CN117813094A

CN117813094A - Combinations of specific BRAF inhibitors (paradox inhibitors) and PD-1 axis binding antagonists for the treatment of cancer

Info

Publication number: CN117813094A
Application number: CN202280040171.XA
Authority: CN
Inventors: J·埃克曼; T·弗里斯; F·赫林; P·F·T·佩塔佐尼; J·维希曼
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2021-06-09
Filing date: 2022-06-07
Publication date: 2024-04-02

Abstract

The present invention relates to a combination of a BRAF inhibitor 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 or solvate thereof, and a PD-1 axis binding antagonist, and the use of said combination as a medicament, in particular for the therapeutic and/or prophylactic treatment of cancer.

Description

Combinations of specific BRAF inhibitors (paradox inhibitors) and PD-1 axis binding antagonists for the treatment of cancer

The present invention relates to a combination of a BRAF inhibitor and a PD-1 axis binding antagonist, as well as uses and pharmaceutical compositions thereof.

The invention provides, inter alia, BRAF inhibitors and PD-1 axis binding antagonists for use in the treatment of cancer, wherein the BRAF inhibitors are compounds of formula (I)

Or a pharmaceutically acceptable salt or solvate thereof.

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

Furthermore, the developed BRAF inhibitors reveal that in BRAF ^V600E The unexpected and "paradoxical" ability to inhibit MAPK signaling in driven tumors, while the same inhibitors exhibited MAPK stimulatory activity in the BRAF wild-type (WT) model (N Engl J Med 2012;366:271-273; and British Journal of Cancer, vol.111, pp.640-645 (2014)).

Then, research on the mechanism of paradoxical RAF proves that oncogenic BRAF ^V600E Phosphorylation of MEK 1/2 in its monomeric cytoplasmic form, whereas WT BRAF and RAF1 activation require complex event stepsWhich includes cell membrane translocation and homo-and/or heterodimerization promoted by activated RAS (KRAS, NRAS, HRAS) (Nature Reviews Cancer, volume 14, pages 455 to 467 (2014)).

Binding of first generation BRAF inhibitors (e.g., vemurafenib, dabrafenib and Kang Naifei ni) to WT BRAF or RAF1 protomers rapidly induces RAF homodimerization and membrane association of newly formed RAF dimers. In the dimeric conformation, one RAF protomer allosterically induces a conformational change in the second RAF protomer, leading to a kinase active state and, importantly, a conformation that is detrimental to inhibitor binding. As a result, dimers induced by drug therapy promote MEK phosphorylation by catalysis operated by unbound protomers and overactivate this pathway.

Tumors almost inevitably evade BRAFi treatment, and the mechanism in most cases involves the ability to trigger acquisition of RAF dimerization. This effect can be offset by MEK inhibitor (MEKi) combination therapy. However, the therapeutic index of these agents is very poor, which limits the dose of MEKi achievable in humans. Thus, resistance to the combination therapy of the first generation BRAFi and MEKi is still mediated by activation of the RAF paradox. Thus, the clinical benefit of these combination therapies remains limited.

Recently, it has been discovered that T cell dysfunction or anergy occurs simultaneously with the inducible and sustained expression of an inhibitory receptor (programmed death 1 polypeptide (PD-1)). One of its ligands, PD-L1, is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al, international. Immun.2007.19 (7): 813) (Thompson RH et al, cancer Res 2006,66 (7): 3381). Interestingly, in contrast to T lymphocytes in normal tissues and peripheral Blood T lymphocytes, most tumor-infiltrating T lymphocytes predominantly express PD-1, suggesting that upregulation of PD-1 on tumor-reactive T cells can lead to impaired anti-tumor immune responses (Blood 2009 1 14 (8): 1537).

The present invention relates to novel combinations of a BRAF inhibitor of formula (I) and a PD-1 axis binding antagonist for use in the treatment of cancer, in particular melanoma, non-small cell lung cancer and leptomeningeal cancer. The compounds of formula (I) are BRAF inhibitors that show negligible activation of MAPK signaling pathways (paradox inhibitors) compared to the first generation BRAF inhibitors on the market: kang Naifei, dabrafenib and vemurafenib (paradox inducers). In addition to this property, the compounds of formula (I) also have very potent brain penetration properties, providing an urgent alternative therapy for the treatment of leptomeningeal cancers or cancers metastasizing into the brain. The present invention discloses a new combination for cancer treatment that has a strong combined activity against BRAF-associated tumors and makes it possible to overcome the rapidly acquired therapeutic resistance often observed in patients treated with first-generation BRAF inhibitors. The combinations disclosed herein for treating cancer exhibit unexpected combined activity far exceeding the additive effect of BRAF inhibitors and PD-1 axis binding antagonist monotherapy.

Drawings

FIG. 1 reports the combination of compound Ia or paradoxical inducer Kang Naifei Ni alone or in combination with MEKi cobicitinib (cobimeinib) against the first generation BRAFi A375 BRAF ^V600E/ NRAS ^Q61K Activity of P-ERK levels in drug resistant cell models.

Fig. 2 shows a schematic diagram of the study design reported in fig. 3 and 4.

FIG. 3 reports the in vivo anti-tumor activity of 5mg/kg of Compound Ia, 12.5mg/kg of anti-mouse PD1 and combinations thereof on allograft tumors derived from the isogenic model YUMM 1.7. Mice were treated until day 31, and drug treatment was then suspended and evidence of tumor regeneration in mice was monitored. The top graph shows the average tumor growth [ mm3] for the different cohorts. The middle graph shows tumor growth in individual mice in different groups of phases. The bottom table summarizes mice with detectable tumors at day 58 in the corresponding cohort.

FIG. 4 reports the in vivo anti-tumor activity of 1mg/kg of Compound Ia, 12.5mg/kg of anti-mouse PD1 and combinations thereof on allograft tumors derived from the isogenic model YUMM 1.7. Mice were treated until day 45, and drug treatment was then suspended and evidence of tumor regeneration in mice was monitored. The top graph shows the average tumor growth [ mm3] for the different cohorts. The middle graph shows tumor growth in individual mice in different groups of phases. The bottom table summarizes mice with detectable tumors at day 58 in the corresponding cohort.

Figure 5 shows a schematic of mouse treatment for the immunophenotype experiments reported in figures 6 and 7.

Figure 6 reports total immune cell (top) and T cell (bottom) infiltration in tumors, as quantified by flow cytometry. Total immune cell infiltration was expressed as a percentage of mCD45 positive to live cells on days 3 and 6, and T cell infiltration was expressed as number of mCD3 positive cells per mm 2.

FIG. 7 reports immunohistochemical analysis of mouse immunocyte marker CD45 for tumors treated for 3 days with any vehicle, 5mg/kg of Compound Ia (daily), 12.5mg/kg of mPD1 (monotherapy) or a combination thereof.

The term "IC50" refers to the concentration of a particular compound required to inhibit a particular measured activity by 50%.

The term "inhibitor" means a compound that competes with a particular ligand for binding to a particular receptor, or reduces or prevents binding of the particular ligand to a particular receptor, or reduces or prevents the function of a particular protein. Specifically, inhibitors as used herein refer to compounds that target, reduce, or inhibit the activity of individual targets, with particular inhibitors having an IC50 value of less than 1 μm, less than 500nM, less than 200nM, less than 100nM, less than 50nM, less than 25nM, less than 10nM, less than 5nM, 2nM, or less than 1 nM.

The term "pharmaceutically acceptable salts" refers to those salts of the compounds of formula (I) which retain the biological effects and properties of the free base or free acid, which salts are not undesirable in biological or other respects. For example, these salts are formed from inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, particularly 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, and the like). In addition, these salts can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts, and the like. 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, polyimide resins, and the like). Particular pharmaceutically acceptable salts of the compounds of formula (I) are the hydrochloride, mesylate and citrate salts.

The term "solvate" refers to a non-covalent stoichiometric or non-stoichiometric combination of solvent and solute. The term "hydrate" refers to a non-covalent stoichiometric or non-stoichiometric combination of water and a solute. For example, the compounds of formula (I) and pharmaceutically acceptable salts thereof may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as anisole, methylene chloride, toluene, 1, 4-dioxane, water, and the like.

The compounds of formula (I) contain an asymmetric centre and may exist as optically pure enantiomers or mixtures of enantiomers, for example, such as racemates.

The asymmetric carbon atom may be in the "R" or "S" configuration according to the Cahn-Ingold-Prelog specification.

For purposes herein, a "recipient human framework" is a framework comprising 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. The recipient human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence as the human immunoglobulin framework or human consensus framework, or it may comprise amino acid sequence changes. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. 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.

"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). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibodies and antigens). The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K _D ) And (3) representing. 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.

An "affinity matured" antibody refers to an antibody having one or more alterations in one or more Complementarity Determining Regions (CDRs) that result in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody that does not have such alterations.

The term "antibody" is used herein in its broadest sense and includes a variety of 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.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ The method comprises the steps of carrying out a first treatment on the surface of the A diabody antibody; a linear antibody; single chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dabs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, nature Biotechnology23:1126-1136 (2005).

As used herein, the term "linker" refers to a peptide linker and is preferably a peptide having an amino acid sequence of at least 5 amino acids in length, preferably 5 to 100 amino acids in length, more preferably 10 to 50 amino acids in length. 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 (G ₄ S) ₂ 。

The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, igG class immunoglobulins are heterotetrameric glycoproteins of about 150,000 daltons, which are composed of two light chains and two heavy chains bonded by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH) (also known as a variable heavy chain domain or heavy chain variable domain) followed by three constant domains (CH 1, CH2, and CH 3) (also known as heavy chain constant regions). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL) (also known as a variable light chain domain or light chain variable domain) followed by a constant light Chain (CL) domain (also known as a light chain constant region). The heavy chain of an immunoglobulin may be assigned to one of five types: known as alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or mu (IgM), some of which may be further divided into subtypes, e.g., gamma ₁ (IgG ₁ )、γ ₂ (IgG ₂ )、γ ₃ (IgG ₃ )、γ ₄ (IgG ₄ )、α ₁ (IgA ₁ ) And alpha ₂ (IgA ₂ ). The light chain of an immunoglobulin can be assigned to one of two types based on the amino acid sequence of its constant domain: referred to as kappa (kappa) and lambda (lambda). Immunoglobulins consist essentially of two Fab molecules and one Fc domain linked by an immunoglobulin hinge region.

