAAGAAAAAGTGACC ATCACATGC (SEQ ID NO: 176)
VLFW1 EIVLTQSPATLSLSPGERATLSC GAAATTGTGTTGACACAGTCTCCAGCCACC
(type b) (SEQ ID NO: 177) CTGTC I I I GTCTCCAGGGGAAAGAGCCACC
CTCTCCTGC (SEQ ID NO: 178)
GAGATCGTGCTGACCCAGTCCCCTGCCAC CCTGTCACTGTCTCCAGGCGAGAGAGCTAC CCTGTCCTGC (SEQ ID NO: 179)
GAGATCGTCCTGACTCAGTCACCCGCTACC CTGAGCCTGAGCCCTGGCGAGCGGGCTAC ACTGAGCTGT (SEQ ID NO: 180)
VLFW1 DIVMTQTPLSLPVTPGEPASISC GATATTGTGATGACCCAGACTCCACTCTCC
(type c) (SEQ ID NO: 181 ) CTGCCCGTCACCCCTGGAGAGCCGGCCTC
CATCTCCTGC (SEQ ID NO: 182)
VLFW1 DVVMTQSPLSLPVTLGQPASISC GATGTTGTGATGACTCAGTCTCCACTCTCC
(type d) (SEQ ID NO: 183) CTGCCCGTCACCCTTGGACAGCCGGCCTC CATCTCCTGC (SEQ ID NO: 184)
VLFW1 DIQMTQSPSSLSASVGDRVTITC GACATCCAGATGACCCAGTCTCCATCCTCC
(type e) (SEQ ID NO: 185) CTGTCTGCATCTGTAGGAGACAGAGTCACC
ATCACTTGC (SEQ ID NO: 186)
VLFW2 WYQQKPGQAPRLLIY TGGTACCAGCAGAAACCTGGCCAGGCTCC
(type a) (SEQ ID NO: 187) CAGGCTCCTCATCTAT (SEQ ID NO: 188)
TGGTATCAGCAGAAGCCCGGCCAGGCCCC CAGACTGCTGATCTAC (SEQ ID NO: 189)
TGGTATCAGCAGAAGCCCGGTCAAGCCCCT AGACTGCTGATCTAC (SEQ ID NO: 190)
VLFW2 WYQQKPGKAPKLLIY TGGTATCAGCAGAAACCAGGGAAAGCTCCT
(type b) (SEQ ID NO: 191 ) AAGCTCCTGATCTAT (SEQ ID NO: 192)
TGGTATCAGCAGAAGCCCGGTAAAGCCCCT AAGCTGCTGATCTAC (SEQ ID NO: 193)
VLFW2 WYLQKPGQSPQLLIY TGGTACCTGCAGAAGCCAGGGCAGTCTCC
(type c) (SEQ ID NO: 194) ACAGCTCCTGATCTAT (SEQ ID NO: 195)
VLFW3 GVPSRFSGSGSGTDFTFTISSLE GGGGTCCCCTCGAGGTTCAGTGGCAGTGG
(type a) AEDAATYYC (SEQ ID NO: 196) ATCTGGGACAGATTTCACCTTTACCATCAGT
AGC CTG G AAGCTG AAG ATG CTG C AAC ATAT TACTGT (SEQ ID NO: 197)
GGCGTGCCCTCTAGATTCTCCGGCTCCGG CTCTGGCACCGAC I I I ACCTTCACCATCTC CAGCCTGGAAGCCGAGGACGCCGCCACCT ACTACTGC (SEQ ID NO: 198)
GGCGTGCCCTCTAGG I I I AGCGGTAGCGG
TAGTGGCACCGACTTCACCTTCACTATCTCT
AGCCTGGAAGCCGAGGACGCCGCTACCTA CTACTGT (SEQ ID NO: 199)
VLFW3 GIPPRFSGSGYGTDFTLTINNIES GGGATCCCACCTCGATTCAGTGGCAGCGG
(type b) EDAAYYFC (SEQ ID NO: 200) GTATGGAACAGA I I I I ACCCTCACAATTAAT
AAC ATAG AATCTG AG G ATG CTGC ATATTACT TCTGT (SEQ ID NO: 201)
VLFW3 GVPSRFSGSGSGTEFTLTISSLQ GGGGTCCCATCAAGGTTCAGCGGCAGTGG
(type c) PDDFATYYC (SEQ ID NO: 202) ATCTGGGACAGAATTCACTCTCACCATCAG
CAGCCTGCAGCCTGATGA I I I I GCAACTTA TTACTGT (SEQ ID NO: 203)
GGCGTGCCCTCTAGATTCTCCGGCTCCGG CTCTGGCACCGAG I I I ACCCTGACCATCTC CAGCCTGCAGCCCGACGACTTCGCCACCT ACTACTGC (SEQ ID NO: 204)
VLFW3 GVPSRFSGSGSGTDFTFTISSLQ GGGGTCCCATCAAGGTTCAGTGGAAGTGG
(type d) PEDIATYYC (SEQ ID NO: 205) ATCTGGGACAGATTTTACTTTCACCATCAGC
AGC CTG C AG CCTG AAG ATATTG C AAC AT AT TACTGT (SEQ ID NO: 206)
GGCGTGCCCTCTAGG I I I AGCGGTAGCGG TAGTGGCACCGACTTCACCTTCACTATCTCT AGCCTGCAGCCCGAGGATATCGCTACCTAC TACTGT (SEQ ID NO: 207)
VLFW4 FGQGTKVEIK (SEQ ID NO: 208) TTCGGCCAAGGGACCAAGGTGGAAATCAAA
(SEQ ID NO: 209)
TTCGGCCAGGGCACCAAGGTGGAAATCAA
G (SEQ ID NO: 210)
TTCGGTCAAGGCACTAAGGTCGAGATTAAG
