Combination therapy for cancer treatment

Abstract

Provided herein are combination therapies comprising a BRAF inhibitor (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof) and an EGFR inhibitor (EGFRi), as well as pharmaceutical compositions, methods, and uses of the combination therapies.

Description

Technical Field

[0001] The present invention relates to a compound of formula (I):

[0002]

[0003] or a pharmaceutically acceptable salt thereof and a combination with an EGFR inhibitor (EGFRi), as well as a pharmaceutical composition, method and use of the combination, wherein the compound of formula (I) is a BRAF inhibitor.

[0004] Sequence Listing

[0005] This application incorporates by reference the computer-readable form (CRF) of the sequence listing in ASCII text format submitted via EFS-Web. The sequence listing text file submitted via EFS-Web, named P38651_SEQ_LISTING_ST26.xml, is 10,068 bytes in size and was created on October 3, 2023. Background Art

[0006] Mutant BRAF is a targetable oncogenic driver, and to date, three BRAF inhibitors (BRAFi) (Vemurafenib, Dabrafenib, Encorafenib) have been marketed, showing efficacy against BRAFV600E-positive melanoma. However, rapid acquisition of drug resistance is almost universally observed, and the duration of the therapeutic benefit of targeted therapies remains limited.

[0007] In addition, the first-generation BRAF inhibitors that have been developed have revealed an unexpected and "paradoxical" ability to inhibit MAPK signaling in BRAF V600E -driven tumors, while the same inhibitors exhibit MAPK-stimulatory activity in BRAF wild-type (WT) models (N. Engl. J. Med., 366:271-273 (2012); and British Journal of Cancer, Volume 111, pages 640 to 645 (2014)).

[0008] Subsequently, mechanistic studies of the RAF paradox have elucidated that oncogenic BRAF V600E phosphorylates MEK1 / 2 in its monomeric cytoplasmic form, while activation of WT BRAF and RAF1 requires a complex series of events, which includes translocation to the cell membrane and homo- and / or hetero-dimerization promoted by activated RAS (KRAS, NRAS, HRAS) (Nature Reviews Cancer, Volume 14, pages 455 to 467 (2014)).

[0009] First-generation BRAF inhibitors (such as vemurafenib, dabrafenib, and encorafenib) bind to WT BRAF or RAF1 protomers rapidly induce RAF homo- and / or hetero-dimerization and membrane association of the newly formed RAF dimers. In the dimeric conformation, one RAF protomer allosterically induces a conformational change in the second RAF protomer, leading to the kinase active state and, importantly, a conformation that is unfavorable for inhibitor binding. As a result, the dimers induced by drug treatment promote MEK phosphorylation through catalysis by the unbound protomers and overactivate the pathway.

[0010] In colorectal cancer (CRC), several preclinical and clinical data suggest that signaling from EGFR contributes to maintaining MAPK activity and to partial activation of RAF dimer formation through the EGFR-RAS pathway. EGFR blockade by anti-EGFR antibodies in combination with first-generation BRAF inhibition has provided some clinical benefits (N. Engl. J. Med., 381:1632-1643 (2019), DOI: 10.1056 / NEJMoa1908075). Although this combination has demonstrated some clinical success, the paradoxical benefits of inducing BRAFi and anti-EGFR antibodies remain limited. Due to the dose-limiting toxicity of first-generation BRAF inhibitors, it is difficult to achieve a C 谷 level covering IC90 or above in this clinical setting. SUMMARY OF THE INVENTION

[0011] Provided herein are solutions to these and other problems in the art.

[0012] In one aspect, the present invention relates to novel combination therapies and uses of compounds of formula (I) (I), or pharmaceutically acceptable salts thereof.

[0013]

[0014] or pharmaceutically acceptable salts thereof. In some embodiments, the compound of formula (I) is (3R)-N-[2-cyano-4-fluoro-3-(3-methyl-4-oxo-quinazolin-6-yl)oxy-phenyl]-3-fluoro-pyrrolidine-1-sulfonamide or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is a BRAF inhibitor. For example, the compound of formula (I) is also described in International Patent Publication No. WO2021116050A1 and is also known as RO7276389.

[0015] On the other hand, the present invention relates to a novel combination of a compound of formula (I) and an EGFR inhibitor, and said combination for the treatment of cancer, in particular colorectal cancer. The compound of formula (I) is a BRAF inhibitor, which, for example, shows negligible paradoxical activation of the MAPK signaling pathway (paradox breaker) when compared to first-generation BRAF inhibitors on the market: encorafenib, dabrafenib, and vemurafenib (paradox inducers). In another example, the compound of formula (I) also has very effective brain penetration properties, thus providing a much-needed alternative therapy for the treatment of leptomeningeal cancer or cancer metastasized to the brain. With excellent safety profiles, the compound of formula (I) allows for much higher individual doses to reach significantly higher C 谷 levels.

[0016] On the other hand, the present invention discloses a novel combination for cancer therapy, which has strong combinatorial activity against BRAF-associated tumors and, for example, has the potential to overcome the rapidly acquired treatment resistance often observed in patients treated with first-generation BRAF inhibitors. In one example, the combination for the treatment of cancer as disclosed in the present invention exhibits unexpected combinatorial activity that exceeds the additive effect of BRAF inhibitor and EGFRi monotherapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For ease of identification of the discussion of any particular element or action, one or more of the most significant digits in the reference numerals refer to the figure number in which the element was first introduced.

[0018] Figure 1 Shows Figures 2 to 5 a schematic diagram of the study design reported in, and prescribes the dosing regimens for different cohorts in a colorectal tumor model with LS411N xenografts according to some embodiments. For example, the same study design was also used to study a colorectal model with HT29 xenografts, as Figures 6 to 9 reported. Both models are CRC models and present the BRAF V600E mutation.

[0019] Figure 2 Reports the in vivo anti-tumor activity of monotherapy of any of the following according to some embodiments against LS411N xenograft tumors in BALB / c nude mice (10 mice / cohort): RO7276389 at 20 mpk (milligrams of compound per kilogram of body weight), 50 mpk, or 140 mpk; or encorafenib at 24 mpk. For example, mice were treated (QD) until day 43, and the graphs represent the mean tumor volume [mm3] for different cohorts.

[0020] Figure 3 The in vivo anti - tumor activity of the combination of RO7276389 at 140 mpk or encorafenib at 24 mpk, in each case with cetuximab at 20 mpk, against LS411N xenograft tumors in BALB / c nude mice (10 mice / cohort) is reported. For example, monotherapy controls are also included, and the mice are treated (QD) until day 43, and the graph represents the mean tumor volume [mm3] for different cohorts. The results are shown on a linear scale (top; Figure 3 a) and on a logarithmic scale (bottom; Figure 3 b).

[0021] Figure 4 The in vivo anti - tumor activity of the combination of RO7276389 at 60 mpk or encorafenib at 24 mpk, in each case with cetuximab at 20 mpk, against LS411N xenograft tumors in BALB / c nude mice (10 mice / cohort) is reported. For example, monotherapy controls are also included, and the mice are treated (QD) until day 43, and the graph represents the mean tumor volume [mm3] for different cohorts. The results are shown on a linear scale (top; Figure 4 a) and on a logarithmic scale (bottom; Figure 4 b).

[0022] Figure 5 The in vivo anti - tumor activity of the combination of RO7276389 at 30 mpk or encorafenib at 24 mpk, in each case with cetuximab at 20 mpk, against LS411N xenograft tumors in BALB / c nude mice (10 mice / cohort) is reported. For example, monotherapy controls are also included, and the mice are treated (QD) until day 43, and the graph represents the mean tumor volume [mm3] for different cohorts. The results are shown on a linear scale (top; Figure 5 a) and on a logarithmic scale (bottom; Figure 5 b).

[0023] Figure 6 The in vivo anti - tumor activity of monotherapy with any of the following, against HT29 xenograft tumors in BALB / c nude mice (10 mice / cohort), is reported: RO7276389 at 20 mpk, 50 mpk or 140 mpk; or encorafenib at 24 mpk. For example, the mice are treated (QD) until day 43, and the graph represents the mean tumor volume [mm3] for different cohorts. The results are shown on a linear scale (top; Figure 6 a) and on a logarithmic scale (bottom; Figure 6 b).

[0024] Figure 7 The in vivo anti - tumor activity of RO7276389 at 140 mpk or encorafenib at 24 mpk in combination with 20 mpk of cetuximab in each case against HT29 xenograft tumors in BALB / c nude mice (10 mice / cohort) according to some embodiments was reported. For example, monotherapy controls were also included, and the mice were treated (QD) until day 43, and the graph represents the mean tumor volume [mm3] for different cohorts. The results are shown on a linear scale (top; Figure 7 a) and on a logarithmic scale (bottom; Figure 7 b).

[0025] Figure 8 The in vivo anti - tumor activity of RO7276389 at 60 mpk or encorafenib at 24 mpk in combination with 20 mpk of cetuximab in each case against HT29 xenograft tumors in BALB / c nude mice (10 mice / cohort) according to some embodiments was reported. For example, monotherapy controls were also included, and the mice were treated (QD) until day 43, and the graph represents the mean tumor volume [mm3] for different cohorts. The results are shown on a linear scale (top; Figure 8 a) and on a logarithmic scale (bottom; Figure 8 b).

[0026] Figure 9 The in vivo anti - tumor activity of RO7276389 at 30 mpk or encorafenib at 24 mpk in combination with 20 mpk of cetuximab in each case against HT29 xenograft tumors in BALB / c nude mice (10 mice / cohort) according to some embodiments was reported. For example, monotherapy controls were also included, and the mice were treated (QD) until day 43, and the graph represents the mean tumor volume [mm3] for different cohorts. The results are shown on a linear scale (top; Figure 9 a) and on a logarithmic scale (bottom; Figure 9 b). Detailed Description

[0027] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, for example: Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention.

[0028] The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the disclosure. All references cited herein are incorporated herein by reference in their entirety.

[0029] Definitions

[0030] As used herein and unless otherwise specified, when the terms “about” and “approximately” refer to the dose, amount, or weight percentage of a component of a composition or dosage form, they mean a dose, amount, or weight percentage that provides a pharmacological effect equivalent to that obtained from the specified dose, amount, or weight percentage, as is well known to one of ordinary skill in the art. The equivalent dose, amount, or weight percentage can be within a range of 30%, 20%, 15%, 10%, 5%, 1%, or less of the specified dose, amount, or weight percentage.

[0031] The term “IC50” refers to the concentration of a specific compound required to inhibit 50% of a specific measured activity. The term “IC90” refers to the concentration of a specific compound required to inhibit 90% of a specific measured activity. The term “IC95” refers to the concentration of a specific compound required to inhibit 95% of a specific measured activity.