By "antibody that binds to the same epitope" as the reference antibody is meant an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, whereas the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. An exemplary competition assay is provided herein.

The term "antigen binding domain" refers to a portion of an antigen binding molecule that comprises a region that specifically binds to and is complementary to a portion or all of an antigen. In the case of larger antigens, the antigen binding molecule may bind only to a specific portion of the antigen, which portion is referred to as an epitope. The antigen binding domain may be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains are derived from a particular source or species, while the remainder of the heavy and/or light chains are derived from a different source or species.

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain of an antibody has. There are five main classes of antibodies: igA, igD, igE, igG and IgM, and some of these antibodies may be further classified into subclasses (isotypes), e.g., igG ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ . In certain aspects, the antibody is an IgG ₁ An isoform. In certain aspects, the antibody is an IgG having P329G, L234A and L235A mutations to reduce Fc region effector function ₁ An isoform. In other aspects, the antibody is an IgG ₂ An isoform. In certain aspects, the antibody is an IgG having an S228P mutation in the hinge region ₄ Isotype to improve IgG ₄ Stability of the antibodies. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (kappa) and lambda (lambda), based on the amino acid sequence of its constant domain.

The term "constant region derived from human" or "human constant region" as used herein refers to the constant heavy chain region and/or constant light chain kappa or lambda region of a human antibody of subclass IgG1, igG2, igG3 or IgG 4. 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, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991) (see, e.g., 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, numbering of amino acid residues in the constant region is according to the EU numbering system, also known as the EU index of Kabat, as described in Kabat, E.A. et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991), NIH Publication 91-3242.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., at ²¹¹ 、I ¹³¹ 、I ¹²⁵ 、Y ⁹⁰ 、Re ¹⁸⁶ 、Re ¹⁸⁸ 、Sm ¹⁵³ 、Bi ²¹² 、P ³² 、Pb ²¹² And a radioisotope of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); a growth inhibitor; enzymes and fragments thereof such as nucleolytic enzymes; an antibiotic; 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 antitumor or anticancer agents disclosed below.

"effector functions" refer to those biological activities attributable to the Fc region of an antibody that vary with the variation of 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; down-regulation of cell surface receptors (e.g., B cell receptors); b cell activation.

As used herein, the term "engineered, engineered" is 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 amino acid sequences, glycosylation patterns, or side chain groups of individual amino acids, as well as combinations of these approaches.

The term "amino acid mutation" as used herein is meant to encompass 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 an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino-terminal and/or carboxy-terminal deletions and insertions of amino acids. A particular amino acid mutation is an amino acid substitution. For the purpose of altering the binding characteristics of, for example, the Fc region, non-conservative amino acid substitutions, i.e., substitution of one amino acid with another amino acid having a different structure and/or chemical nature, are particularly preferred. Amino acid substitutions include substitution 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 may be used to generate amino acid mutations. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is also contemplated that methods of altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, are useful. Various names may be used herein to indicate identical amino acid mutations. For example, substitution of proline at position 329 of the Fc domain for glycine can be expressed as 329G, G329, G ₃₂₉ P329G or Pro329Gly.

An "effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount that is effective to achieve a desired therapeutic or prophylactic result at the requisite dosage over the requisite period of time.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a 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 carboxy terminus of the heavy chain. However, antibodies produced by the host cell may 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 expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain, or the antibody may comprise a cleaved variant of a full-length heavy chain. This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering). Thus, the C-terminal lysine (Lys 447) or C-terminal glycine (Gly 446) and lysine (Lys 447) of the Fc region may or may not be present. The amino acid sequence of the heavy chain comprising the Fc region is denoted herein as absent a C-terminal glycine-lysine dipeptide, if not otherwise indicated. In one aspect, a heavy chain comprising an Fc region as specified herein, said heavy chain comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system) is comprised in an antibody according to the invention. In one aspect, a heavy chain comprising an Fc region as specified herein, said heavy chain comprising an additional C-terminal glycine residue (G446, numbering according to the EU index) is comprised in an antibody according to the invention. Unless otherwise specified herein, 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, 5 th edition Public Health Service, national Institutes of Health, bethesda, MD, 1991.

A "modification that facilitates association of a first subunit and a second subunit of an Fc domain" is manipulation of the peptide backbone or post-translational modification of an Fc domain subunit that reduces or prevents a polypeptide comprising an Fc domain subunit from associating with the same polypeptide to form a homodimer. As used herein, "modification to promote association" specifically includes 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 promote association of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both of the Fc domain subunits in order to render their association sterically or electrostatically advantageous, respectively. Thus, (hetero) dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may be different in the sense that the additional components fused to each subunit (e.g., antigen binding portion) are not identical. In some embodiments, the modification that facilitates association includes an amino acid mutation, particularly an amino acid substitution, in the Fc domain. In a particular embodiment, the modification that facilitates association comprises a separate amino acid mutation, in particular an amino acid substitution, for each of the two subunits of the Fc domain.

"framework" or "FR" refers to the variable domain residues other than the Complementarity Determining Regions (CDRs). The FR of the variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, CDR and FR sequences typically occur in VH (or VL) with the following sequences: FR1-CDR-H1 (CDR-L1) -FR2-CDR-H2 (CDR-L2) -FR3-CDR-H3 (CDR-L3) -FR4.

The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a natural antibody or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. 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 completely identical to the nucleic acid content of the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the original transformed cell.

A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or an amino acid sequence derived from a non-human antibody that utilizes a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues.

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

A "human consensus framework" is a framework that represents the amino acid residues that are 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 subset of variable domain sequences. In general, a subset of sequences is as described in Kabat et al, sequences of Proteins of Immunological Interest, fifth edition, NIH Publication 91-3242, bethesda MD (1991), volumes 1-3. In one aspect, for VL, the subgroup is subgroup κI as in Kabat et al, supra. In one aspect, for VH, the subgroup is subgroup III as in Kabat et al, supra.

"humanized" antibody refers to chimeric antibodies comprising amino acid residues from non-human CDRs and amino acid residues from human FR. In certain aspects, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a non-human antibody, in "humanized form" refers to an antibody that has been humanized.

The term "hypervariable region" or "HVR" as used herein refers to the individual regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, e.g., the "complementarity determining regions" ("CDRs").

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:

(a) A highly variable loop present at the following amino acid residues: 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) (Chothia and Lesk, J.mol. Biol.196:901-917 (1987));

(b) CDRs occurring 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, 5 th edition Public Health Service, national Institutes of Health, bethesda, MD (1991)). And

(c) Antigen contact points occur 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)).

The CDRs are determined according to the method described by Kabat et al, supra, unless otherwise indicated. Those skilled in the art will appreciate that CDR names may also be determined according to the method described by Chothia, supra, mccallium, supra, or any other scientifically accepted naming system.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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.

An "isolated" antibody is an antibody that has been isolated from a component of its natural environment. In some aspects, the antibodies are purified to greater than 95% or 99% purity as determined by, for example, 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 of assessing antibody purity, see, e.g., flatman et al, J.chromatogr.B 848:79-87 (2007).

The term "nucleic acid molecule" or "polynucleotide" includes any compound and/or substance comprising a nucleotide polymer. Each nucleotide consists of a base, in particular 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. In general, nucleic acid molecules are described by a sequence of bases, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually expressed from 5 'to 3'. Herein, 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 mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. Furthermore, the term nucleic acid molecule includes sense and antisense strands, as well as single 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 nucleotide bases 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 the antibodies of the invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the coding molecule such that mRNA can be injected into a subject to produce in vivo antibodies (see, e.g., stadler et al, nature Medicine 2017, 6/12 on-line publication, doi:10.1038/nm.4356 or EP 2 101 823 B1).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more positions in a host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., individual antibodies comprising the population have identity and/or bind to the same epitope, except possibly variant antibodies (e.g., containing naturally occurring mutations or produced during production of a monoclonal antibody preparation, such variants typically 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 the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. The naked antibody may be present in a pharmaceutical composition.

"Natural antibody" refers to naturally occurring immunoglobulin molecules having different structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminal to the C-terminal, each heavy chain has a variable domain (VH), also known as a variable heavy chain domain or heavy chain variable region, followed by three constant heavy chain domains (CH 1, CH2 and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL), also known as a variable light chain domain or light chain variable region, followed by a constant light Chain (CL) domain.

A "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, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of an antigen. For example, the anti-PD-Ll antibodies of the invention block signaling through PD-1 in order to restore the functional response (e.g., proliferation, cytokine production, target cell killing) by T cells from a dysfunctional state to antigen stimulation.

An "antagonist" or activating antibody is an antibody that enhances or initiates signaling through the antigen to which it binds. In some embodiments, the antagonist antibody causes or activates signaling in the absence of the natural ligand.

The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products that contain information concerning the indication, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

By "insubstantial cross-reaction" is meant that a molecule (e.g., an antibody) does not recognize or specifically bind an antigen that differs from the actual target antigen of the molecule (e.g., an antigen closely related to the target antigen), particularly when compared to the target antigen. For example, an antibody may bind less than about 10% to less than about 5% of an antigen other than the actual target antigen, or may bind the antigen other than 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%.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the candidate sequence to the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity for the purposes of the alignment. The alignment for determining the percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, clustal W, megalign (DNASTAR) software, or FASTA packages. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared. Alternatively, the percent identity value may be generated using the sequence comparison computer program ALIGN-2. ALIGN-2 sequence comparison computer program was written by GeneTek corporation and the source code has been submitted with the user document to U.S. Copyright Office, washington D.C.,20559, registered there with U.S. copyright accession number TXU510087 and described in WO 2001/007511.

For purposes herein, the BLOSUM50 comparison matrix is used to generate values for percent amino acid sequence identity using the ggsearch program of FASTA package version 36.3.8c or higher, unless otherwise specified. FASTA packages are described by W.R.Pearson and D.J.Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; R.Pearson (1996) "Effective protein sequence comparison" meth.enzymol.266:227-258; and Pearson et al, (1997) Genomics 46:24-36 and are publicly available from www.fasta.bioch.virginia.edu/fasta_www2/fasta_down. Shtml or www.ebi.ac.uk/Tools/sss/fasta. Alternatively, sequences may be compared using a public server accessible at fasta. Bioch. Virginia. Edu/fasta_www2/index. Cgi, using a ggsearch (global protein: protein) program and default options (BLOSUM 50; open: -10; ext: -2; ktup=2) to ensure that global rather than local alignment is performed. The percentage amino acid identity is given in the output alignment header.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation that is in a form that allows for the biological activity of the active ingredient contained therein to be effective, and that is free of additional components that have unacceptable toxicity to the subject to whom the pharmaceutical composition is to be administered.