(SEQ ID NO: 21 1 ) Table 3. Constant region amino acid sequences of human IgG heavy chains and human kappa light chain
HC lgG4 (S228P) mutant constant region amino acid sequence (EU Numbering)
ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS W SGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 212)
LC Human kappa constant region amino acid sequence
RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC (SEQ ID NO: 213)
HC lgG4 (S228P) mutant constant region amino acid sequence lacing C-terminal lysine (K) (EU Numbering)
ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG (SEQ ID NO: 214)
HC lgG1 wild type
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 215)
HC lgG1 (N297A) mutant constant region amino acid sequence (EU Numbering) ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYA STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 216)
HC lgG1 (D265A, P329A) mutant constant region amino acid sequence (EU Numbering)
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVAVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LAAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 217)
HC lgG1 (L234A, L235A) mutant constant region amino acid sequence (EU Numbering)
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 218)
Table 4. Amino acid sequences of the heavy and light chain leader sequences for humanized mAbs BAP049-Clone-B and BAP049-Clone-E
BAP049-Clone-B HC MAWVWTLPFLMAAAQSVQA (SEQ ID NO: 221 )
LC MSVLTQVLALLLLWLTGTRC (SEQ ID NO: 222)
BAP049-Clone-E HC MAWVWTLPFLMAAAQSVQA (SEQ ID NO: 221 ) LC MSVLTQVLALLLLWLTGTRC (SEQ ID NO: 222)

Claims

Claims
1 . A pharmaceutical combination comprising an anti-PD-1 antibody molecule and N-(5- cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in
pharmaceutically acceptable salt form.
2. A pharmaceutical combination according to claim 1 , wherein the anti-PD-1 antibody molecule is an isolated antibody molecule capable of binding to a human Programmed Death-1 (PD-1) comprising a heavy chain variable region (VH) comprising a HCDR1 , a HCDR2 and a HCDR3 amino acid sequence of BAP049-Clone-B or BAP049-Clone-E as described in Table 1 and a light chain variable region (VL) comprising a LCDR1 , a LCDR2 and a LCDR3 amino acid sequence of BAP049-Clone-B or BAP049-Clone-E as described in Table 1 .
3. A pharmaceutical combination according to claim 1 , wherein the anti-PD-1 antibody molecule comprises:
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33; or
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32.
4. A pharmaceutical combination according to claim 1 or 2 or 3, wherein the anti-PD-1 antibody molecule comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70.
5. A pharmaceutical combination according to any one of claims 1 to 4, wherein the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72.