[0032] The term “inhibitor” refers to a compound that competes with a specific ligand for binding to a specific receptor, or reduces or prevents the binding of that specific ligand to the specific receptor, or that reduces or prevents the function of a specific protein. Specifically, an inhibitor as used herein refers to a compound that targets, reduces, or inhibits the activity of each target, and a specific inhibitor has an IC50 value of less than 1 μM, less than 500 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, 2 nM, or less than 1 nM.

[0033] The term "pharmaceutically acceptable salt" refers to salts of those compounds of formula (I) that retain the biological effects and properties of the free base or free acid and are not otherwise undesirable biologically. For example, such salts are formed from inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., especially hydrochloric acid) and organic acids (such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, gentisic acid, etc.). In addition, such salts can be prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts, etc. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimine resins, etc.). Particular pharmaceutically acceptable salts of the compounds of formula (I) are the hydrochloride, mesylate and citrate salts.

[0034] The term "FOLFOX" refers to a combination chemotherapy regimen used for the treatment of colorectal cancer. It includes the drugs calcium folinate (folinic acid), fluorouracil and oxaliplatin, and is usually administered via intravenous infusion. In some embodiments, the FOLFOX regimen includes i) administering oxaliplatin in an amount of about 50 mg / m 2 to about 200 mg / m 2 ; ii) administering calcium folinate in an amount of about 200 mg / m 2 to about 600 mg / m 2 ; iii) administering 5-fluorouracil (5-FU) in an amount of about 1200 mg / m 2 to about 3600 mg / m 2 . FOLFOX administration is carried out according to the prescribed and approved treatment methods. Generally, the administration of the FOLFOX regimen is distributed on days 1 and 2 of a two-week cycle. For the purposes of the present invention, modified and adjusted FOLFOX regimens are considered to be covered. The administration of FOLFOX in combination with the administration of cetuximab can be either simultaneous or subsequent, especially subsequent.

[0035] The term "FOLFIRI" refers to a combination chemotherapy regimen used for the treatment of colorectal cancer. It includes the drugs calcium folinate (folinic acid), fluorouracil and irinotecan. In some embodiments, the FOLFIRI regimen includes i) administering irinotecan in an amount of about 150 mg / m 2 to about 250 mg / m 2 , especially about 180 mg / m 2administering irinotecan in an amount of; ii) administering leucovorin in an amount of from about 200 mg / m 2 to about 600 mg / m 2 administering 5-fluorouracil (5-FU) in an amount of from about 1200 mg / m 2 to about 3600 mg / m 2 FOLFIRI administration is carried out according to a prescribed and approved treatment method. Generally, the administration of the FOLFIRI regimen is distributed on days 1 and 2 of a two-week cycle. For the purposes of the present invention, a modified and adjusted FOLFIRI regimen is considered to be covered. The administration of FOFIRI in combination with the administration of cetuximab can be either simultaneous or subsequent, particularly subsequent.

[0036] The term "patient" refers to a human patient. The patient can be an adult.

[0037] The term "antibody" as used herein specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired biological activity. In one case, the antibody is a full-length monoclonal antibody.

[0038] As used herein, the term IgG "isotype" or "subclass" refers to any subclass of immunoglobulins defined by the chemical and antigenic characteristics of the immunoglobulin constant region.

[0039] Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. Immunoglobulins are mainly divided into five classes: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and are described generally, for example, in Abbas et al., Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody can be part of a larger fusion molecule that is formed by the covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0040] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein and refer to an antibody in its substantially intact form rather than an antibody fragment as defined below. The term refers to an antibody that includes the Fc region.

[0041] The term "Fc region" as used herein is defined as the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by a host cell can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain can include the full-length heavy chain, or the antibody can include a cleavage variant of the full-length heavy chain. This can be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447). Thus, the C-terminal lysine (K447) or C-terminal glycine (G446) and lysine (K447) of the Fc region may or may not be present. Unless otherwise indicated, the amino acid sequence of the heavy chain including the Fc region is represented herein as lacking the C-terminal lysine (K447). In one aspect, the heavy chain including the Fc region as specified herein is included in an antibody according to the disclosure herein, the heavy chain including an additional C-terminal glycine-lysine dipeptide (G446 and K447). In one aspect, the heavy chain including the Fc region as specified herein is included in an antibody according to the disclosure herein, the heavy chain including an additional C-terminal glycine residue (G446). In one aspect, the heavy chain including the Fc region as specified herein is included in an antibody according to the disclosure herein, the heavy chain including an additional C-terminal lysine residue (K447). In one embodiment, the Fc region contains a single amino acid substitution N297A. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0042] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in a pharmaceutical composition.

[0043] An "antibody fragment" comprises a portion of a full-length antibody, preferably comprising its antigen-binding region. In some instances, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0044] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or arising during the production of a monoclonal antibody preparation, such variants generally being present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies according to the present invention can be prepared by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus.

[0045] As used herein, the term "hypervariable region" or "HVR" refers to each region within the variable domain of an antibody that is highly variable in sequence and determines antigen-binding specificity, e.g., "complementary determining regions" ("CDRs").

[0046] Typically, an antibody comprises six CDRs; three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

[0047] (a) hypervariable loops present at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 - 917 (1987));

[0048] (b) CDRs present at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0049] (c) antigen contact points occurring at the following amino acid residues: 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2), and 93 to 101 (H3) (MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996)).

[0050] In some embodiments, respectively, CDR - H1 is referred to as HVR - H1, CDR - H2 is referred to as HVR - H2, CDR - H3 is referred to as HVR - H3, CDR - L1 is referred to as HVR - L1, CDR - L2 is referred to as HVR - L2, and CDR - L3 is referred to as HVR - L3.

[0051] Unless otherwise specified, CDRs are determined by the method described by Kabat et al. supra. Those skilled in the art will understand that CDR names can also be determined according to Chothia supra, McCallum supra, or any other scientifically accepted naming system.

[0052] "Framework" or "FR" refers to the variable domain residues other than the complementarity - determining regions (CDRs). The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, CDR and FR sequences typically occur in VH (or VL) in the following sequence: FR1 - CDR - H1 (CDR - L1) - FR2 - CDR - H2 (CDR - L2) - FR3 - CDR - H3 (CDR - L3) - FR4.

[0053] The term "Kabat - numbered variable domain residues" or "Kabat - numbered amino acid positions" and variations thereof refer to the numbering system proposed for the heavy - chain variable domain or light - chain variable domain in the above - cited Kabat et al. literature. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FRs or HV Rs of the variable domain. For example, the heavy - chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat numbering) and insertion residues after residue 82 of the heavy - chain FR (e.g., residues 82a, 82b, and 82c, etc. according to Kabat numbering). The Kabat numbering of residues of a given antibody can be determined by aligning the antibody sequence with the homologous regions of the "standard" Kabat - numbered sequence.

[0054] The term "package insert" is used to refer to the instructions that are typically included in the commercial packaging of a therapeutic product and that contain information regarding indications, usage, dosage, administration, combination therapies, contraindications, and / or warnings related to the use of such therapeutic product.

[0055] As used herein, "in combination with" means that, in addition to administering one treatment modality, another treatment modality is also administered. For example, it includes a treatment regimen that includes administering an EGFR inhibitor described herein (e.g., osimertinib or cetuximab) and a compound of formula (I) or a pharmaceutically acceptable salt thereof. Thus, "in combination with" means that another treatment modality is administered before, during, or after administering one treatment modality to a patient.

[0056] A drug administered "concurrently" with one or more other drugs is administered within the same treatment cycle, on the same day of treatment with one or more other drugs, and optionally concurrently with one or more other drugs. For example, for a cancer treatment administered every three weeks, the concurrently administered drugs are administered on day 1 of the three-week cycle, respectively.

[0057] Non-limiting examples of EGFR inhibitors include cetuximab panitumumab osimertinib (merelectinib, ) erlotinib gefitinib necitumumab (PortrazzaTM), neratinib lapatinib vandetanib and brigatinib Additional examples of EGFR inhibitors are known in the art. In one embodiment, the EGFR inhibitor is a monoclonal anti-EGFR antibody. In one embodiment, the EGFR inhibitor is an orthosteric EGFR inhibitor. In one embodiment, the EGFR inhibitor is an allosteric EGFR inhibitor.

[0058] In some embodiments of the present invention, the EGFR inhibitor is cetuximab Cetuximab is a chimeric monoclonal IgG1 antibody produced by recombinant DNA technology in a mammalian cell line (Sp2 / 0). Cetuximab can be prepared by methods known to those skilled in the art and described in WO2001032712. Cetuximab is commercially available and has the following CAS registration number: 205923-56-4. As used herein, "Cetuximab" refers to a recombinant human / mouse chimeric monoclonal antibody that specifically binds to the extracellular domain of the human epidermal growth factor receptor (EGFR). Cetuximab consists of the Fv region of a murine anti-EGFR antibody with human IgG1 heavy chain and κ light chain constant regions and has a molecular weight of approximately 152 kDa. Cetuximab is produced in mammalian (murine myeloma) cell cultures. Cetuximab is also described in: WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), proposed INN: List 82, Volume 13, Number 4, 1999, published on December 9, 1999 (see page 269). In one embodiment, Cetuximab is sold under the trade name for sale.

[0059] In some embodiments of the present invention, the EGFR inhibitor is panitumumab Panitumumab is a fully humanized monoclonal IgG2 antibody. Panitumumab can be prepared by methods known to those skilled in the art and described in WO2006069202. Panitumumab is commercially available and has the following CAS registration number: 339177-26-3. The term "panitumumab" refers to the antibody described in WHO Drug Information, Volume 18, Number 2, 2004; proposed INN: List 91; amendment: List 96.

[0060] In one embodiment, the anti-EGFR antibody comprises:

[0061] (a) a heavy chain variable region (VH) comprising the following amino acid sequence:

[0062] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO:1), and

[0063] (b) a light chain variable region (VL) comprising the following amino acid sequence:

[0064] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO:2).

[0065] In some cases, the anti-EGFR antibody comprises (a) a VH that comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO:1, or comprises the sequence of SEQ ID NO:1; (b) a VL that comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO:2, or comprises the sequence of SEQ ID NO:2; or (c) a VH as described in (a) and a VL as described in (b).

[0066] In one embodiment, the anti-EGFR antibody comprises cetuximab, which comprises:

[0067] (a) The following heavy chain amino acid sequence:

[0068] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:3), and

[0069] (b) The following light chain amino acid sequence:

[0070] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA (SEQ ID NO:4).