"pharmaceutically acceptable carrier" refers to ingredients of a pharmaceutical composition or formulation other than the active ingredient, which are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The term "PD-1 axis binding antagonist" is a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners to eliminate T cell dysfunction caused by signaling on the PD-1 signaling axis, with the result that T cell function { e.g., proliferation, cytokine production, target cell killing) is restored or enhanced. As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

The term "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling resulting from the interaction of PD-1 with one or more of its binding partners, e.g., PD-L1, PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In particular aspects, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signaling resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist reduces a negative co-stimulatory signal mediated by or through signaling by PD-1 mediated by a cell surface protein expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., increasing the response of an effector to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect, the PD-1 binding antagonist is MDX-1106 as described herein. In another specific aspect, the PD-1 binding antagonist is Merck 3745 as described herein. The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling resulting from the interaction of PD-Ll with one or more of its binding partners (such as PD-1, B7-1). In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L L to its binding partner. In a specific aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signaling resulting from interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In one embodiment, the PD-L1 binding antagonist may reduce a negative co-stimulatory signal mediated by or through signaling by PD-L L mediated by a cell surface protein expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., increasing the response of an effector to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L L antibody. In a specific aspect, the anti-PD-L1 antibody is yw243.55.s70 as described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1 105 as described herein. In yet another specific aspect, the anti-PD-L1 antibody is MPDL3280A as described herein.

The term "PD-L2 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signaling resulting from interaction of PD-L2 with one or more of its binding partners (such as PD-1). In one embodiment, the PD-L2 binding antagonist reduces a negative co-stimulatory signal mediated by or through signaling through PD-L2 mediated by a cell surface protein expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., increasing the response of an effector to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

A "PD-1 oligopeptide", "PD-L1 oligopeptide" or "PD-L2 oligopeptide" is an oligopeptide that preferably specifically binds to a PD-1, PD-L1 or PD-L2 negative co-stimulatory polypeptide, respectively, comprising a receptor, ligand or signaling component, respectively, as described herein. Such oligopeptides may be chemically synthesized using known methods of oligopeptide synthesis or may be prepared and purified using recombinant techniques. Such oligopeptides are typically at least about 5 amino acids in length, alternatively at least about 6,7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more. Such oligopeptides may be identified using well known techniques. In this regard, it is noted that techniques for screening libraries of oligopeptides capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Pat. nos. 5,556,762,5,750,373,4,708,871,4,833,092,5,223,409,5,403,484,5,571,689,5,663,143; pct publication nos. WO 84/03506 and WO84/03564; geysen et al, proc.Natl. Acad. Sci.U.S. A.,81:3998-4002 (1984); geysen et al, proc.Natl.Acad.Sci.S. A.,82:178-182 (1985), geysen et al in Synthetic Peptides as Antigens,130-149 (1986), geysen et al J.Immunol. Metk,102:259-274 (1987), schoofs et al J.Immunol., 140:61-616 (1988), cwirla, S.E. et al, proc.Natl.Acad.Sci.USA,87:6378 (1990), lowman, H.B. et al, biochemistry,30:10832 (1991), clackson, T.et al Nature,352:624 (1991), marks, J.D. et al J.mol. Biol.,222:581 (1991), kang, A.S. et al Proc.Natl.Acad.Sci.USA., 88, and Smin (1991) J.M.H.B., J.S. 35 (1991), and Smin 6:668.S. J.S. 1991).

The term "anergy" refers to the non-responsive state to antigen stimulation caused by incomplete or insufficient signaling through T cell receptors (e.g., intracellular Ca in the absence of ras activation) ⁺² Increase). Stimulation of the antigen in the absence of co-stimulation also results in the production of T-cell failure, resulting inEven in the case of co-stimulation, the cells still become refractory to subsequent antigen activation. The presence of interleukin-2 can often overcome this non-responsive state. The non-potent T cells do not undergo clonal expansion and/or acquire effector function.

The term "depletion" refers to T cell depletion, a T cell dysfunctional state that results from sustained TCR signaling during many chronic infections and cancers. Depletion differs from anergy in that depletion is not caused by incomplete or inadequate signaling, but rather by sustained signaling. Depletion is defined as poor effector function, sustained expression of inhibitory receptors, and a transcriptional state that differs from that of functional effector or memory T cells. Depletion results in less than optimal control of infection and tumors. Depletion can be caused by either external negative regulatory pathways (e.g., immunomodulatory cytokines) or by intracellular negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).

"enhancing T cell function" refers to inducing, causing or stimulating T cells to have sustained or increased biological function, or restoring or reactivating depleted or inactive T cells. Examples of enhancing T cell function include: gamma interferon from CD8 relative to pre-intervention levels ⁺ Increased secretion of T cells, increased proliferation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance). In one embodiment, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 1, 20%, 150%, 200%. The manner in which this enhancement is measured is known to those of ordinary skill in the art.

"tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding (tumor binding), tumor shrinkage, and tumor elimination.

"immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic and increasing tumor immunogenicity aids in the elimination of tumor cells by an immune response. Examples of enhancing tumor immunogenicity include treatment with anti-PDL antibodies and ME inhibitors.

"sustained response" refers to the sustained effect on reducing tumor growth after cessation of treatment. For example, the tumor size may remain the same or smaller than the size at the beginning of the dosing phase. In some embodiments, the duration of the sustained response is at least the same as the duration of the treatment, at least 1.5 times, 2.o times, 2.5 times, or 3.O times the length of the duration of the treatment.

As used herein, "treatment" (and grammatical variations thereof) refers to a clinical intervention that attempts to alter the natural course of a disease in an individual being treated, and that may be performed for prophylaxis or that may be performed during a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating a disease state, and alleviating or improving prognosis. In some aspects, the antibodies of the invention are used to delay the progression of a disease or to slow the progression of a disease.

As used herein, the term "cancer" refers to a proliferative disease, such as cancer that is colorectal cancer, sarcoma, head and neck cancer, squamous cell carcinoma, breast cancer, pancreatic cancer, gastric cancer, non-small cell lung cancer, and mesothelioma, including refractory versions of any of the foregoing cancers, or a combination of one or more of the foregoing cancers. In one embodiment, the cancer is colorectal cancer and optionally the chemotherapeutic agent is irinotecan. In embodiments where the cancer is a sarcoma, optionally, the sarcoma is chondrosarcoma, leiomyosarcoma, gastrointestinal stromal tumor, fibrosarcoma, osteosarcoma, liposarcoma, or malignant fibrous histiocytoma.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). (see, e.g., kit et al, kuby Immunology, 6 th edition, w.h. freeman and co., page 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 VH or VL domains, respectively, from antibodies that bind that antigen to screen libraries 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).

The term "vector" as used herein refers to a nucleic acid molecule capable of carrying another nucleic acid linked thereto. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that integrate into the genome of a host cell into which they have 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".

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

The term "antigen binding site of an antibody" as used herein refers to the amino acid residues in an antibody that are responsible for antigen binding. The antigen binding portion of an antibody comprises amino acid residues from a "complementarity determining region" or "CDR". "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 comprise 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 most conducive to antigen binding and defines antibody properties. CDR and FR regions are defined according to the standard definition of Kabat et al (Sequences of Proteins ofImmunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991)) and/or those residues from "hypervariable loops".

Antibodies specifically refer to the selective recognition of a particular epitope of an antigen by an antibody. For example, natural antibodies are monospecific. A "bispecific antibody" according to the invention is an antibody having two different antigen binding specificities. The antibodies of the invention are specific for two different antigens, namely DR5 is the first antigen and FAP is the second antigen.

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

The term "bispecific" refers to an antigen binding molecule that is capable of specifically binding to at least two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises at least two antigen binding sites, each of which is specific for a different epitope. In certain embodiments, the bispecific antigen binding molecule is capable of binding two epitopes simultaneously, in particular two epitopes expressed on two unique cells.

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 "pestle and mortar" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic manipulation effects to prepare antibody Fc-heterodimer molecules (WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al Science,229:81 (1985)); bispecific antibodies were generated using leucine zippers (see, e.g., kostelny et al, j. Immunol.,148 (5): 1547-1553 (1992)); bispecific antibody fragments were prepared using the "diabody" technique (see, e.g., hollinger et al, proc. Natl. Acad. Sci. USA,90:6444-6448 (1993)); and the use of single chain Fv (sFv) dimers (see, e.g., gruber et al, J.Immunol.,152:5368 (1994)); and trispecific antibodies were prepared as described, for example, in Tutt et al J.Immunol.147:60 (1991).

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

Antibodies or fragments herein also include a "dual acting FAb" or "DAF" comprising at least one antigen binding site that binds to FAP or DR5 and another, different antigen (see, e.g., US 2008/0069820).

The term "valency" as used in this application means the presence of a specified number of binding sites in an antibody molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" denote the presence of two binding sites, four binding sites and six binding sites, respectively, in an antibody molecule. Bispecific antibodies according to the invention are at least "bivalent" and may be "trivalent" or "multivalent" (e.g. "tetravalent" or "hexavalent").

The antibodies of the invention have two or more binding sites and are bispecific. That is, an antibody may be bispecific even in the presence of more than two binding sites (i.e., the antibody is trivalent or multivalent). Bispecific antibodies of the 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, linked to other antigen binding sites (e.g., single chain Fv, VH and/or VL domains, fab or (Fab) 2) via one or more peptide linkers. The antibodies may be full length antibodies from a single species, or may be chimeric or humanized.

The term "amino acid" as used in this application means a group of naturally occurring carboxy alpha-amino acids comprising: alanine (three-letter code: ala, one-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).

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include offspring. Thus, the words "transfectants" and "transfected cells" include primary test cells and cultures derived from such cells irrespective of the number of transfers. It should also be appreciated that all offspring may not be exactly identical in DNA content due to deliberate or unintended mutations. Including variant progeny that have the same function or biological activity as screened in the original transformed cell.