6. The pharmaceutical combination according to any one of claims 1 to 5 for use as a medicament.
7. A pharmaceutical combination according to any one of claims 1 to 5 for use in the treatment of cancer.
8. A pharmaceutical combination according to any one of claims 1 to 5 for use in the treatment of liver cancer (HCC).
9. A pharmaceutical combination according to any one of claims 1 to 5 for use
according to claim 8 wherein the HCC is characterized by positive FGFR4 and KLB expression.
10. A pharmaceutical combination according to any one of claims 1 to 5 for use
according to claim 8 or 9 wherein the HCC has progressed during or after discontinuation of sorafenib treatment or wherein the patient suffering from the HCC is intolerant to sorafenib treatment.
1 1 . A pharmaceutical combination comprising an FGFR4 inhibitor and an anti-PD-1
antibody molecule for use in the treatment of liver cancer.
12. A method of treating cancer in a subject in need thereof comprising administering to said subject simultaneously or sequentially N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule comprises: (a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70; or
(f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72.
13. A method according to claim 9, wherein the cancer is liver cancer.
14. A therapeutic regimen comprising the simultaneous or sequential administration of N- (5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form and an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule comprises:
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of
SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70; or
(f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72.
15. A therapeutic regimen according to claim 14, wherein the anti-PD-1 antibody
molecule is administered at a dose of about 300 mg once every three weeks.
16. A therapeutic regimen according to claim 14, wherein the anti-PD-1 antibody
molecule is administered at a dose of about 400 mg once every four weeks.
17. A therapeutic regimen according to any one of claims 14 to 16 wherein N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide is administered at a dose of about 80 to 120 mg daily.
18. The therapeutic regimen of claim 17 wherein N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide is administered at a dose of about 80 mg daily.
A therapeutic regimen according to any one of claims 14 to 18 for the treatment of liver cancer.
20. Use of a pharmaceutical combination according to any one of claims 1 to 5 for the preparation of a medicament for the treatment of liver cancer.
21 . A kit comprising a pharmaceutical combination according to any one of claims 1 to 5 with instructions for simultaneous or sequential administration of N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide in free form or in pharmaceutically acceptable salt form and the anti-PD-1 antibody molecule.
22. A pharmaceutical combination comprising N-(5-cyano-4-((2- methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 -yl)methyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide, or a pharmaceutically acceptable salt thereof, and an anti-PD-1 antibody molecule, wherein the anti-PD-1 antibody molecule comprises:
(a) a heavy chain variable region (VH) comprising a HCDR1 amino acid sequence of SEQ ID NO: 4, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(b) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 1 ; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID
NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(c) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224, a HCDR2 amino acid sequence of SEQ ID NO: 5, and a HCDR3 amino acid sequence of SEQ ID
NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 13, a LCDR2 amino acid sequence of SEQ ID NO: 14, and a LCDR3 amino acid sequence of SEQ ID NO: 33;
(d) a VH comprising a HCDR1 amino acid sequence of SEQ ID NO: 224; a HCDR2 amino acid sequence of SEQ ID NO: 2; and a HCDR3 amino acid sequence of SEQ ID
NO: 3; and a VL comprising a LCDR1 amino acid sequence of SEQ ID NO: 10, a LCDR2 amino acid sequence of SEQ ID NO: 1 1 , and a LCDR3 amino acid sequence of SEQ ID NO: 32;
(e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 70; or
(f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 72,
for use in treating HCC, wherein the anti-PD-1 antibody molecule is administered in a dose of about 300 mg to 400 mg once every three weeks or once every four weeks.
23. A pharmaceutical combination for use according to claim 22, wherein the anti-PD-1 antibody molecule is administered at a dose of about 300 mg once every three weeks.
24. A pharmaceutical combination for use according to claim 22, wherein the anti-PD-1 antibody molecule is administered at a dose of about 400 mg once every four weeks.
25. A pharmaceutical combination for use according to any one of claims 22 to 24
wherein N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1 -yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide is administered at a dose of about 50 to 150 mg daily.
26. A pharmaceutical combination for use according to claim 25 wherein N-(5-cyano-4- ((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1 - yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide is administered at a dose of about 80 mg daily or 120 mg daily.
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