[0071] In some embodiments, the EGFR inhibitor is an EGFR-specific antagonist that is an anti-EGFR antibody. A variety of anti-EGFR antibodies are contemplated and described herein. In certain embodiments, the isolated anti-EGFR antibody can bind to, for example, human EGFR (also known as ErbB-1 and HER1) (as shown by UniProtKB / Swiss-Prot accession number P00533) or variants thereof. In some embodiments, the anti-EGFR antibody is capable of inhibiting the binding between EGF and EGFR. In some embodiments, the anti-EGFR antibody is a monoclonal antibody. In some embodiments, the anti-EGFR antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the anti-EGFR antibody is a humanized antibody. In some embodiments, the anti-EGFR antibody is a chimeric antibody. In some embodiments, the anti-EGFR antibody is a human antibody. Exemplary anti-EGFR antibodies include cetuximab and panitumumab. Examples of anti-EGFR antibodies useful in the methods of the present invention and methods for their preparation are described in U.S. Patent Nos. 5,558,864, 6,217,866, 7,060,808, and 7,598,350, which are hereby incorporated by reference in their entireties.

[0072] In some embodiments, the anti-EGFR antibody comprises:

[0073] (a) HVR-H1, HVR-H2, and HVR-H3 sequences that are GFSLTNYG (SEQ ID NO:5), WSGGN (SEQ ID NO:6), and LTYYDYE (SEQ ID NO:7), respectively, and

[0074] (b) HVR-L1, HVR-L2, and HVR-L3 sequences that are SQSIGTN (SEQ ID NO:8), KYASE (SEQ ID NO:9), and NNNWPT (SEQ ID NO:10), respectively.

[0075] In one embodiment, the anti-EGFR antibody comprises:

[0076] (a) A heavy chain variable region (VH) comprising the following amino acid sequence:

[0077] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO:1), and

[0078] (b) A light chain variable region (VL) comprising the following amino acid sequence:

[0079] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO:2).

[0080] In some embodiments, the anti-EGFR antibody comprises (a) a VH that comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO:1, or comprises the sequence of SEQ ID NO:1; (b) a VL that comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO:2, or comprises the sequence of SEQ ID NO:2; or (c) a VH as described in (a) and a VL as described in (b).

[0081] In one embodiment, the anti-EGFR antibody comprises:

[0082] (a) The following heavy chain amino acid sequence:

[0083] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:3), and

[0084] (b) The following light chain amino acid sequence:

[0085] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA (SEQ ID NO:4).

[0086] In some embodiments, the anti-EGFR antibody comprises a cleavable moiety or linker that, when cleaved (e.g., by proteases in the tumor microenvironment), activates the antibody antigen-binding domain to bind its antigen, e.g., by removing a non-binding spacer moiety.

[0087] In some embodiments, the anti-EGFR antibody comprises six HVR sequences (e.g., three heavy-chain HVRs and three light-chain HVRs) and / or heavy-chain variable domain and light-chain variable domain from an anti-EGFR antibody described in the following patents: U.S. Patent Nos. 5,558,864, 6,217,866, 7,060,808 or 7,598,350.

[0088] In a further embodiment, the anti-EGFR antibody has reduced or minimal effector function. In another specific aspect, the minimal effector function results from a "null effector Fc mutation" or a glycosylation mutation. In a further instance, the null effector Fc mutation is the N434S or M428L / N434S substitution in the constant region. In a further instance, the null effector Fc mutation is the N434S substitution in the constant region. In some cases, the isolated anti-EGFR antibody is glycosylated. Glycosylation of the antibody is generally N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of galactosamine N-acetyl, galactose or xylose to a hydroxy amino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used). By altering the amino acid sequence to remove one of the above tripeptide sequences (for N-linked glycosylation sites), glycosylation sites can be conveniently removed from the antibody. Mutations can be made by substituting the asparagine, serine or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine or a conservative substitution).

[0089] As used herein, "treatment" includes effective cancer treatment with an effective amount of a therapeutic agent (e.g., a compound of formula (I), cetuximab or panitumumab) or a combination of therapeutic agents (e.g., a compound of formula (I) in combination with cetuximab or panitumumab). Treatment can be first-line treatment (e.g., the patient may not have been previously treated or have not received a prior systemic therapy), or second-line or subsequent treatment. For example, a patient is successfully "treated" if one or more symptoms associated with the cancer described herein are alleviated or eliminated, including but not limited to reducing cancer cell proliferation (or destroying cancer cells), alleviating symptoms caused by the disease, improving the quality of life of the patient suffering from the disease, reducing the dosage of other medications required to treat the disease and / or prolonging the survival of the patient.

[0090] The term "delayed progression" of a disease refers to delaying, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the cancer described herein. Such delay can have different time lengths, depending on the medical history of the cancer described herein and / or the patient to be treated. It will be apparent to those skilled in the art that sufficient or significant delay can actually encompass prevention, as the patient will not develop cancer.

[0091] As used herein, "effective amount" means the amount of a therapeutic agent (e.g., a compound of formula (I) and / or cetuximab) described herein that achieves a therapeutic result. In some instances, the effective amount of a therapeutic agent or combination of therapeutic agents is the amount of the agent or combination of agents that achieves the clinical endpoints provided herein. The effective amount herein can vary depending on factors such as the disease state, age, sex, and weight of the patient, and the ability of the agent to elicit the desired response in the patient. The effective amount is also the amount where the therapeutic beneficial effects outweigh any toxic or detrimental effects of the treatment. In some embodiments, an effective amount of a drug can have the following effects: reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slowing or stopping) the infiltration of cancer cells into surrounding organs; inhibiting (i.e., slowing or stopping) tumor metastasis; inhibiting (i.e., slowing or stopping) tumor growth; and / or alleviating one or more of the symptoms associated with the disease. The effective amount can be administered one or more times. The effective amount of a drug, compound, pharmaceutical composition, or combination therapy described herein can be an amount sufficient to effect treatment directly or indirectly.

[0092] "QD" means administering the agent described herein once daily.

[0093] "BID" means administering the agent described herein twice daily.

[0094] "Q2W" means administering the agent described herein once every two weeks.

[0095] "PO" means administering the agent described herein orally.

[0096] "IV" means administering any agent described herein intravenously.

[0097] In one embodiment of the combination therapy described herein, the EGFR inhibitor is administered according to the package insert.

[0098] In one embodiment of the combination therapy described herein, the EGFR inhibitor is cetuximab administered in a fixed-dose Q2W regimen.

[0099] In another embodiment, the combination therapy described herein includes cetuximab, wherein cetuximab is administered in an amount of about 200 to 400 mg / m2.

[0100] The compound of formula (I) contains an asymmetric center and can exist as optically pure enantiomers or a mixture of enantiomers, for example, in the form of a racemate such as a racemic mixture.

[0101] According to the Cahn-Ingold-Prelog convention, the asymmetric carbon atom can have an "R" or "S" configuration.

[0102] "Acceptor human framework" for the purposes herein is a framework that contains an amino acid sequence derived from a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework of a human immunoglobulin framework or a human consensus framework as defined below. An "acceptor human framework" derived from a human immunoglobulin framework or a human consensus framework can contain the same amino acid sequence as the human immunoglobulin framework or the human consensus framework, or it can contain amino acid sequence variations. In some aspects, the number of amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0103] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

[0104] An "affinity matured" antibody is an antibody that has one or more alterations in one or more complementarity determining regions (CDRs), which result in an improvement in the affinity of the antibody for an antigen as compared to the parental antibody that does not have such alterations.

[0105] The term "antibody" as used herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0106] "Antibody fragment" refers to a molecule other than a full antibody, which comprises a portion of a full antibody and binds to an antigen to which the full antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0107] As used herein, the term "linker" refers to a peptide linker and is preferably a peptide having an amino acid sequence with a length of at least 5 amino acids, preferably a length of 5 to 100 amino acids, more preferably 10 to 50 amino acids. In one embodiment, the peptide linker is (G x S) n or (G x S) n G m , where G = glycine, S = serine, and (x = 3, n = 3, 4, 5 or 6, and m = 0, 1, 2 or 3) or (x = 4, n = 2, 3, 4 or 5 and m = 0, 1, 2 or 3), preferably x = 4 and n = 2 or 3, more preferably x = 4 and n = 2. In one embodiment, the peptide linker is (G4S)2.

[0108] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, an immunoglobulin of the IgG class is a heterotetrameric glycoprotein of approximately 150,000 daltons, which is composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also known as the variable heavy chain domain or heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3) (also known as the heavy chain constant regions). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also known as the variable light chain domain or light chain variable domain), followed by a constant light chain (CL) domain (also known as the light chain constant region). The heavy chains of immunoglobulins can be assigned to one of the following five types: called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of the following two types based on the amino acid sequence of their constant domains: called kappa (κ) and lambda (λ). An immunoglobulin substantially consists of two Fab molecules and an Fc domain linked by an immunoglobulin hinge region.

[0109] An "antibody that binds to the same epitope as the reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of this antibody to its antigen by 50% or more in a competition assay. An exemplary competition assay is provided herein.

[0110] The term "antigen-binding domain" refers to a part of an antigen-binding molecule that contains a region that specifically binds and is complementary to a part or all of an antigen. In the case of a large antigen, the antigen-binding molecule can bind only to a specific part of the antigen, which is called an epitope. The antigen-binding domain can be provided by, for example, one or more antibody variable domains (also known as antibody variable regions). Preferably, the antigen-binding domain contains an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0111] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0112] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain aspects, the antibody is of the IgG1 isotype. In certain aspects, the antibody is of the IgG1 isotype with P329G, L234A, and L235A mutations to reduce Fc region effector function. In other aspects, the antibody is of the IgG2 isotype. In certain aspects, the antibody is of the IgG4 isotype with an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The constant domains of the heavy chains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types based on the amino acid sequence of its constant domain, and these two types are called kappa (κ) and lambda (λ).

[0113] As used herein, the term "human-derived constant region" or "human constant region" refers to the constant heavy chain region and / or the constant light chain kappa or lambda region of a human antibody of the subclasses IgG1, IgG2, IgG3, or IgG4. Such constant regions are well known in the art and are described, for example, by Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A. et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, the numbering of amino acid residues in the constant regions is according to the EU numbering system, also known as Kabat's EU index, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0114] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., radioactive isotopes of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and Lu); chemotherapeutic agents or drugs (e.g., oxaliplatin, fluorouracil, methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), daunorubicin, melphalan, mitomycin C, chlorambucil, idarubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as ribonucleases; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer drugs disclosed hereinafter.

[0115] "Effector function" refers to those biological activities attributable to the Fc region of an antibody that vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0116] As used herein, the terms "engineered, engineered, engineering" are considered to include any manipulation of the peptide backbone, or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, glycosylation pattern or side chain groups of individual amino acids, and combinations of these methods.

[0117] As used herein, the term "amino acid mutation" encompasses amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as reduced binding to Fc receptors or increased association with another peptide. Amino acid sequence deletions and insertions include amino-terminal and / or carboxyl-terminal deletions and insertions of amino acids. A specific amino acid mutation is an amino acid substitution. For the purpose of altering, for example, the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., substituting one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Genetic or chemical methods well known in the art can be used to generate amino acid mutations. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, are also contemplated as useful. Various names may be used herein to denote the same amino acid mutation. For example, substitution of proline at position 329 of the Fc domain with glycine can be denoted as 329G, G329, G 329 , P329G, or Pro329Gly.