"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). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibodies and antigens). The affinity of a molecule X for its partner Y can generally be expressed by a 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.

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

Binding of antibodies to death receptors can be studied by BIAcore assay (GE-Healthcare Uppsala, sweden). The affinity of binding is defined by the terms ka (association rate constant of antibody from antibody/antigen complex), kD (dissociation constant) and kD (kD/ka)

"reduced binding" (e.g., reduced binding to Fc receptor) refers to reduced 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 assay method), i.e., eliminating interactions altogether. Conversely, "increased binding" refers to an increase in binding affinity for the corresponding interaction.

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

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

As used herein, the terms "first" and "second" with respect to antigen binding portions and the like are used to facilitate differentiation when each type of portion is more than one.

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three dimensional structural characteristics and/or specific charge characteristics. An epitope is the region of an antigen to which an antibody binds.

As used herein, the term "epitope" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a stretch of contiguous amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids) to which an antigen binding portion binds, thereby forming 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 the serum, and/or in the extracellular matrix (ECM). Unless otherwise indicated, proteins referred to herein as antigens (e.g., PD-1 and PD-L1) may be any native form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human protein. When referring to a particular protein herein, the term encompasses "full length", unprocessed proteins, as well as any form of protein resulting from intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

As used herein, the term "engineering, engineered, engineering", particularly the term with the prefix "sugar-" 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 glycosylation changes in glycoproteins expressed in the cell. Furthermore, glycosylation engineering includes mutations and the effect of cellular environment on glycosylation. In one embodiment, glycosylation engineering is engineered to be a change in glycosyltransferase activity. In a particular embodiment, the engineering results in altered glucosaminyl transferase activity and/or fucosyl transferase activity.

The combination therapy according to the invention has a synergistic effect. A "synergistic effect" of two compounds is one in which the combined effect of the two agents is greater than the sum of their respective effects, and is statistically different from the control and single agent. In another embodiment, the combination therapies disclosed herein have an additive effect. The "additive effect" of two compounds is one in which the combined effect of the two agents is the sum of their respective effects and is statistically different from the control and/or single agent.

In one aspect, the invention provides a BRAF inhibitor and a PD-1 axis binding antagonist for use in the treatment of cancer, wherein the BRAF inhibitor is a compound of formula (I)

Or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments of the invention, the compound of formula (I) is a compound according to formula (Ia):

in some embodiments of the invention, the compound of formula (I) is a compound according to formula (Ib):

non-limiting examples of PD-1 axis binding antagonists for use in accordance with the invention include the Semiphene Li Shan antibodyPembrolizumab +.>Nawuzumab +.>RN888 (PF-06801591), alemtuzumab +.>Avermectin->And Devaluzumab (ImfinziTM).

In some embodiments of the invention, the PD-1 axisThe binding antagonist is selected from the group consisting of cimetidine Li ShanPembrolizumab +.>Nawuzumab +.>And PD-1 binding antagonists of RN888 (PF-06801591). In some embodiments of the invention, the PD-1 axis binding antagonist is selected from alemtuzumab +.>AbamectinAnd PD-L1 binding antagonists of dewaruzumab (ImfinziTM).

Measurement program

Material

DMEM supplemented with L-glutamine was purchased from phenol red free medium (Thermo Fisher Scientific). Fetal Bovine Serum (FBS) was purchased from VWR. Advanced ERK phosphate-T202/Y204 kit-10,000 tests were purchased from Cisbio catalog number 64AERPEH. A375 was initially obtained from ATCC and stored by Roche (Roche) store. 384 well microplates were purchased from Greiner Bio-One,384 wells (with lid, hiBase, low volume catalogue 784-080).

HTRF assay for P-ERK determination in A375 cells or HCT116 cells

A375 is a cell cancer model for BRAF expressing the V600E mutation, and HCT116 is a cell cancer model for WT BRAF. First generation BRAF inhibitors (such as, for example, dabrafenib) induce paradox effects on tumor cells, as they inhibit the growth of V600E mutated BRAF cells (such as, for example, a 375) while they activate the growth of WT BRAF cells (such as, for example, HCT 116). ERK 1,2 phosphorylation (the end member of the phosphorylation cascade of the MAPK pathway) is reported hereinafter as the primary reading of the activation state of the MAPK pathway. Prior to assay, the A375 and HCT116 cell lines were maintained in DMEM phenol red free medium supplemented with 10% Fetal Bovine Serum (FBS). After compound treatment, P-ERK levels were determined by measuring FRET fluorescent signals induced by selective binding of 2 antibodies (Cisbio catalog No. 64 AERPEH) provided in the above kit to ERK protein upon phosphorylation at Thr202/Tyr 204. Briefly, 8000 cells/well of 12 μl of medium/well were seeded in 384-well plates and placed overnight in an incubator (at 37 ℃ and 5% CO2 humidified atmosphere), and the plates were treated in duplicate with test compounds dabrafenib and PLX8394 (the latter two as controls) at the following final drug concentrations on the following day: 10. Mu.M-3. Mu.M-1. Mu.M-0.3. Mu.M-0.1. Mu.M-0.03. Mu.M, 01. Mu.M-0.003. Mu.M, all wells were normalized to DMSO and drug incubation occurred for 1 hour. Then, 4 μl of 4X lysis buffer supplied by the kit was added to the wells, then the plates were centrifuged for 30 seconds (300 rcf) and incubated on a plate shaker for 1h at room temperature.

At the end of incubation, 4. Mu.L/well of higher P-ERK antibody solution (prepared according to manufacturer's instructions) was added to the test wells, followed by 4. Mu.L/well of script P-ERK antibody solution (prepared according to manufacturer's instructions) (Cisbio catalog number 64 AERPEH).

For the data to normalize correctly, the non-drug treatment control wells reported in the table below are always contained in each plate (according to manufacturer's instructions):

control and experimental p-ERK HTRF pore content (μl):

plates were then centrifuged at 300rcf for 30 seconds, sealed to prevent evaporation, and incubated overnight at room temperature in the dark.

The plates were then analyzed and fluorescence emission values were collected by a Pherastast FSX (BMG Labtech) device at 665nM and 620 nM.

The fluorescence values obtained are according to the formula: ratio = signal (620 nm)/signal (625 nm) 10000, then subtracting all values from the average of the ratios on the blank.

In the case of a375 cells (BRAF inhibition), the average of the ratios derived from DMSO-only treated cells (minus blank) was taken as 100%, and the average of the ratios derived from 10 μΜ darifenacin treated cells (minus blank) was taken as 0%, the data were normalized. The average of the normalized points was fitted to an S-curve and the IC50 was determined. The results are shown in Table 1.

In the case of HCT116 cells (BRAF activation), the average of the ratios derived from DMSO-only treated cells (minus the blank) was taken as 0%, and the average of the ratios derived from darafenib-treated cells (which were at the concentration that provided the highest signal) (minus the blank) was taken as 100%, with the data normalized. Individual points were fitted to sigmoid or bell-shaped curves and the percent activation compared to the maximum activation mediated by dabrafenib was determined. EC50 is the concentration at which activation equivalent to 50% of the maximum achieved by dabrafenib is obtained. The results are shown in Table 2.

If the activation does not reach 50% of the maximum achieved by Lafenib, then the EC50 calculation is not applicable.

The percentage of the maximum paradoxical induction effect from darifenacin was determined by evaluating the percentage of the test compound that induced its maximum P-ERK signal as the percentage of the highest signal produced by darifenacin within the tested dose range.

WO2012/118492 discloses reference compounds AR-25 as example 25, AR-30 as example 30 and AR-31 as example 31.

Table 1: example 1 ((3R) -N- [ 2-cyano-4-fluoro-3- (3-methyl-4-oxo-quinazolin-6-yl) oxy-phenyl ] -3-fluoro-pyrrolidine-1-sulfonamide) has high affinity for RAF kinase and high selectivity for C-terminal Src kinase (CSK) and lymphocyte-specific tyrosine protein kinase (LCK) when compared to AR-30 and AR-31.

Table 2: example 1 ((3R) -N- [ 2-cyano-4-fluoro-3- (3-methyl-4-oxo-quinazolin-6-yl) oxy-phenyl ] -3-fluoro-pyrrolidine-1-sulfonamide) is inhibiting paradoxical RAF activation in WT BRAF expressing HCT-116 cancer cells. Compared to dabrafenib or AR-25, the maximal spurious induction is reduced to less than 50%.

The preparation of the compounds of formula (I) according to the invention can be carried out sequentially or in a concurrent synthetic route. The synthesis of the present invention is shown in the following general scheme. The skills required to carry out the reactions and purification of the resulting product are known to those skilled in the art.

In more detail, the compounds of formula (I) may be prepared by the methods given below, by the methods given in the examples or by similar methods. Suitable reaction conditions for the individual reaction steps are known to the person skilled in the art. The reaction sequence is not limited to the sequence shown in scheme 1, but the sequence of the reaction steps may be freely changed depending on the starting materials and their respective reactivities. Starting materials are commercially available or may be prepared by methods similar to those given below, by methods described in the specification or in the references cited in the exemplary procedures below, or by methods known in the art.

Scheme 1

It will be appreciated that the compounds of formula I of the present invention may be derivatized at functional groups to provide derivatives that are capable of conversion back to the parent compound in vivo.

Experimental procedure

(3R) -N- [ 2-cyano-4-fluoro-3- (3-methyl-4-oxo-quinazolin-6-yl) oxy-phenyl ] -3-fluoro-pyrrolidine-1-sulfonamide (compound of formula (Ia)) and (3S) -N- [ 2-cyano-4-fluoro-3- (3-methyl-4-oxo-quinazolin-6-yl) oxy-phenyl ] -3-fluoro-pyrrolidine-1-sulfonamide (compound of formula (Ib))

6-hydroxy-3-methyl-quinazolin-4-one

2-amino-5-hydroxybenzoic acid (10 g,65.3mmol, eq: 1.0) and N-methylformamide (30 g,29.9mL,503mmol, eq: 7.7) were heated at 145℃for 21h 45min and then cooled to room temperature. The reaction mixture was taken up with 50mL H ₂ Dilute with O and stir at room temperature for 20min. The resulting precipitate was collected by filtration. The pale brown solid was washed 3 times with 20mL of water. The solid was taken up in toluene and evaporated to dryness (3×). The solid was dried under high vacuum overnight at 40 ℃ to give the title compound as a light brown solid (10.3 g,89% yield). MS (ESI) m/z:177.1[ M+H ]] ⁺ 。

3, 6-difluoro-2- (3-methyl-4-oxo-quinazolin-6-yl) oxy-benzonitrile

Cesium carbonate (3.22 g,9.79mmol, eq: 1.15) was added to a solution of 6-hydroxy-3-methylquinazolin-4-one (1500 mg,8.51mmol, eq: 1.0) in N, N-dimethylformamide (35 mL) at room temperature. The mixture was stirred at room temperature for 30min, then 2,3, 6-trifluorobenzonitrile (1.47 g,1.08ml,9.37mmol, eq: 1.1) was added. After 1h, the reaction was cooled on ice and diluted with water (120 mL). The resulting solid was collected by filtration, washed with ice water (100 mL) and heptane (100 mL) and dried by suction. The solid was taken up in toluene and evaporated to dryness (3×) and then dried in vacuo overnight to give the title compound as a light brown solid (2.58 g,97% yield). MS (ESI) m/z:314.1[ M+H ] +.

(3R) -3-fluoropyrrolidine-1-sulfonamide

(R) -3-fluoropyrrolidine hydrochloride (1.8 g,14.3mmol, eq: 1.2) was added to a solution of diamide sulfate (1.148 g,11.9mmol, eq: 1.0) and triethylamine (2.42 g,3.33mL,23.9mmol, eq: 2) in dioxane (10 mL). The reaction was stirred in a sealed tube at 115 ℃ for 15.5h, then cooled to room temperature and concentrated in vacuo. The residue was diluted with DCM, evaporated to dryness on silica gel and transferred to a column. Purification by flash chromatography (40 g silica, 80% EtOAc) afforded the title compound as a white crystalline solid (1.82 g,91% yield). MS (ESI) m/z:169.1[ M+H ]] ⁺ 。

(3S) -3-fluoropyrrolidine-1-sulfonamide

Triethylamine (304 mg, 419. Mu.l, 3.01mmol, eq: 2.0) was added to a suspension of diamide sulfate (146 mg,1.5mmol, eq: 1.0) and (S) -3-fluoropyrrolidine hydrochloride (234 mg,1.8mmol, eq: 1.2) in dioxane (1.3 ml). The reaction was stirred in a sealed tube at 115 ℃ for 16h 35min, then concentrated in vacuo. The residue was diluted with MeOH and evaporated to dryness with silica gel and transferred to a column. Purification by flash chromatography (40 g silica, 0 to 8% MeOH/DCM) afforded the title compound as a pale yellow solid (193 mg,75% yield). MS (ESI) m/z:169.1[ M+H ] ] ⁺ 。

(3R) -N- [ 2-cyano-4-fluoro-3- (3-methyl-4-oxo-quinazolin-6-yl) oxy-phenyl ] -3-fluoro-pyrrolidine-1-sulfonamide (a compound of formula (Ia))

(R) -3-fluoropyrrolidine-1-sulfonamide (1.26 g,7.51mmol, eq: 2.1) and cesium carbonate (2.56 g,7.87mmol, eq: 2.2) were suspended in dry DMF (10.2 ml) under argon. The reaction mixture was stirred at 50℃for 30min. The reaction mixture was cooled to room temperature and 3, 6-difluoro-2- ((3-methyl-4-oxo-3, 4-dihydroquinazolin-6-yl) oxy) benzonitrile (1.12 g,3.58mmol, eq: 1.0) in DMF (25.5 ml) was addedA solution. The reaction mixture was stirred at 100 ℃ for 15h and then concentrated in vacuo. The resulting mixture was treated with saturated NH ₄ Aqueous Cl (100 mL) and EtOAc (100 mL) were taken up. The phases were separated and the aqueous layer was further extracted with 2x100ml EtOAc. The combined organic layers were washed with water (200 mL) and brine (200 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated in vacuo. The aqueous layer was back extracted with EtOAc (3X 100 mL). The combined organic extracts were washed with brine (200 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated in vacuo. The residue was diluted with DCM and MeOH and concentrated onto silica. Purification by flash chromatography (120 g,0.5-2% MeOH/DCM) afforded an off-white solid, which was triturated with 1:1 heptane/DCM (20 mL) and then dried in vacuo to afford the title compound as a colorless solid (1.087 g,66% yield). MS (ESI) m/z:426.2[ M+H ] ] ⁺ . Chiral SFC: rt= 4.594min[Chiralpak IC column, 4.6x250mm,5 μm particle size (Daicel); contains 0.2% NHEt ₂ A 20 to 40% meoh gradient for 8min; flow rate: 2.5mL/min;140bar back pressure]。

(3S) -N- [ 2-cyano-4-fluoro-3- (3-methyl-4-oxo-quinazolin-6-yl) oxy-phenyl ] -3-fluoro-pyrrolidine-1-sulfonamide (a compound of formula (Ib))

(S) -3-fluoropyrrolidine-1-sulfonamide (181 mg,1.08mmol, eq: 2.1) was dissolved in DMF (1.6 ml). Cesium carbonate (268 mg,1.13mmol, eq: 2.2) was added at room temperature, and the reaction mixture was stirred at 50℃for 30min. The reaction mixture was cooled to room temperature, and a solution of 3, 6-difluoro-2- ((3-methyl-4-oxo-3, 4-dihydroquinazolin-6-yl) oxy) benzonitrile (160.8 mg, 513. Mu. Mol, eq: 1.0) in DMF (4 ml) was added. The reaction mixture was stirred at 105 ℃ for 2h 50min, then concentrated in vacuo. The residue was taken up in DCM and taken up with saturated NH ₄ The aqueous Cl solution was washed. The aqueous layer was back extracted twice with DCM. The combined organic layers were purified by Na ₂ SO ₄ Dried, filtered and evaporated. The residue (brown oil) was diluted with DCM and transferred to column. Pure by flash chromatographyThe reaction was quenched (80 g,0 to 100% EtOAc in DCM) to give a solid, which was further purified by SFC to give the title compound as a pale yellow solid (119 mg,50% yield). MS (ESI) m/z:426.2[ M+H ] ] ⁺ . Chiral SFC: rt= 4.411min[Chiralpak IC column, 4.6x250mm,5 μm particle size (Daicel); contains 0.2% NHEt ₂ A 20 to 40% MeOH gradient for 8min; flow rate: 2.5mL/min;140bar back pressure]。

The invention relates in particular to:

a combination of a BRAF inhibitor and a PD-1 axis binding antagonist, wherein the BRAF inhibitor is a compound of formula (I)

Or a pharmaceutically acceptable salt or solvate thereof;

a combination of a BRAF inhibitor and a PD-1 axis binding antagonist as described herein, 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;

a combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to the invention, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist;

the combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to the invention, wherein the PD-1 axis binding antagonist is selected from the group consisting of a cimrpu Li Shan antibodyPembrolizumab +.>Nawuzumab +.>RN888 (PF-06801591), alemtuzumab +.>Abametric sheet

Anti-cancer agentAnd Dewaruzumab (ImfinziTM);

the combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to the invention, wherein the PD-1 axis binding antagonist is selected from the group consisting of a cimrpose Li Shan antibody Pembrolizumab +.>Nawuzumab +.>And PD-1 binding antagonists of RN888 (PF-06801591);

the combination of a BRAF inhibitor and a PD-1 axis binding according to the invention, wherein the PD-1 axis binding antagonist is selected from the group consisting of alemtuzumabAvermectin->And PD-L1 inhibitors of dewaruzumab (ImfinziTM);

the combination of a BRAF inhibitor and a PD-1 axis binding according to the invention, wherein the PD-1 axis binding antagonist is alemtuzumab

A combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to the invention herein for use as a medicament;

a combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to the invention for use in the therapeutic and/or prophylactic treatment of cancer;

use of a combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to the invention for the manufacture of a medicament for the treatment or prevention of cancer;

a method for treating or preventing cancer, in particular melanoma, non-small cell lung cancer or leptomeningeal cancer, comprising administering to a patient in need thereof an effective amount of a combination of a BRAF inhibitor according to the invention and a PD-1 axis binding antagonist;

a pharmaceutical composition comprising a combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to the invention, and one or more pharmaceutically acceptable excipients;

The combination, use, method or pharmaceutical composition according to the invention, wherein the BRAF inhibitor is administered orally and the PD-1 axis binding antagonist is administered by injection, such as intravenous or subcutaneous injection;

the combination, use, method or pharmaceutical composition according to the invention, wherein the BRAF inhibitor is administered simultaneously with the PD-1 axis binding antagonist;

a combination, use, method or pharmaceutical composition according to the invention, wherein a BRAF inhibitor and a PD-1 axis binding antagonist are co-formulated;

the combination, use, method or pharmaceutical composition according to the invention, wherein the BRAF inhibitor and the PD-1 axis binding antagonist are administered sequentially;

the combination, use, method or pharmaceutical composition according to the invention, the BRAF inhibitor is administered orally daily, and wherein the PD-1 axis binding antagonist is administered once weekly by intravenous or subcutaneous injection;

the combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to the invention, wherein the cancer is thyroid cancer, colorectal cancer, melanoma, brain cancer, leptomeningeal cancer or non-small cell lung cancer;

a combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to the invention, wherein the cancer is with BRAF ^V600X Mutations are associated;

the combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to the invention, wherein the cancer is BRAF ^V600X Mutation-positive unresectable or metastatic cancer;

the combination BRAF inhibitor and PD-1 axis binding antagonist for use according to the invention, wherein the BRAF is determined using a method comprising ^V600X Mutation: (a) PCR or sequencing nucleic acids (e.g., DNA) extracted from tumor tissue and/or body fluid samples of a patient; (b) determining BRAF in the sample ^V600 Is expressed by (a);

a combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to or for use according to the invention, comprising one or more additional anti-cancer agents, in particular one or more anti-cancer agents selected from the group consisting of MEK inhibitors, MEK degradants, EGFR inhibitors, EGFR degradants, HER2 and/or HER3 inhibitors, HER2 and/or HER3 degradants, SHP2 inhibitors, SHP2 degradants, axl inhibitors, axl degradants, ALK inhibitors, ALK degradants, PI3K inhibitors, PI3K degradants, SOS1 inhibitors, SOS1 degradants, signal transduction pathway inhibitors, checkpoint inhibitors, modulators of the apoptotic pathway, cytotoxic chemotherapeutic agents, angiogenesis targeting therapies, immune targeting agents and antibody drug conjugates;

a combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to the invention, which additionally comprises a MEK inhibitor;

A combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to the invention, which additionally comprises a MEK inhibitor selected from cobicitinib, bimetinib (binimetinib) and trametinib (trametinib);

a combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to the invention, which additionally comprises cobicitinib;

use of a BRAF inhibitor and a PD-1 axis binding antagonist in combination according to the invention for the manufacture of a medicament for the treatment or prevention of cancer, wherein the BRAF inhibitor 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;

use of a combination of a BRAF inhibitor according to the invention herein and a PD-1 axis binding, wherein the PD-1 axis binding antagonist is selected from the group consisting of cimrpe Li Shan anti-PembrolizumabNawuzumab +.>RN888 (PF-06801591), alemtuzumab +.>Avermectin->And Dewaruzumab (ImfinziTM);

use of a combination of a BRAF inhibitor according to the invention and a PD-1 axis binding antagonist for the preparation of a medicament according to the invention, wherein the PD-1 axis binding antagonist is alemtuzumab

A method for treating or preventing cancer, the method comprising administering to a patient in need thereof an effective amount of a BRAF inhibitor as described herein and a PD-1 axis binding antagonist, wherein the BRAF inhibitor 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;

The method of treating or preventing cancer according to the present invention, wherein the cancer is selected from the group consisting of thyroid cancer, colorectal cancer, melanoma, brain cancer, pia mater cancer, and non-small cell lung cancer;

the method for treating or preventing cancer according to the present invention, wherein the PD-1 axis binding antagonist is selected from the group consisting of a cimrpu Li Shan antagonistPembrolizumab +.>Nawuzumab +.>RN888 (PF-06801591), alemtuzumab +.>Avermectin->And Dewaruzumab (ImfinziTM);

a pharmaceutical composition as described herein wherein the PD-1 axis binding antagonist is selected from the group consisting of a cimrpress Li Shan antagonistPembrolizumab +.>Nawuzumab +.>And PD-1 binding antagonists of RN888 (PF-06801591);

the pharmaceutical composition as described herein wherein the PD-1 axis binding antagonist is selected from the group consisting of atuzumabAvermectin->And PD-L1 binding antagonists of dewaruzumab (ImfinziTM);

a pharmaceutical composition as described herein wherein the PD-1 axis binding antagonist is alemtuzumab

A pharmaceutical composition as described herein 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;

a pharmaceutical composition as described herein wherein the compound of formula (I) is (3S) -N- [ 2-cyano-4-fluoro-3- (3-methyl-4-oxo-quinazolin-6-yl) oxy-phenyl ] -3-fluoro-pyrrolidine-1-sulfonamide;

A pharmaceutical composition as described herein for use in the treatment or prevention of cancer, in particular thyroid cancer, colorectal cancer, melanoma, brain cancer, leptomeningeal cancer or non-small cell lung cancer;

a pharmaceutical composition as described herein for use in the treatment or prevention of cancer, in particular thyroid cancer, colorectal cancer, melanoma, brain cancer, pia mater cancer or non-small cell lung cancer, wherein a BRAF inhibitor and a PD-1 axis binding antagonist are administered orally;

a pharmaceutical composition as described herein for use in the treatment or prevention of cancer, in particular thyroid cancer, colorectal cancer, melanoma, brain cancer or non-small cell lung cancer, wherein the first composition is administered simultaneously with the second composition;

a pharmaceutical composition as described herein for use in the treatment or prevention of cancer, in particular thyroid cancer, colorectal cancer, melanoma, brain cancer, leptomeningeal cancer or non-small cell lung cancer, wherein the first composition and the second composition are co-formulated;

a pharmaceutical composition as described herein for use in the treatment or prevention of cancer, in particular thyroid cancer, colorectal cancer, melanoma, brain cancer, leptomeningeal cancer or non-small cell lung cancer, wherein the first composition and the second composition are administered sequentially;

a pharmaceutical composition as described herein for use in the treatment or prevention of cancer, wherein the cancer is associated with a BRAF mutation;

a pharmaceutical composition as described herein for use in the treatment or prevention of cancer, wherein the cancer is BRAF ^V600X Mutation-positive unresectable or metastatic cancers, particularly BRAF ^V600E Or BRAF ^V600K Mutation-positive unresectable or metastatic cancer;

a pharmaceutical composition as described herein for use in the treatment or prevention of cancer, in particular thyroid cancer, colorectal cancer, melanoma, brain cancer or non-small cell lung cancer, wherein the determination is made using a method comprisingBRAF ^V600X Mutation: (a) PCR or sequencing nucleic acids (e.g., DNA) extracted from tumor tissue and/or body fluid samples of a patient; (b) determining BRAF in the sample ^V600 Is expressed by (a);

use of a pharmaceutical composition as described herein in the treatment or prevention of cancer, in particular thyroid cancer, colorectal cancer, melanoma, brain cancer, leptomeningeal cancer or non-small cell lung cancer; and

use of a pharmaceutical composition as described herein in the manufacture of a medicament for the treatment or prophylaxis of cancer, in particular thyroid cancer, colorectal cancer, melanoma, brain cancer, pia matera cancer or non-small cell lung cancer.

Further embodiments of the invention are:

an embodiment of the invention relates to a method of treating or preventing cancer, particularly thyroid cancer, colorectal cancer, melanoma, brain cancer, leptomeningeal cancer or non-small cell lung cancer, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition as described herein;

certain embodiments of the invention relate to pharmaceutical compositions as described herein for use as a medicament for the therapeutic and/or prophylactic treatment of brain metastases;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use as a medicament for the therapeutic and/or prophylactic treatment of brain metastases, wherein the primary tumor is melanoma or non-small cell lung cancer;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use in the treatment and/or prevention of cancer, particularly melanoma or non-small cell lung cancer, wherein the patient has not received targeted therapy;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use in the treatment and/or prevention of cancer, particularly melanoma or non-small cell lung cancer, wherein the patient has not received targeted therapy and wherein the patient has checkpoint inhibitor treatment experience;

Certain embodiments of the invention relate to a pharmaceutical composition as described herein for use in the treatment and/or prevention of cancer, in particular melanoma or non-small cell lung cancer, wherein the patient has a therapeutic experience with respect to targeted therapies, and wherein the patient has a checkpoint inhibitor therapeutic experience;

certain embodiments of the invention relate to pharmaceutical compositions as described herein for use in the treatment and/or prevention of cancer, particularly melanoma or non-small cell lung cancer, wherein the cancer was previously treated by surgery;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use in the treatment and/or prevention of cancer, particularly melanoma or non-small cell lung cancer, wherein the patient has not received treatment for BRAF inhibitor treatment;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use as a medicament for the therapeutic and/or prophylactic treatment of a BRAF inhibitor-treated resistant tumor;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use in the treatment and/or prevention of cancer, particularly melanoma, non-small cell lung cancer or leptomeningeal cancer, wherein the patient has not been treated with a therapeutic inhibitor for MEK;

Certain embodiments of the invention relate to a pharmaceutical composition as described herein for use in the treatment and/or prevention of cancer, particularly melanoma or non-small cell lung cancer, wherein the patient is untreated for PD-1 axis binding antagonist treatment;

certain embodiments of the invention relate to pharmaceutical compositions as described herein for use as a medicament for the therapeutic and/or prophylactic treatment of a tumor that is resistant to treatment by a MEK inhibitor; and

certain embodiments of the invention relate to pharmaceutical compositions as described herein for use as a medicament for the therapeutic and/or prophylactic treatment of a PD-1 axis binding antagonist for the treatment of a resistant tumor.

Certain embodiments of the invention relate to a pharmaceutical composition as described herein for use as a medicament for the therapeutic and/or prophylactic treatment of cancer in a subject previously treated with a BRAF inhibitor selected from Kang Naifei, dabrafenib and Kang Naifei, and a MEK inhibitor selected from bimatinib, trimatinib and cobicitinib;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use as a medicament for the therapeutic and/or prophylactic treatment of cancer in a subject previously treated with a BRAF inhibitor selected from Kang Naifei, dabrafenib and Kang Naifei, and a PD-1 axis binding antagonist;

Certain embodiments of the invention relate to a pharmaceutical composition as described herein for use as a medicament in therapeutic and/or prophylactic treatment of a subject previously treated with Kang Naifei ni and bimatinib;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use as a medicament in therapeutic and/or prophylactic treatment of a subject previously treated with dabrafenib and trimetinib;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use as a medicament in the therapeutic and/or prophylactic treatment of a subject previously treated with vemurafenib and cobicitinib;

certain embodiments of the invention relate to a pharmaceutical composition as described herein for use as a medicament in the therapeutic and/or prophylactic treatment of cancer (particularly melanoma or non-small cell lung cancer) in a subject previously treated with a checkpoint inhibitor;

certain embodiments of the invention relate to pharmaceutical compositions as described herein for use as a medicament in the therapeutic and/or prophylactic treatment of cancer (particularly melanoma or non-small cell lung cancer) in a subject previously treated with a PD-1 axis binding antagonist;

certain embodiments of the invention relate to BRAF inhibitors and PD-1 axis binding antagonists as described herein for use in the therapeutic and/or prophylactic treatment of cancer, particularly melanoma or non-small cell lung cancer, wherein the patient has not received targeted therapy;

Certain embodiments of the invention relate to BRAF inhibitors and PD-1 axis binding antagonists as described herein for use in the therapeutic and/or prophylactic treatment of cancer, particularly melanoma or non-small cell lung cancer, wherein the patient has not been treated for targeted therapy, and wherein the patient has checkpoint inhibitor treatment experience;

certain embodiments of the invention relate to BRAF inhibitors and PD-1 axis binding antagonists as described herein for use in the therapeutic and/or prophylactic treatment of cancer, wherein the patient has treatment experience with respect to targeted therapy, and wherein the patient has checkpoint inhibitor treatment experience;

certain embodiments of the invention relate to a BRAF inhibitor and a PD-1 axis binding antagonist as described herein for use in the therapeutic and/or prophylactic treatment of cancer, particularly melanoma or non-small cell lung cancer, wherein the patient is untreated for treatment with the BRAF inhibitor;

certain embodiments of the invention relate to a BRAF inhibitor and a PD-1 axis binding antagonist as described herein for use as a medicament for the therapeutic and/or prophylactic treatment of a BRAF inhibitor-treated resistant tumor;

certain embodiments of the invention relate to a BRAF inhibitor and a PD-1 axis binding antagonist as described herein for use in the therapeutic and/or prophylactic treatment of cancer, particularly melanoma or non-small cell lung cancer, wherein the patient is untreated with respect to the treatment with the PD-1 axis binding antagonist;