[0118] An "effective amount" of an agent (e.g., a pharmaceutical composition) is an amount that is capable of effectively achieving the desired therapeutic or prophylactic result at the required dose and for the required period of time.

[0119] The term "Fc region" as used herein defines the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by a host cell can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expressing a particular nucleic acid molecule encoding a full-length heavy chain can include the full-length heavy chain, or the antibody can include a cleaved variant of the full-length heavy chain. This can be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) of the Fc region or the C-terminal glycine (Gly446) and lysine (Lys447) may or may not be present. Unless otherwise specified, the amino acid sequence of the heavy chain containing the Fc region is represented herein as lacking the C-terminal glycine-lysine dipeptide. In one aspect, a heavy chain comprising an Fc region as specified herein is included in an antibody according to the invention, said heavy chain comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, a heavy chain comprising an Fc region as specified herein is included in an antibody according to the invention, said heavy chain comprising an additional C-terminal glycine residue (G446, according to the EU index numbering). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0120] "Modifications that facilitate association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent association of polypeptides containing Fc domain subunits with the same polypeptide to form a homodimer. As used herein, modifications that facilitate association specifically include individual modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, wherein the modifications are complementary to each other to facilitate association of the two Fc domain subunits. For example, modifications that facilitate association can alter the structure or charge of one or both of the Fc domain subunits such that their association is spatially or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing the first Fc domain subunit and a polypeptide containing the second Fc domain subunit, which may be different in the sense that the additional components (e.g., antigen-binding portions) fused to each subunit are not the same. In some embodiments, modifications that facilitate association include amino acid mutations in the Fc domain, particularly amino acid substitutions. In one particular embodiment, modifications that facilitate association include individual amino acid mutations, particularly amino acid substitutions, to each of the two subunits of the Fc domain.

[0121] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining regions (CDRs). The FRs of a variable domain generally consist of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally occur in VH (or VL) in the following sequence: FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0122] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein.

[0123] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived from the primary transformed cell, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the original transformed cell are included herein.

[0124] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or an amino acid sequence of a non-human-derived antibody that is derived from a human antibody library or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0125] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells (e.g., NS0 or CHO cells) or from animals transgenic for human immunoglobulin genes (e.g., mice), or antibodies expressed by transfection of recombinant expression vectors into host cells. Such recombinant human antibodies have rearranged variable and constant regions. The recombinant human antibodies according to the invention have undergone somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not be present in the in vivo human antibody germline repertoire under natural conditions.

[0126] "Human consensus framework" refers to a framework that represents the amino acid residues most commonly present in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. In general, the subgroup of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one aspect, for VL, the subgroup is subgroup κI as described in Kabat et al., supra. In one aspect, for VH, the subgroup is subgroup III as described in Kabat et al., supra.

[0127] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may contain at least a portion of an antibody constant region derived from a human antibody. An antibody in a "humanized form", e.g., a non-human antibody, refers to an antibody that has been humanized.

[0128] As used herein, the term "hypervariable region" or "HVR" refers to each region within the variable domain of an antibody that is hypervariable in sequence and determines antigen-binding specificity, e.g., "complementary determining region" ("CDR").

[0129] Typically, an antibody comprises six CDRs; three in VH (CDR-H1, CDR-H2, CDR-H3), and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

[0130] (a) Hypervariable loops present at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 - 917 (1987));

[0131] (b) CDRs present at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0132] (c) Antigen - contact points occurring at the following amino acid residues: 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2), and 93 to 101 (H3) (MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996)).

[0133] Unless otherwise specified, CDRs are determined by the method described by Kabat et al. supra. Those skilled in the art will understand that CDR names can also be determined according to Chothia supra, MacCallum supra, or any other scientifically - accepted naming system.

[0134] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0135] An "individual" or "subject" is a mammal. Mammals include but are not limited to domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non - human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

[0136] An "isolated" antibody is one that has been separated from the components of its natural environment. In some aspects, the antibody is purified to greater than 95% or 99% purity as determined by, for example, methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0137] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base (specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U))), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Generally, nucleic acid molecules are described by their base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Typically, the sequence of bases is represented from 5' to 3'. In the present context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and hybrid polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleobases having derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression in vitro and / or in vivo (e.g., in a host or patient) of an antibody of the invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule such that the mRNA can be injected into a subject to produce an antibody in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0138] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules that are contained within a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0139] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding an antibody heavy chain and light chain (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0140] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or arising during the production of the monoclonal antibody preparation, such variants generally being present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies according to the present invention can be prepared by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0141] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in a pharmaceutical composition.

[0142] "Native antibody" refers to naturally occurring immunoglobulin molecules having different structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also known as the variable heavy chain domain or heavy chain variable domain, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also known as the variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain.

[0143] "Blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. For example, the anti-EGFR antibodies of the present invention block signal transduction through EGFR.

[0144] "Antagonist" or activating antibody is an antibody that enhances or initiates signal transduction through the antigen to which it binds. In some embodiments, the antagonist antibody causes or activates signal transduction in the absence of the native ligand.

[0145] The term "package insert" is used to refer to the instructions that are typically included in the commercial packaging of a therapeutic product and that contain information regarding indications, usage, dosage, administration, combination therapy, contraindications and / or warnings relating to the use of such therapeutic product.

[0146] "No substantial cross-reactivity" means that a molecule (e.g., an antibody) does not recognize or specifically bind an antigen that is different from the actual target antigen of that molecule (e.g., an antigen that is closely related to the target antigen), particularly when compared to that target antigen. For example, an antibody may bind less than about 10% to less than about 5% of antigens that are different from the actual target antigen, or may bind the antigens that are different from the actual target antigen in an amount consisting of less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1%, preferably less than about 2%, 1% or 0.5%, and most preferably less than about 0.2% or 0.1% of the antigens that are different from the actual target antigen.

[0147] The "percent amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and for the purposes of the alignment without considering any conservative substitutions as part of the sequence identity. The alignment used to determine the percent amino acid sequence identity can be achieved in various ways within the skill in the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate the percent identity value. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001 / 007611.

[0148] Unless otherwise indicated, for the purposes of this text, the ggsearch program of the FASTA package version 36.3.8c or later is used to generate values of the percentage of amino acid sequence identity using the BLOSUM50 comparison matrix. The FASTA program package was created by W.R. Pearson and D.J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W.R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36 and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare sequences, using the ggsearch (global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global rather than a local alignment. The percentage of amino acid identity is given in the output alignment header.

[0149] The term “pharmaceutical composition” or “pharmaceutical preparation” refers to a preparation that is in a form that permits the bioactivity of the active ingredient(s) contained therein to be effective and that contains no additional components that are toxic to the subject to whom the pharmaceutical composition will be administered.

[0150] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to the components of a pharmaceutical composition or preparation other than the active ingredient(s), which are non-toxic to the subject. Pharmaceutically acceptable carriers include, for example, any and all materials compatible with the administration of a drug, including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other materials and compounds compatible with the administration of a drug. It is contemplated for use in the compositions of the present invention except in cases where any conventional medium or agent is incompatible with the active compound. Supplementary active compounds can also be incorporated into the composition. In some instances, pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0151] As used herein, "treatment" (and its grammatical variants such as "treat" or "treating") refers to an attempt to alter the natural course of a disease in an individual being treated and can be performed prophylactically or as a clinical intervention that can be performed during a clinical pathological process. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. In some aspects, the antibodies of the invention are used to delay the development or slow the progression of a disease.

[0152] As used herein, the term "cancer" refers to a proliferative disease such as colorectal cancer, sarcoma, head and neck cancer, squamous cell carcinoma, breast cancer, pancreatic cancer, gastric cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, and mesothelioma, including refractory versions of any of the foregoing cancers, or combinations of one or more of the foregoing cancers. In one embodiment, the cancer is colorectal cancer.

[0153] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have similar structures, each domain including four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using, respectively, the VH or VL domain from an antibody that binds that antigen to screen a library of complementary VL or VH domains. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0154] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0155] As used herein, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and their derivatives, such as fragments thereof.

[0156] As used herein, the term "antigen-binding site of an antibody" refers to the amino acid residues in an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody contains amino acid residues from "complementary determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues defined herein. Thus, the light chain variable domain and the heavy chain variable domain of an antibody contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding and defines the properties of the antibody. The CDR regions and FR regions are determined according to the criteria of Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and / or those residues from "hypervariable loops".

[0157] Antibody specifically refers to the selective recognition of a particular epitope of an antigen by an antibody. For example, a natural antibody is monospecific. A "bispecific antibody" as described in the present invention is an antibody having two different antigen-binding specificities. The antibody of the present invention is specific for two different antigens, namely DR5 as the first antigen and FAP as the second antigen.

[0158] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen.

[0159] The term "bispecific" means that an antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants. Generally, a bispecific antigen-binding molecule contains at least two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two distinct cells.

[0160] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)) and "milling" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic manipulation effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

[0161] Engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies", are also included herein (see, e.g., US2006 / 0025576A1).

[0162] The antibodies or fragments herein also include "dual action FAb" or "DAF", which contain at least one antigen-binding site that binds to FAP or DR5 and another different antigen (see, e.g., US 2008 / 0069820).

[0163] As used herein, the term "valence" refers to the number of binding sites present in an antibody molecule. Thus, the terms "bivalent", "tetravalent", and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antibody molecule. The bispecific antibodies according to the invention are at least "bivalent" and can be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent").

[0164] The antibodies of the present invention have two or more binding sites and are bispecific. That is, even in the presence of more than two binding sites (i.e., the antibody is trivalent or multivalent), the antibody can be bispecific. The bispecific antibodies of the present invention include, for example, multivalent single-chain antibodies, diabodies and triabodies, as well as antibodies having the constant domain structure of a full-length antibody, which are linked to other antigen-binding sites (e.g., single-chain Fv, VH domain and / or VL domain, Fab or (Fab)2) by one or more peptide linkers. The antibody can be a full-length antibody from a single species, or can be chimeric or humanized.

[0165] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0166] As used in this application, the term "amino acid" refers to the group of naturally occurring carboxy α-amino acids including the following: alanine (three-letter code: ala, single-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0167] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably and all such names include progeny. Thus, the words "transfectant" and "transfected cell" include the primary subject cell and cultures derived from that cell without regard to the number of transfers. It should also be understood that all progeny may not be precisely identical in DNA content due to either deliberate or inadvertent mutations. Variant progeny having the same function or biological activity as screened in the original transformed cell are included.