Certain embodiments of the invention relate to a BRAF inhibitor and a PD-1 axis binding antagonist treatment as described herein for use in the therapeutic and/or prophylactic treatment of a PD-1 axis binding antagonist treatment resistant tumor;

certain embodiments of the invention relate to a BRAF inhibitor and PD-1 axis binding antagonist treatment as described herein for use as a medicament in the therapeutic and/or prophylactic treatment of cancer in a subject previously treated with a BRAF inhibitor (selected from Kang Naifei, dabrafenib and Kang Naifei) and/or a MEK axis binding antagonist (selected from cobratinib, bimatinib and trimitinib);

certain embodiments of the invention relate to a BRAF inhibitor and PD-1 axis binding antagonist treatment as described herein for use as a medicament in the therapeutic and/or prophylactic treatment of a subject previously treated with Kang Naifei ni and bimatinib;

certain embodiments of the invention relate to BRAF inhibitors and PD-1 axis binding antagonist treatments as described herein for use as a medicament in therapeutic and/or prophylactic treatment of a subject previously treated with dabrafenib and trimetinib;

certain embodiments of the invention relate to a BRAF inhibitor and PD-1 axis binding antagonist treatment as described herein for use as a medicament in the therapeutic and/or prophylactic treatment of a subject previously treated with vemurafenib and cobicitinib;

Certain embodiments of the invention relate to BRAF inhibitors and PD-1 axis binding antagonist treatments as described herein for use in medicaments in therapeutically and/or prophylactically treated cancers (particularly melanoma or non-small cell lung cancer) in a subject previously treated with a checkpoint inhibitor;

in one embodiment, the invention provides a kit comprising a BRAF inhibitor and a PD-1 axis binding antagonist treatment as described herein, prescription information also referred to as "instructions", blister packaging or bottles (HDPE or glass) and containers. The prescription information preferably includes advice to the patient regarding administration of a combination of a BRAF inhibitor and a PD-1 axis binding antagonist treatment as described herein;

in one embodiment, the subject being treated becomes refractory to the prior treatment periods described herein; and

in one embodiment, the subject being treated develops brain metastasis during the prior treatment described herein.

Certain embodiments of the invention relate to a combination according to the invention or a pharmaceutical composition comprising a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, wherein at least one substituent comprises at least one radioisotope. Specific examples of radioisotopes are ² H、 ³ H、 ¹³ C、 ¹⁴ C and C ¹⁸ F。

Furthermore, the present invention includes all optical isomers (as far as applicable) of the compounds of formula (I), i.e. diastereomers, diastereomeric mixtures, racemic mixtures, all corresponding enantiomers and/or tautomers thereof, and solvates thereof.

If desired, the racemic mixture of the compounds of the present invention may be separated such that the individual enantiomers are separated. The separation may be carried out by methods well known in the art, such as coupling a racemic mixture of compounds with an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography.

In some embodiments that provide optically pure enantiomers, optically pure enantiomers 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 end product or alternatively a suitable intermediate may be subjected to enantiomeric separation.

In one embodiment, one or more additional anticancer agents are used in combination with the BRAF inhibitors and MEK inhibitors described herein, wherein the additional anticancer agent is selected from the group consisting of MEK inhibitors, MEK degradation agents, EGFR inhibitors, EGFR degradation agents, inhibitors of HER2 and/or HER3, degradation agents of HER2 and/or HER3, SHP2 inhibitors, SHP2 degradation agents, axl inhibitors, axl degradation agents, ALK inhibitors, ALK degradation agents, PI3K inhibitors, PI3K degradation agents, SOS1 inhibitors, SOS1 degradation agents, signal transduction pathway inhibitors, checkpoint inhibitors, modulators of apoptotic pathways, cytotoxic chemotherapeutic agents, angiogenesis targeting therapies, immune targeting agents, and antibody drug conjugates.

In some embodiments, one of the additional anti-cancer agents is a MEK inhibitor. Non-limiting examples of MEK inhibitors include cobicitinibBimetinib->And trametinib->Additional examples of MEK inhibitors are known in the art.

In some embodiments, one of the additional anti-cancer agents is an EGFR inhibitor. Non-limiting examples of EGFR inhibitors include cetuximab (cetuximab)Panitumumab (panitumumab)>Octreotide (osimertinib) (merlecitinib,/i>) Erlotinib (erlotinib) >Gefitinib (gefitinib)>Xitumumab (Portrazza) and lenatinib (nepatinib)>Lapatinib (lapatinib)>Vandetanib (vanretanib)And bunatinib (briglatinib)>Additional examples of EGFR inhibitors are known in the art. In some embodiments, the EGFR is an allosteric EGFR inhibitor.

In some embodiments, one of the additional anti-cancer agents is an inhibitor of HER2 and/or HER 3. Non-limiting examples of HER2 and/or HER3 inhibitors include lapatinib (Iapatinib), canertinib (canertinib), (E) -2-methoxy-N- (3- (4- (3-methyl-4- (6-methylpyridin-3-yloxy) phenylamino) quinazolin-6-yl) allyl) acetamide (GP-724714), sapertinib (sapitinib), 7- [ [4- [ (3-ethynylphenyl) amino ] -7-methoxy-6-quinazolinyl ] oxy ] -N-hydroxy-heptanamide (CUDC-101), xylotinib (mubritinib), 6- [4- [ (4-ethylpiperazin-1-yl) methyl ] phenyl ] -N- [ (1R) -1-phenylethyl ] -7H-pyrrolo [2,3-d ] pyrimidin-4-amine (AEE 788), tocarbotinib (iritinib) (tucatinib), botinib (pozitab), N- [4- (4-acetyl-1-cyclohexyl ] -7-quinazolinyl ] oxy ] -N-hydroxy-heptanamide (CUDC-101), xylotinib), 6- [4- [ (4-ethylpiperazin-1-yl) methyl ] -N- [ (1-ethyl-1-phenyl ] -7H-pyrrolo [2,3-d ] pyrimidin-4-amine (AEE 788) 7-cyclopentyl-5- (4-phenoxyphenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-ylamine (KIN 001-051), 6, 7-dimethoxy-N- (4-phenoxyphenyl) quinazolin-4-amine (KIN 001-30), dasatinib (dasatinib), and bosutinib (bosutinib).

In some embodiments, one of the additional anti-cancer agents is an inhibitor of SHP 2. Non-limiting examples of SHP2 inhibitors include 6- (4-amino-4-methylpiperidin-1-yl) -3- (2, 3-dichlorophenyl) pyrazin-2-amine (SHP 099), [3- [ (3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [4.5] decan-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, WO 2015/107495, WO2019/075265, PCT/U82019/056786 and PCT/l 82020/053019.

In some embodiments, one of the additional anti-cancer agents is a PI3K inhibitor. Non-limiting examples include bupiriib (BKM 120), aperilisib (BYL 719), sha Tuoli plug (samotolisib) (LY 3023414), 8- [ (1R) -1- [ (3, 5-difluorophenyl) amino ] ethyl ] -N, N-dimethyl-2- (morpholin-4-yl) -4-oxo-4H-chromen-6-carboxamide (AZD 8186), tenacissib (tenalaisib) (RP 6530), fu Shali stat (voxtalisib hydrochloride) hydrochloride (SAR-245409), ji Dali plug (gedatoliib) (PF-05212384), panulib (P-7170), tasilib (tasselisib) (GDC-0032), trans-2-amino-8- [4- (2-hydroxyethoxy) cyclohexyl ] -6- (6-methoxypyridin-3-yl) -4-methylpyrido [2,3-d ] pyrimidine-7 (8H) -one (04691502), and bivaldeco (bv) (PF-37954), N2- [ 4-oxo-4- [4- (4-oxo-8-phenyl-4H-1-benzopyran-2-) yl) morpholin-4-ium-4-ylmethoxy ] butanoyl ] -L-arginyl-glycyl-L-serine acetate (SF-1126), picilib (GDC-0941), 2-methyl-1- [ 2-methyl-3- (trifluoromethyl) benzyl ] -6- (morpholin-4-yl) -1H-benzimidazole-4-carboxylic acid (GSK 2636771), idola (idelalisib) (GS-1101), erbitux tosylate (umbralisib tosylate) (TGR-1202), picilib (GDC-0941), kupannix hydrochloride (copanlisib hydrochloride) (BAY 84-1236), dacrilib (dazolib) (BEZ-235), 1- (4- [5- [ 5-amino-6- (5-tert-butyl-1, 3, 4-oxadiazol-2-yl) -2H-benzimidazole-4-carboxylic acid (GSK 2636771), idol-argil tosylate (TGR-1202), picilib (GDC-0941), kupannix hydrochloride (copanlisib hydrochloride) (dazol-1236), dactyl (dazolib-235), 1- (5-amino-1, 3, 4-oxadiazol-2-yl) -2-pyrazin-2-yl ] -2-hydroxy-triazol-1-3-yl) -1-hydroxy-1-3-triazolyl-1-3-hydroxy-1-triazolyl-1-3-hydroxy-propan-yl-1-yl-propano-1-hydroxy-methyl-1-methyl-hydroxy-methyl-carbonyl-L-carboxylate 5- [6, 6-dimethyl-4- (morpholin-4-yl) -8, 9-dihydro-6H- [1,4] oxazinyl [4,3-e ] purin-2-yl ] pyrimidin-2-amine (GDC-0084) everolimus (everolimus), rapamycin (rapamycin), piperifusine (perifosine), sirolimus (sirolimus) and temsirolimus (temsirolimus).