[0168] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0169] As used herein, the terms "bind" or "specifically bind" refer to the binding of an antibody to an antigen epitope in an in vitro assay, preferably in a surface plasmon resonance assay (SPR, BIAcore, GE-Healthcare Uppsala, Sweden). The affinity of the binding is defined by the terms ka (the association rate constant of the antibody from the antibody / antigen complex), kD (the dissociation constant), and KD (kD / ka). Binding or specifically binding refers to a binding affinity (KD) of 10 -8 mol / l or less, preferably 10 - 9 M to 10 -13 mol / l.

[0170] The binding of an antibody to a death receptor can be studied by a BIAcore assay (GE-Healthcare Uppsala, Sweden). The affinity of the binding is defined by the terms ka (the association rate constant of the antibody from the antibody / antigen complex), kD (the dissociation constant), and KD (kD / ka)

[0171] "Reduced binding" (e.g., reduced binding to an Fc receptor) refers to a decrease in the affinity for the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes reducing the affinity to zero (or below the detection limit of the analytical method), i.e., completely eliminating the interaction. Conversely, "increased binding" refers to an increase in the binding affinity for the corresponding interaction.

[0172] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

[0173] As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, such as a cell in a tumor (such as a cancer cell or a cell of the tumor stroma). In particular, "target cell antigen" refers to folate receptor 1.

[0174] As used herein, the terms "first" and "second" with respect to antigen-binding portions and the like are used for convenience in distinguishing when there is more than one of each type of portion.

[0175] The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemical reactive surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge characteristics. An epitope is the antigenic region that binds to an antibody.

[0176] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to a site on a polypeptide macromolecule (e.g., a continuous stretch of amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids) to which an antigen-binding portion binds to form an antigen-binding portion-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, in free substances in serum and / or in the extracellular matrix (ECM). Unless otherwise specified, a protein referred to herein as an antigen (e.g., EGFR) can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In one particular embodiment, the antigen is a human protein. When referring to a specific protein herein, the term encompasses the "full-length", unprocessed protein, as well as any form of the protein produced by intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

[0177] As used herein, the terms "engineer, engineered, engineering", particularly terms with the prefix "glyco-" and the term "glycosylation engineering" are considered to include any manipulation of the glycosylation pattern of a naturally occurring or recombinant polypeptide or fragment thereof. Glycosylation engineering includes metabolic engineering of the glycosylation machinery of a cell, including genetic manipulation of oligosaccharide synthesis pathways to effect changes in the glycosylation of glycoproteins expressed in the cell. In addition, glycosylation engineering includes the effects of mutations and the cellular environment on glycosylation. In one embodiment, glycosylation engineering is an alteration in glycosyltransferase activity. In one particular embodiment, the engineering results in an altered glucosaminyltransferase activity and / or fucosyltransferase activity.

[0178] The combination therapies according to some embodiments of the present invention have a synergistic effect. The "synergistic effect" of two compounds is where the combined effect of the two agents is greater than the sum of their individual effects and is statistically different from the effect of a control and of a single drug.

[0179] Combination therapy

[0180] Provided herein is a combination therapy (or composition) comprising a compound of formula (I):

[0181]

[0182] or a pharmaceutically acceptable salt thereof and an EGFR inhibitor as described herein. In some embodiments, the compound of formula (I) is (3R)-N-[2-cyano-4-fluoro-3-(3-methyl-4-oxo-quinazolin-6-yl)oxy-phenyl]-3-fluoro-pyrrolidine-1-sulfonamide or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is the free base. In some embodiments, the compound of formula (I) is a pharmaceutically acceptable salt of the compound. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is a BRAF inhibitor.

[0183] Some embodiments of the present invention include:

[0184] A combination of a BRAF inhibitor and an EGFR inhibitor, wherein the BRAF inhibitor is a compound of formula (I)

[0185]

[0186] or a pharmaceutically acceptable salt thereof;

[0187] A combination of a BRAF inhibitor and an EGFR inhibitor, wherein the BRAF inhibitor is a compound of formula (I)

[0188]

[0189] or a pharmaceutically acceptable salt thereof, wherein the EGFR inhibitor is cetuximab;

[0190] A combination of a BRAF inhibitor and an EGFR inhibitor as described herein, wherein the EGFR inhibitor is a monoclonal anti-EGFR antibody;

[0191] A combination of a BRAF inhibitor and an EGFR inhibitor as described herein, wherein the EGFR inhibitor is cetuximab or panitumumab;

[0192] A combination of a BRAF inhibitor and an EGFR inhibitor as described herein, wherein the EGFR inhibitor is cetuximab;

[0193] A combination of a BRAF inhibitor and an EGFR inhibitor as described herein, wherein the EGFR inhibitor is panitumumab;

[0194] A combination of a BRAF inhibitor and an EGFR inhibitor as described herein, which is used as a medicament;

[0195] A combination of a BRAF inhibitor and an EGFR inhibitor as described herein, which is used for the therapeutic and / or prophylactic treatment of cancer, particularly colorectal cancer;

[0196] A combination of a BRAF inhibitor and an EGFR inhibitor as described herein, which is used for the therapeutic treatment of cancer, particularly colorectal cancer;

[0197] In another embodiment, the present invention relates to a pharmaceutical product for treating colorectal cancer (the colorectal cancer involves a tumor containing b-Raf with a V600E mutation), the pharmaceutical product comprising (A) a first component, which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active agent; and (B) a second component, which comprises cetuximab as an active agent; the amount of the active agent being such that the combination is therapeutically effective in the treatment of the cancer;

[0198] In one embodiment, cetuximab is initially administered at a dose of 400 mg / m 2 in the form of an intravenous infusion over 120 minutes, and then weekly at a dose of 250 mg / m in an intravenous infusion over 60 minutes one week later. 2 All the medicaments can be administered, for example, until disease progression or unacceptable toxicity occurs.

[0199] Use of a combination of a BRAF inhibitor and an EGFR inhibitor as described herein for the preparation of a medicament for treating or preventing cancer, particularly colorectal cancer;

[0200] Use of a combination of a BRAF inhibitor and an EGFR inhibitor as described herein for the preparation of a medicament for treating cancer, particularly colorectal cancer;

[0201] A method for treating or preventing cancer, particularly colorectal cancer, the method comprising administering to a patient in need thereof an effective amount of a combination of a BRAF inhibitor and an EGFR inhibitor as described herein, particularly, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof and cetuximab are administered simultaneously, and more particularly, wherein the compound of formula (I) and cetuximab are administered sequentially;

[0202] A method for treating cancer, particularly colorectal cancer, the method comprising administering to a patient in need thereof an effective amount of a combination of a BRAF inhibitor and an EGFR inhibitor as described herein, particularly, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof and cetuximab are administered simultaneously, and more particularly, wherein the compound of formula (I) and cetuximab are administered sequentially;

[0203] A method for treating a patient suffering from cancer, especially colorectal cancer, the method comprising administering to a patient in need thereof a combination of an effective amount of a BRAF inhibitor of formula (I) as described herein or a pharmaceutically acceptable salt thereof and an EGFR inhibitor, in particular, wherein the compound of formula (I) and the EGFR inhibitor are administered simultaneously, and more particularly, wherein the compound of formula (I) and the EGFR inhibitor are administered sequentially;

[0204] A method for treating a patient suffering from cancer, especially colorectal cancer, the method comprising administering to a patient in need thereof a combination of an effective amount of a BRAF inhibitor of formula (I) or a pharmaceutically acceptable salt thereof and cetuximab, in particular, wherein the compound of formula (I) and cetuximab are administered simultaneously, and more particularly, wherein the compound of formula (I) and cetuximab are administered sequentially;

[0205] A method for treating a patient suffering from cancer, especially colorectal cancer, the method comprising administering to a patient in need thereof a combination of an effective amount of a BRAF inhibitor of formula (I) or a pharmaceutically acceptable salt thereof, in particular, wherein the compound of formula (I) and panitumumab are administered simultaneously, and more particularly, wherein the compound of formula (I) and panitumumab are administered sequentially;

[0206] A pharmaceutical composition comprising: a combination of a BRAF inhibitor and an EGFR inhibitor as described herein, and one or more pharmaceutically acceptable excipients;

[0207] The combination, use, method or pharmaceutical composition as described herein, wherein the BRAF inhibitor is administered orally and the EGFR inhibitor is administered by injection such as intravenous injection or subcutaneous injection;

[0208] The combination, use, method or pharmaceutical composition as described herein, wherein the BRAF inhibitor is administered orally and the EGFR inhibitor is administered by intravenous injection;

[0209] The combination, use, method or pharmaceutical composition as described herein, wherein the BRAF inhibitor is administered orally and the EGFR inhibitor is administered by subcutaneous injection;

[0210] The combination, use, method or pharmaceutical composition according to any one of claims 1 to 9, wherein the BRAF inhibitor and the EGFR inhibitor are administered simultaneously;

[0211] The combination, use, method or pharmaceutical composition as described herein, wherein the BRAF inhibitor and the EGFR inhibitor are administered sequentially;

[0212] A combination of a BRAF inhibitor and an EGFR inhibitor for use as described herein, wherein the cancer is associated with a BRAF V600X mutation, especially BRAF V600E or BRAFV600K mutations, and more particularly BRAF V600E mutations;

[0213] A combination of a BRAF inhibitor and an EGFR inhibitor for use as described herein, wherein the cancer is BRAF V600X mutation-positive unresectable or metastatic cancer;

[0214] A combination of a BRAF inhibitor and an EGFR inhibitor for use as described herein, wherein the cancer is BRAF V600E mutation-positive unresectable or metastatic cancer;

[0215] A combination of a BRAF inhibitor and an EGFR inhibitor for use as described herein, wherein the cancer is BRAF V600X mutation-positive unresectable or metastatic colorectal cancer;

[0216] A combination of a BRAF inhibitor and an EGFR inhibitor for use as described herein, wherein the cancer is BRAF V600E mutation-positive unresectable or metastatic colorectal cancer;

[0217] A combination of a BRAF inhibitor and an EGFR inhibitor for use as described herein, wherein a method comprising the following is used to determine BRAF V600X mutation: (a) performing PCR or sequencing on nucleic acid (e.g., DNA) extracted from a sample of a patient's tumor tissue and / or body fluid; and (b) determining the expression of BRAF in the sample; and V600 and

[0218] A combination of a BRAF inhibitor and an EGFR inhibitor for use as described herein, comprising one or more additional anticancer agents selected from MEK inhibitors, MEK degraders, HER2 and / or HER3 inhibitors, HER2 and / or HER3 degraders, SHP2 inhibitors, SHP2 degraders, Axl inhibitors, Axl degraders, ALK inhibitors, ALK degraders, PI3K inhibitors, PI3K degraders, SOS1 inhibitors, SOS1 degraders, signal transduction pathway inhibitors, checkpoint inhibitors, regulators of the apoptosis pathway, cytotoxic chemotherapeutic agents, angiogenesis-targeted therapies, immunotargeting agents, and antibody-drug conjugates.

[0219] Further embodiments of the invention include:

[0220] In one embodiment, a kit is provided that includes a BRAF inhibitor and an EGFR inhibitor of formula (I) as described herein, prescribing information (also referred to as "instructions"), a blister pack or bottle (HDPE or glass), and a container. The prescribing information preferably includes advice to the patient regarding administration of the combination of the BRAF inhibitor and the EGFR inhibitor for treatment as described herein;

[0221] In one embodiment, the subject being treated has become refractory during the prior treatment as described herein; and

[0222] In one embodiment, the subject being treated has developed brain metastases during the prior treatment as described herein.

[0223] The structures described herein are also intended to include compounds that differ only in the presence of one or more isotope - rich atoms. For example, those in which one or more hydrogen atoms are replaced by deuterium ( 2 H), or one or more carbon atoms are replaced by carbon enriched in 13 C or 14 C are within the scope of the present invention.

[0224] In addition, embodiments of the present invention include all optical isomers (where applicable) of the compounds of formula (I), namely, diastereoisomers, mixtures of diastereomers, racemic mixtures, all their corresponding enantiomers and / or tautomers, and their solvates.

[0225] If desired, for example, a racemic mixture of the compounds of the present invention can be separated to isolate the individual enantiomers. The separation can be carried out by methods well - known in the art, such as coupling the racemic mixture of the compound to an enantiomerically pure compound to form a mixture of diastereomers, and then separating the individual diastereomers by standard methods such as fractional crystallization or chromatography.

[0226] In some embodiments providing optically pure enantiomers, an optically pure enantiomer means that the compound contains >90% by weight of the desired isomer, particularly >95% by weight of the desired isomer, or more particularly >99% by weight of the desired isomer, the weight percentages being based on the total weight of the isomers of the compound. Chiral - pure compounds or chiral - enriched compounds can be prepared by chiral - selective synthesis or by separation of enantiomers. The separation of enantiomers can be carried out on the final product, or alternatively on a suitable intermediate.

[0227] In one embodiment, one or more additional anti-cancer agents are used in combination with a BRAF inhibitor and an EGFR inhibitor as described herein, wherein the additional anti-cancer agents are selected from MEK inhibitors, MEK degraders, inhibitors of HER2 and / or HER3, degraders of HER2 and / or HER3, SHP2 inhibitors, SHP2 degraders, Axl inhibitors, Axl degraders, ALK inhibitors, ALK degraders, PI3K inhibitors, PI3K degraders, SOS1 inhibitors, SOS1 degraders, signal transduction pathway inhibitors, checkpoint inhibitors, regulators of the apoptosis pathway, cytotoxic chemotherapeutic agents, angiogenesis-targeted therapies, immune-targeted agents, and antibody-drug conjugates.

[0228] In some embodiments, one of the additional anti-cancer agents is a MEK inhibitor. Non-limiting examples of MEK inhibitors include cobimetinib binimetinib and trametinib. Additional examples of MEK inhibitors are known in the art.

[0229] In some embodiments, one of the additional anti-cancer agents is an inhibitor of HER2 and / or HER3. Non-limiting examples of HER2 and / or HER3 inhibitors include lapatinib, canertinib, (E)-2-methoxy-N-(3-(4-(3-methyl-4-(6-methylpyridin-3-yloxy)phenylamino)quinazolin-6-yl)allyl)acetamide (GP-724714), sapitinib, 7-[[4-[(3-ethynylphenyl)amino]-7-methoxy-6-quinazolinyl]oxy]-N-hydroxy-heptanamide (CUDC-101), mubritinib, 6-[4-[(4-ethylpiperazin-1-yl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE788), tucatinib, poziotinib, N-[4-[1-[4-(4-acetyl-1-piperazinyl)cyclohexyl]-4-amino-3-pyrazolo[3,4-d]pyrimidinyl]-2-methoxyphenyl]-1-methyl-2-indolecarboxamide (KIN001-111), 7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine (KIN001-051), 6,7-dimethoxy-N-(4-phenoxyphenyl)quinazolin-4-amine (KIN001-30), dasatinib, and bosutinib.

[0230] In some embodiments, one of the additional anti-cancer agents is an inhibitor of SHP2. Non-limiting examples of SHP2 inhibitors include 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099), [3-[(3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl]-6-(2,3-dichlorophenyl)-5-methylpyrazin-2-yl]methanol (RMC-4550), RMC-4630, TNO155, and the compounds disclosed in WO 2015 / 107493, WO 2015 / 107494, WO2015 / 107495, WO 2019 / 075265, PCT / U82019 / 056786 and PCT / l82020 / 053019.

[0231] In some embodiments, one of the additional anti-cancer agents is a PI3K inhibitor. Non-limiting examples include buparlisib (BKM120), alpelisib (BYL719), samtolisib (LY3023414), 8-[(1R)-1-[(3,5-difluorophenyl)amino]ethyl]-N,N-dimethyl-2-(morpholin-4-yl)-4-oxo-4H-chromene-6-carboxamide (AZD8186), tenalisib (RP6530), voxtalisib hydrochloride (SAR-245409), gedatolisib (PF-05212384), panulisib (P-7170), taselisib (GDC-0032), trans-2-amino-8-[4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one (PF-04691502), duvelisib (ABBV-954), N2-[4-oxo-4-[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholin-4-ium-4-ylmethoxy]butanoyl]-L-arginyl-glycyl-L-aspartyl-L-serine acetate (SF-1126), pictilisib (GDC-0941), 2-methyl-1-[2-methyl-3-(trifluoromethyl)benzyl]-6-(morpholin-4-yl)-1H-benzimidazole-4-carboxylic acid (GSK2636771), idelalisib (GS-1101), umbralisib tosylate (TGR-1202), pictilisib (GDC-0941), copanlisib hydrochloride (BAY 84-1236), dactolisib (BEZ-235), 1-(4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1-H-1,2,4-triazol-3-yl]piperidin-1-yl)-3-hydroxypropan-1-one (AZD-8835), 5-[6,6-dimethyl-4-(morpholin-4-yl)-8,9-dihydro-6H-[1,4]oxazino[4,3-e]Purin-2-yl]pyrimidin-2-amine (GDC-0084), everolimus, rapamycin, perifosine, sirolimus, and temsirolimus.,

[0232] In some embodiments, one of the additional anti-cancer agents is an ALK inhibitor. Non-limiting examples include crizotinib (PF-02341066), ceritinib (LDK378), alectinib (alecensa), brigatinib (AP26113), lorlatinib (PF-6463922), ensartinib (X-396), entrectinib (RXDX-101), reprotectinib (TPX-0005), belizatinib (TSR-011), alkotinib (ZG-0418), foritinib (SAF-189), CEP-37440, TQ-B3139, PLB1003, and TPX-0131

[0233] In some embodiments, one of the additional anti-cancer agents is a checkpoint inhibitor. In some examples, the checkpoint inhibitor is a CTLA-4 binding antagonist, a PD-1 binding antagonist, or a PD-L1 binding antagonist. In some embodiments, the CTLA-4 inhibitor is ipilimumab or tremelimumab (GP-675,206). In some examples, the PD-1 binding antagonist is selected from cemiplimab pembrolizumab nivolumab and RN888 (PF-06801591). In some examples, the PD-L1 binding antagonist is selected from atezolizumab avelumab and durvalumab (Imfinzi TM ).

[0234] In some embodiments, one of the additional anti-cancer agents is an antibody-drug conjugate. Non-limiting examples of antibody-drug conjugates include gemtuzumab ozogamicin (MylotargTM), inotuzumab ozogamicin brentuximab vedotin ado - trastuzumab emtansine (TDM - f; )、mirvetuximab soravtansine (IMGN853) and anetumab ravtansine.

[0235] In some embodiments, one of the additional anti - cancer agents is an antibody, such as bevacizumab (MvastiTM, )、trastuzumab avelumab rituximab (MabTheraTM、 )、edrecolomab (Panorex)、daratumuab olaratumab (LartruvoTM)、ofatumumab alemtuzumab cetuximab oregovomab、cemiplimab pembrolizumab dinutiximab obinutuzumab )、trametinib (GP - 675,206)、ramucirumab ublituximab (TG - 1101)、panitumumab elotuzumab (EmplicitiT'V')、necitumumab (PortrazzaT'V')、cirmtuzumab (UC - 961)、ibritumomab isatuximab (SAR650984)、nimotuzumab、fresolimumab (GC1008)、irilumab (INN)、mogamulizumab Ficlatuzumab (AV-299), denosumab Ganitumab, urelumab, pidilizumab, amatuximab, blinatumomab (AMG103; ) or midostaurin (Rydapt).

[0236] In another aspect, the present disclosure provides a method of treating colorectal cancer (CRC) in a patient in need thereof, the method comprising administering, during a treatment period, an effective amount of a combination therapy comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof and (b) an EGFR inhibitor as described herein. In some embodiments, the colorectal cancer is metastatic CRC expressing EGFR or BRAF V600E mutation-positive metastatic CRC. In some embodiments, the cancer is a cancer that has acquired an on-target EGFR resistance mutation. In a preferred aspect, the EGFR inhibitor is cetuximab or panitumumab. In a more preferred aspect, the EGFR inhibitor is cetuximab. In another more preferred aspect, the EGFR inhibitor is panitumumab.

[0237] Pharmaceutical formulation

[0238] The pharmaceutical formulations of the antibodies (e.g., EGFR inhibitor antibodies) as described herein are made in the form of lyophilized formulations or aqueous solutions by mixing such antibodies of desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)). The pharmaceutical carriers are generally non-toxic to the recipient at the dosages and concentrations employed, including but not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., cetrimonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20( Baxter International, Inc.). Certain exemplary sHASEGP and methods of use (including rHuPH20) are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0239] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0240] The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0241] The active ingredient can be encapsulated in, for example, microcapsules prepared by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively); encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in a coarse emulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980).

[0242] Sustained-release preparations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of shaped articles such as membranes or microcapsules, for example.

[0243] Preparations for in vivo administration are generally sterile. For example, sterility can be readily achieved by filtration through sterile filtration membranes.

[0244] The antibody can be administered by any suitable means, including parenterally, intralungally, and intranasally, and, if local treatment is desired, intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be effected by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is to be brief or long-term. A variety of dosing schedules are contemplated herein, including but not limited to single or multiple administrations, bolus administration, and pulsed infusion at various time points.

[0245] Another embodiment of the invention provides a pharmaceutical composition containing one or more compositions, wherein each composition contains one or more compounds used according to the invention and one or more therapeutically inert carriers, diluents, or excipients, and a method for preparing such a pharmaceutical composition. In one example, a compound of formula (I) can be formulated into a galenical administration form by mixing it with a physiologically acceptable carrier (i.e., a carrier that is non-toxic to the recipient at the doses and concentrations used) at ambient temperature at an appropriate pH and desired purity. The pH of the formulation depends mainly on the specific use and concentration of the compound, but is preferably in the range of about 3 to about 8. In one example, the compound of formula (I) is formulated in an acetate buffer at pH 5. In another embodiment, the compound of formula (I) is sterile. The compound can be stored, for example, as a solid or amorphous composition, as a lyophilized formulation, or as an aqueous solution.

[0246] The composition is formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the medicament, the method of administration, the timing of administration, and other factors known to the practicing physician.

[0247] The pharmaceutical composition can be obtained by processing the BRAF inhibitor described herein together with a pharmaceutically acceptable inorganic or organic carrier or excipient. For example, lactose, corn starch or its derivatives, talc, stearic acid or its salts, etc. can be used as such carriers for tablets, coated tablets, dragees and hard gelatin capsules. Suitable carriers for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols, etc. However, depending on the nature of the active substance, a carrier is often not required in the case of soft gelatin capsules. Suitable carriers for the production of solutions and syrups are, for example, water, polyols, glycerol, vegetable oils, etc. Suitable carriers for suppositories are, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, etc.

[0248] In addition, the pharmaceutical composition may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for altering the osmotic pressure, buffers, masking agents or antioxidants. They may also contain other therapeutically valuable substances.

[0249] The pharmaceutical compositions (alone or in combination) of BRAF inhibitors can be prepared for storage in the form of a lyophilized preparation or an aqueous solution by mixing the active ingredient having the desired purity with an optional pharmaceutical carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. (ed.) (1980)). Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and include buffers, such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as TWEEN™, PLURONICSTM or polyethylene glycol (PEG).

[0250] Pharmaceutical compositions of BRAF inhibitors include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will generally be the amount of BRAF inhibitor or EGFR inhibitor treatment that produces a therapeutic effect. Generally, on a percentage basis, the amount will range from about 1% to about 90% of the active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%. Methods of preparing these compositions include the step of associating the BRAF inhibitor with a carrier and optionally one or more accessory ingredients. In general, pharmaceutical compositions may be prepared by uniformly and intimately associating the BRAF inhibitor with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product. Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavoured base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouthwashes, etc., each containing a predetermined amount of BRAF inhibitor as an active ingredient. The BRAF inhibitor may also be administered as boluses, electuaries or pastes.

[0251] In one embodiment of the invention, the BRAF inhibitor and the EGFR inhibitor are formulated as two separate pharmaceutical compositions.

[0252] The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively), be entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. (ed.) (1980).

[0253] Preparations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0254] The dosage can be varied within a wide range, but of course will have to be adjusted according to the individual requirements of each specific case. In the case of oral administration, the dosage for adults can vary, and general formula (I) compounds in an amount of about 5 mg to about 4000 mg per day or the corresponding amount of a pharmaceutically acceptable solvate can be used. The daily dosage can be administered as a single dose or in multiple divided doses. In addition, when there is an indication that the upper limit can be exceeded, the upper limit can also be exceeded.

[0255] The following examples illustrate but do not limit the invention and are merely representative of the invention. Pharmaceutical compositions, such as tablets or coated tablets, conveniently contain about 5 mg to 500 mg, especially about 100 mg to 500 mg, of the compound of formula (I).

[0256] In some embodiments of this example, the compound of formula (I) is administered twice a day at a dose of about 500 mg to about 1000 mg, especially at a dose of about 800 mg.

[0257] In some embodiments of this example, the compound of formula (I) is administered twice a day at a dose of about 1500 mg to about 2000 mg, especially at a dose of about 1600 mg.

[0258] In some embodiments of this example, the compound of formula (I) is administered three times a day at a dose of about 1000 mg to about 1300 mg, especially at a dose of about 1200 mg.

[0259] In one embodiment, the compound of formula (I) is orally administered three times a day at a dose of about 1200 mg, while cetuximab is administered at a dose of about 400 mg / m 2 to about 500 mg / m 2 as an initial dose and then at a dose of about 200 mg / m 2 to about 300 mg / m 2 intravenously or subcutaneously once a week.

[0260] In one embodiment, the compound of formula (I) is orally administered three times a day at a dose of about 1200 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 250 mg / m 2 intravenously or subcutaneously once a week.

[0261] In one embodiment, the compound of formula (I) is orally administered three times a day at a dose of about 1200 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 250 mg / m 2The dose is administered intravenously or subcutaneously once a week, while FOLFOX or FOLFIRI chemotherapy is also administered once every two weeks.

[0262] In one embodiment, the compound of formula (I) is administered orally three times a day at a dose of about 1200 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 500 mg / m 2 administered intravenously or subcutaneously once every two weeks, while FOLFOX or FOLFIRI chemotherapy is also administered once every two weeks.

[0263] In one embodiment, the compound of formula (I) is administered orally three times a day at a dose of about 1200 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 250 mg / m 2 administered intravenously or subcutaneously once a week, while FOLFOX chemotherapy is also administered once every two weeks.

[0264] In one embodiment, the compound of formula (I) is administered orally three times a day at a dose of about 1200 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 250 mg / m 2 administered intravenously or subcutaneously once a week, while FOLFIRI chemotherapy is also administered once every two weeks.

[0265] In one embodiment, the compound of formula (I) is administered orally twice a day at a dose of about 800 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 250 mg / m 2 administered intravenously or subcutaneously once a week.

[0266] In one embodiment, the compound of formula (I) is administered orally twice a day at a dose of about 800 mg, while cetuximab is administered at a dose of about 400 mg / m 2 to about 500 mg / m 2 as an initial dose and then at a dose of about 200 mg / m 2 to about 300 mg / m 2 administered intravenously or subcutaneously once a week.

[0267] In one embodiment, the compound of formula (I) is administered orally twice a day at a dose of about 800 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 250 mg / m 2 administered intravenously or subcutaneously once a week.

[0268] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1600 mg, while cetuximab is administered at a dose of about 400 mg / m 2 to about 500 mg / m 2 as an initial dose and then intravenously or subcutaneously once a week at a dose of about 200 mg / m 2 to about 300 mg / m 2 .

[0269] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1600 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then intravenously or subcutaneously once every other week at a dose of about 250 mg / m 2 .

[0270] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1600 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then intravenously or subcutaneously once a week at a dose of about 250 mg / m 2 , while FOLFOX or FOLFIRI chemotherapy is also administered once every other week.

[0271] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1600 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then intravenously or subcutaneously once every other week at a dose of about 500 mg / m 2 , while FOLFOX or FOLFIRI chemotherapy is also administered once every other week.

[0272] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1600 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then intravenously or subcutaneously once a week at a dose of about 250 mg / m 2 , while FOLFOX chemotherapy is also administered once every other week.

[0273] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1600 mg, while cetuximab is administered at a dose of about 400 mg / m 2 as an initial dose and then intravenously or subcutaneously once a week at a dose of about 250 mg / m 2 , while FOLFIRI chemotherapy is also administered once every other week.

[0274] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1800 mg, while cetuximab is administered as an initial dose at a dose of about 400 mg / m 2 and then intravenously or subcutaneously once weekly at a dose of about 250 mg / m 2 .

[0275] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1800 mg, while cetuximab is administered as an initial dose at a dose of about 400 mg / m 2 and then intravenously or subcutaneously once weekly at a dose of about 250 mg / m 2 , while FOLFOX or FOLFIRI chemotherapy is also administered once every other week.

[0276] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1800 mg, while cetuximab is administered as an initial dose at a dose of about 400 mg / m 2 and then intravenously or subcutaneously once every other week at a dose of about 500 mg / m 2 , while FOLFOX or FOLFIRI chemotherapy is also administered once every other week.

[0277] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1800 mg, while cetuximab is administered as an initial dose at a dose of about 400 mg / m 2 and then intravenously or subcutaneously once weekly at a dose of about 250 mg / m 2 , while FOLFOX chemotherapy is also administered once every other week.

[0278] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1800 mg, while cetuximab is administered as an initial dose at a dose of about 400 mg / m 2 and then intravenously or subcutaneously once weekly at a dose of about 250 mg / m 2 , while FOLFIRI chemotherapy is also administered once every other week.

[0279] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 2000 mg, while cetuximab is administered as an initial dose at a dose of about 400 mg / m 2 and then intravenously or subcutaneously once weekly at a dose of about 250 mg / m 2 .

[0280] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 2000 mg, while cetuximab is administered intravenously or subcutaneously once weekly at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 250 mg / m 2 and FOLFOX chemotherapy is also administered once every other week.

[0281] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 2000 mg, while cetuximab is administered intravenously or subcutaneously once weekly at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 250 mg / m 2 and FOLFOX or FOLFIRI chemotherapy is also administered once every other week.

[0282] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 2000 mg, while cetuximab is administered intravenously or subcutaneously once every other week at a dose of about 500 mg / m 2 as an initial dose and then at a dose of about 500 mg / m 2 and FOLFOX or FOLFIRI chemotherapy is also administered once every other week.

[0283] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 2000 mg, while cetuximab is administered intravenously or subcutaneously once weekly at a dose of about 400 mg / m 2 as an initial dose and then at a dose of about 250 mg / m 2 and FOLFIRI chemotherapy is also administered once every other week.

[0284] In one embodiment, the compound of formula (I) is orally administered three times daily at a dose of about 1200 mg, while panitumumab is administered intravenously at a dose of about 6 mg / kg as an initial dose and then at a dose of about 6 mg / kg once every two weeks (every 14 days).

[0285] In one embodiment, the compound of formula (I) is orally administered three times daily at a dose of about 1200 mg, while panitumumab is administered intravenously at a dose of about 6 mg / kg as an initial dose and then at a dose of about 6 mg / kg once every two weeks (every 14 days), and FOLFOX or FOLFIRI chemotherapy is also administered once every two weeks.

[0286] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1600 mg, while panitumumab is intravenously administered as an initial dose at about 6 mg / kg and then at about 6 mg / kg once every two weeks (once every 14 days).

[0287] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1600 mg, while panitumumab is intravenously administered as an initial dose at about 6 mg / kg and then at about 6 mg / kg once every two weeks (once every 14 days), and FOLFOX or FOLFIRI chemotherapy is also administered once every two weeks.

[0288] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1800 mg, while panitumumab is intravenously administered as an initial dose at about 6 mg / kg and then at about 6 mg / kg once every two weeks (once every 14 days).

[0289] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 1800 mg, while panitumumab is intravenously administered as an initial dose at about 6 mg / kg and then at about 6 mg / kg once every two weeks (once every 14 days), and FOLFOX or FOLFIRI chemotherapy is also administered once every two weeks.

[0290] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 2000 mg, while panitumumab is intravenously administered as an initial dose at about 6 mg / kg and then at about 6 mg / kg once every two weeks (once every 14 days).

[0291] In one embodiment, the compound of formula (I) is orally administered twice daily at a dose of about 2000 mg, while panitumumab is intravenously administered as an initial dose at about 6 mg / kg and then at about 6 mg / kg once every two weeks (once every 14 days), and FOLFOX or FOLFIRI chemotherapy is also administered once every two weeks.

[0292] Examples

[0293] The following examples are provided to illustrate the invention and are not of a limiting character. Examples of the compositions according to the invention include:

[0294] Example A

[0295] Tablets of the following compositions are manufactured in a conventional manner.

[0296] Table 3: Possible tablet compositions

[0297]

[0298] Preparation procedure

[0299] 1. Mix components 1, 2, 3 and 4 and granulate with purified water.

[0300] 2. Dry the granules at 50 °C.

[0301] 3. Pass the granules through a suitable grinding device.

[0302] 4. Add component 5, mix for three minutes; press on a suitable press.

[0303] Example B-1

[0304] Manufacture capsules having the following composition.

[0305] Table 4: Possible capsule ingredient compositions

[0306]

[0307] Preparation procedure

[0308] 1. Mix components 1, 2 and 3 in a suitable mixer for 30 minutes.

[0309] 2. Add components 4 and 5 and mix for 3 minutes.

[0310] 3. Fill into suitable capsules.

[0311] First, mix the compound of formula (I), lactose and corn starch in a mixer, and then mix in a grinder. Return the mixture to the mixer; add talc powder thereto and mix well. Load the mixture into suitable capsules, such as hard gelatin capsules, using a machine.

[0312] Example B-2

[0313] Manufacture soft gelatin capsules having the following composition.

[0314] Table 5: Possible soft gelatin capsule ingredient compositions

[0315] Ingredient mg / capsule Compound of formula (I) 5 Yellow wax 8 Hydrogenated soybean oil 8 Partially hydrogenated vegetable oil 34 Soybean oil 110 Total 165

[0316] Table 6: Possible soft gelatin capsule compositions

[0317] Ingredient mg / capsule Gelatin 75 Glycerol 85% 32 Karion 83 8 (dry matter) Titanium dioxide 0.4 Yellow iron oxide 1.1 Total 116.5

[0318] Preparation procedure

[0319] Dissolve the compound of formula (I) in the warm melt of the other ingredients and fill the mixture into soft gelatin capsules of suitable size. Process the filled soft gelatin capsules according to the general procedure.

[0320] Example C

[0321] Manufacture suppositories having the following composition:

[0322] Table 7: Possible suppository composition

[0323] Ingredient mg / suppository Compound of formula (I) 15 Suppository base 1285 Total 1300

[0324] Preparation procedure

[0325] Melt the suppository base in a glass or steel container, mix well and cool to 45 °C. At this time, add the fine powder of the compound of formula (I) thereto and stir until the compound is completely dispersed. Pour the mixture into a suppository mold of appropriate size, let it stand and cool; then remove the suppositories from the mold and individually wrap them in wax paper or metal foil.

[0326] Example D

[0327] Manufacture injection solutions having the following composition.

[0328] Table 8: Possible injection solution composition

[0329] Ingredient mg / injection solution Compound of formula (I) 3 Polyethylene glycol 400 150 Acetic acid Q.s., pH adjusted to 5.0 Water for injection Made up to 1.0 ml

[0330] Preparation procedure

[0331] Dissolve the compound of formula (I) in a mixture of polyethylene glycol 400 and part of the water for injection. Adjust the pH to 5.0 with acetic acid. Adjust the volume to 1.0 ml by adding the remaining amount of water. Filter the solution, fill it into vials with appropriate overfill and sterilize.

[0332] Example E

[0333] Manufacture sachets having the following composition.

[0334] Table 9: Possible sachet composition

[0335] Ingredient mg / bag Compound of formula (I) 50 Lactose, fine powder 1015 Microcrystalline cellulose (AVICEL PH 102) 1400 Sodium carboxymethyl cellulose 14 Polyvinylpyrrolidone K 30 10 Magnesium stearate 10 Flavoring additive 1 Total 2500

[0336] Preparation procedure

[0337] Mix the compound of formula (I) with lactose, microcrystalline cellulose and sodium carboxymethylcellulose and granulate it together with a mixture of polyvinylpyrrolidone in water. Mix the granules with magnesium stearate and flavoring additives and fill them into sachets.

[0338] Biological example

[0339] Test agent

[0340] (3R)-N-[2-cyano-4-fluoro-3-(3-methyl-4-oxo-quinazolin-6-yl)oxy-phenyl]-3-fluoro-pyrrolidine-1-sulfonamide (referred to herein as RO7276389) was provided in powder form by Roche, Basel, Switzerland and re-suspended before use. Cetuximab was provided by WuXi (China).

[0341] Cell lines and culture conditions

[0342] Cell lines were obtained from ATCC and maintained in a humidified incubator with 5% CO2 under standard conditions and passaged twice a week. The culture conditions are reported in the table below:

[0343]

[0344] Experimental procedures

[0345] Experiments were conducted at WuXi (China), which provided female BALB / c nude mice and cetuximab. Subcutaneous implants were made on one flank of the mice and the mice were randomly grouped when tumors were established (between 100 and 200 mm 3 ), and were treated orally daily with RO7276389 or intravenously with cetuximab once every two weeks, as Figure 1 reported

[0346] Example 1

[0347] Mice were implanted with the cell line LS411N with BRAF V600E mutation (5.0 x 10 6 cells / mouse). After tumors were established (150 mm 3 ), the mice were randomly grouped and received either RO7276389 (140 mpk, 60 mpk or 30 mpk) or encorafenib (24 mpk) orally (PO) once daily (QD: quaque die; once a day), either alone or in combination with cetuximab (2QW) via IV injection. A schematic of the study design is shown in Figure 1 . Figure 2 The results reported in Figures 3 to 5The results reported in [study name] demonstrate that the combination of RO7276389 and EGFRi has the potential to significantly reduce tumor growth in CRC when compared to the current standard of care (SOC), encorafenib in combination with cetuximab. Notably, treatment with the combination of higher doses of 140 mpk and 60 mpk RO7276389 with cetuximab even led to a reduction in tumor volume.

[0348] Example 2

[0349] Mice were implanted with the cell line HT29 bearing the BRAF V600E mutation (5.0 x 10 6 cells / mouse). After tumor establishment (approximately 150 mm 3 ), the mice were randomly grouped and received either RO7276389 (140 mpk, 60 mpk, or 30 mpk) or encorafenib (24 mpk) orally (PO) once daily (QD), either alone or in combination with cetuximab (2QW) via IV injection. Figure 6 The results reported in [study name] demonstrate that RO7276389 monotherapy achieved significantly higher dose-dependent tumor growth inhibition when compared to encorafenib. Figures 7 to 9 The results reported in [study name] demonstrate that the combination of RO7276389 and EGFRi has the potential to significantly reduce tumor growth in CRC when compared to the current SOC, encorafenib in combination with cetuximab. Notably, treatment with the combination of RO7276389 with cetuximab even led to a reduction in tumor volume.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with an EGFR inhibitor.

2. The combination according to claim 1, wherein the compound of formula (I) or the pharmaceutically acceptable salt is a BRAF inhibitor.

3. The combination according to claim 1 or 2, wherein the compound of formula (I) is (3R)-N-[2-cyano-4-fluoro-3-(3-methyl-4-oxo-quinazolin-6-yl)oxy-phenyl]-3-fluoro-pyrrolidine-1-sulfonamide or a pharmaceutically acceptable salt thereof.

4. The combination according to claims 1 to 3, wherein the compound of formula (I) is the free base.

5. The combination according to claims 1 to 4, wherein the EGFR inhibitor is a monoclonal anti-EGFR antibody.

6. The combination according to any one of claims 1 to 5, wherein the EGFR inhibitor is cetuximab.

7. The combination according to any one of claims 1 to 6, for use as a medicament.

8. The combination according to any one of claims 1 to 7, for the therapeutic and / or prophylactic treatment of cancer, particularly colorectal cancer.

9. Use of the combination according to any one of claims 1 to 8 for the manufacture of a medicament for the treatment or prevention of cancer, particularly colorectal cancer.

10. A method for the treatment or prevention of cancer, particularly colorectal cancer, the method comprising administering to a patient in need thereof an effective amount of the combination according to any one of claims 1 to 8, in particular, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof and cetuximab are administered simultaneously, and more particularly, wherein the compound of formula (I) and cetuximab are administered sequentially.

11. A pharmaceutical composition comprising: the combination according to any one of claims 1 to 8, and one or more pharmaceutically acceptable excipients.

12. The combination, use, method or pharmaceutical composition according to any one of claims 1 to 11, wherein the BRAF inhibitor is administered orally and the EGFR inhibitor is administered by injection such as intravenous injection or subcutaneous injection.

13. The combination, use, method or pharmaceutical composition according to any one of claims 1 to 12, wherein the BRAF inhibitor is administered simultaneously with the EGFR inhibitor.

14. The combination, use, method or pharmaceutical composition according to any one of claims 1 to 12, wherein the BRAF inhibitor is administered sequentially with the EGFR inhibitor.

15. The combination, use, method or pharmaceutical composition according to any one of claims 1 to 14, wherein the cancer is associated with a BRAF V600X mutation, in particular a BRAF V600E mutation.

16. The combination, use, method or pharmaceutical composition according to any one of claims 1 to 15, wherein the cancer is BRAF V600X mutation-positive unresectable or metastatic cancer, especially BRAF V600E mutation-positive unresectable or metastatic cancer, more especially BRAF V600E mutation-positive unresectable or metastatic colorectal cancer.

17. The combination, use, method or pharmaceutical composition according to any one of claims 1 to 16, wherein a method comprising the following is used to determine BRAF V600X mutation: (a) performing PCR or sequencing on nucleic acid (e.g., DNA) extracted from a sample of the patient's tumor tissue and / or body fluid; and (b) determining the expression of BRAF V600 in the sample.

18. The combination, use, method or pharmaceutical composition according to any one of claims 1 to 17, which comprises one or more additional anti-cancer agents selected from MEK inhibitors, MEK degraders, inhibitors of HER2 and / or HER3, degraders of HER2 and / or HER3, SHP2 inhibitors, SHP2 degraders, Axl inhibitors, Axl degraders, ALK inhibitors, ALK degraders, PI3K inhibitors, PI3K degraders, SOS1 inhibitors, SOS1 degraders, signal transduction pathway inhibitors, checkpoint inhibitors, regulators of apoptosis pathways, cytotoxic chemotherapeutic agents, angiogenesis-targeted therapies, immune-targeting agents and antibody-drug conjugates.

19. The combination, use, method or pharmaceutical composition according to any one of claims 1 to 18, which is combined with FOLFOX or FOLFIRI chemotherapy, particularly FOLFOX chemotherapy.

20. The present invention as described in the specification.

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