In some embodiments, one of the additional anti-cancer agents is an ALK inhibitor. Non-limiting examples include crizotinib (PF-0234766), ceritinib (LDK 378), ai Laiti, alecitib (alecensa), buntinib (AP 26113), laratinib (lorelatinib) (PF-6463922), ensartinib (enartinib) (X-396), entacritinib (enterretinib) (RXD-101), reprotective tinib (reprotetinib) (TPX-0005), bei Liza tinib (belizatinib) (TSR-011), alcotinib (Alkotinib) (ZG-0418), repetinib (foritinib) (SAF-189), CEP-37440, TQ-B3139, PLB1003 and TPX-0131)

In some embodiments, one of the additional anti-cancer agents is a checkpoint inhibitor. In some embodiments, 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 (ipilimumab)Or tremelimumab (GP-675,206). In some embodiments, the PD-1 binding antagonist is selected from the group consisting of cimiput Li Shan anti +.>PembrolizumabNawuzumab +.>And RN888 (PF-06801591). In some embodiments, the PD-L1 binding antagonist is selected from alemtuzumab +. >Avermectin->And Devaluzumab (Imfinzi) ^TM )。

In some embodiments, one of the additional anti-cancer agents is an antibody drug conjugate. Non-limiting examples of antibody drug conjugates include gemtuzumab ozagrel (gemtuzumab ozogamicin) (mylotarg tm), oxtuzumab ozogamicin (inotuzumab ozogamicin)Bentuxi Shan Kangwei statin conjugate (brentuximab vedotin)>Trastuzumab-maytansinoid conjugate (ado-trastuzumab emtansine) (TDM-f;) Rituximab-grommet conjugate (mirvetuximab soravtansine) (IMGN 853) and acil Shan Kanglei-star conjugate (anetumab ravtansine).

In some embodiments, one of the additional anti-cancer agents is an antibody, such as bevacizumab (MvastiTM,) Trastuzumab depicting>AbamectinRituximab (MabTheraTM, < >>) Edeclomab (Panorex), daratumumab (daratumuab)>Olympic mab (olarruvotm), olmesamab (ofatumumab)>Alemtuzumab (alemtuzumab) in>Cetuximab->Ago Fu Shan anti (orenovomab), cimip Li Shan anti +.>Pembrolizumab +.>Denotuximab (dinutiximab)Abitumomab (obinutuzumab) as well as methods of using the same >Tramadol mab (GP-675,206), ramucirumab (ramucirumab)/(limumab)>Wu Tuo Acximab (ublituximab) (TG-1101), panitumumabErltuzumab (eltuzumab) (EmplicitiT 'V'), cetuximab (portrazat 'V'), cetuximab (cirmtuzumab) (UC-961), ibritumomab (ibritumomab)Ai Satuo Acximab (isatuximab) (SAR 650984), nituzumab (nimotuzumab), non-hematoxylin mab (fresolimumab) (GC 1008), li Ruilu mab (Iirilumab) (INN), mo Geli bead mab (mogamulizumab)>Non-clarituximab (AV-299), denosumab (denosumab)>Ganitumumab, wu Ruilu mab (urelumab), pimelimumab, amatuximab, bei Lintuo outuzumab (blinatumomab) (AMG 103;)>) Or midostaurin (Rydapt).

In some embodiments, the isolated anti-PD-L1 antibody is glycosylated.

Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxy amino acid, most typically serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used. Glycosylation sites can be conveniently removed from antibodies by altering the amino acid sequence to remove one of the tripeptide sequences described above (for N-linked glycosylation sites). Variations may be made by substituting an asparagine, serine or threonine residue within a glycosylation site with another amino acid residue (e.g., glycine, alanine or conservative substitutions).

In any of the embodiments herein, the isolated anti-PD-L1 antibody can bind to human PD-L1, e.g., human PD-L1 as shown in UniProtKB/Swiss-Prot accession No. Q9NZQ7.1, or a variant thereof.

In still further embodiments, the invention provides a composition comprising an anti-PD-L1, anti-PD-1 or anti-PD-L2 antibody, or antigen-binding fragment thereof, as provided herein, and at least one pharmaceutically acceptable carrier. In some embodiments, an anti-PD-L1, anti-PD-1, or anti-PD-L2 antibody, or antigen-binding fragment thereof, administered to an individual is a composition comprising one or more pharmaceutically acceptable carriers.

Any pharmaceutically acceptable carrier described herein or known in the art may be used.

In some embodiments, an anti-PD-L1 antibody described herein is in a formulation comprising an amount of antibody of about 60mg/mL, histidine acetate at a concentration of about 20mM, sucrose at a concentration of about 120mM, and polysorbate (e.g., polysorbate 20) at a concentration of 0.04% (w/v), and the pH of the formulation is about 5.8. In some aspects, an anti-PD-L1 antibody described herein is in a formulation comprising an amount of antibody of about 125mg/mL, histidine acetate at a concentration of about 20mM, sucrose at a concentration of about 240mM, and polysorbate (e.g., polysorbate 20) at a concentration of 0.02% (w/v), and the pH of the formulation is about 5.5.

Antibody preparation

As described above, in some embodiments, the PD-1 binding antagonist is an antibody (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody). The antibodies described herein may be prepared using techniques available in the art for producing antibodies, exemplary methods of which are described in more detail in the following sections. Antibodies are directed against a target "antigen". For example, the antibody may be directed against PD-1 (such as human PD-1), PD-L1 (such as human PD-L1), PD-L2 (such as human PD-L2). Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to a mammal having a disorder may produce a therapeutic benefit in that mammal.

In certain embodiments, the dissociation constant (Kd) of an antibody described herein is 1 μm, 150nM, 100nM, 50nM, 10nM, 1nM, 0.1nM, 0.01nM, or 0.001nM (e.g., 10 to 8M or less, e.g., from 10 to 8M to 10 to 13M, e.g., from 10 to 9M to 10 to 13M).

In one embodiment, kd is measured by radiolabeled antigen binding assay (RIA) with the antibody of interest and its antigen in Fab form as described in the assay below. The solution binding affinity of Fab to antigen was measured by equilibrating Fab with a minimum concentration (125I) of labeled antigen in the presence of unlabeled antigen titration series, followed by capture of bound antigen with an anti-Fab antibody coated plate (see, e.g., chen et al, j. Mol. Biol.293:865-881 (1999)). To determine the conditions for the assay, the capture anti-Fab antibodies (Cappel Labs) were coated with 5. Mu.g/ml in 50mM sodium carbonate (pH 9.6) Microplates (Thermo Scientific) were left overnight and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (about 23 ℃). In a non-adsorbed plate (Nunc# 269620), 100pM or 26pM [125I ]]Antigen is mixed with serial dilutions of Fab of interest. Then incubating the target Fab overnight; however, incubation may last longer (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture was transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution was then removed and 0.1% polysorbate 20 +.>The plates were washed eight times. When the plate has dried, 150. Mu.l/well of scintillator (MICROSICINT-20. TM.; packard) is added and the plate is counted for several tens of minutes on a TOPCON. TM. Gamma. Counter (Packard). The concentration of each Fab that gave less than or equal to 20% of maximum binding was selected for use in the competitive binding assay.

According to another embodiment, the immobilized antigen CM5 chip is used at about 10 Response Units (RU) at 25℃Or->Kd was measured by surface plasmon resonance (BIAcore, inc., piscataway, N.J.). Briefly, carboxymethylated dextran biosensor chips (CM 5, BIACORE, inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen was diluted to 5. Mu.g/ml (about 0.2. Mu.M) with 10mM sodium acetate pH 4.8, followed by injection at a flow rate of 5. Mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After antigen injection, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, double serial dilutions of Fab (0.78 nM to 500 nM) were injected in PBS containing 0.05% polysorbate 20 (TWEEN-20. TM.) surfactant (PBST) at 25℃at a flow rate of about 25. Mu.l/min. By simultaneously fitting and combining and dissociating the sensorgrams, a simple one-to-one Langmuir combining model is used The rate of binding (kon) and rate of dissociation (koff) were calculated by the evaluation software version 3.2. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, e.g., chen, Y et al, J.mol. Biol.293:865-881 (1999). If the association rate exceeds 106M-1s-1 as determined by the above surface plasmon resonance assay, the association rate can be determined by using a fluorescence quenching technique, i.e. by measuring the increase or decrease in fluorescence emission intensity (excitation wavelength=295 nM; emission wavelength=340 nM, bandpass=16 nM) of a 20nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25 ℃ in the presence of increasing concentrations of antigen as measured in a spectrometer such as a spectrophotometer equipped with a flow stop device (Aviv Instruments) or a 8000 series SLM-aminoco (TM) spectrophotometer (thermo spectronic).

Antibody fragments

In certain embodiments, the antibodies described herein are antibody fragments. Antibody fragments include, but are not limited to, fab '-SH, F (ab') 2, fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al, nat.Med.9:129-134 (2003). For a review of scFv fragments, see, e.g.,described in The Pharmacology of Monoclonal Antibodies, volume 113, rosenburg and Moore editions (Springer-Verlag, new York), pages 269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. For a discussion of Fab fragments and F (ab') 2 fragments that include salvage receptor binding epitope residues and have an extended in vivo half-life, see U.S. patent No. 5869046.

Diabodies are antibody fragments having two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, nat.Med.9:129-134 (2003); and Hollinger et al, proc.Natl. Acad. Sci. USA 90:6444-6448 (1993). Trisomy and tetrasomy antibodies are also described in Hudson et al, nat.Med.9:129-134 (2003). A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (domntis, inc., waltham, MA; see, e.g., U.S. patent No. 6,248,516B1). Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E.coli or phage), as described herein.

Chimeric and humanized antibodies

In certain embodiments, the antibodies described herein are chimeric antibodies. Some chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567 and Morrison et al, proc.Natl. Acad.Sci.USA,81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class switch" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, the chimeric antibody is a humanized antibody. Typically, the non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed in, for example, almagro and Franson, front. Biosci.13:1619-1633 (2008), and further described, for example, in Riechmann et al, nature 332:323-329 (1988); queen et al, proc.Nat' l Acad.Sci.USA 86:10029-10033 (1989); U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; kashmiri et al Methods 36:25-34 (2005) (describing SDR (a-CDR) porting); padlan, mol. Immunol.28:489-498 (1991) (describing "surface reshaping"); dall' Acqua et al, methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al, methods 36:61-68 (2005) and Klimka et al, br.J.cancer,83:252-260 (2000) (describing "guide selection" Methods for FR shuffling).

Human framework regions useful for humanization include, but are not limited to: the framework regions were selected using the "best fit" method (see, e.g., sims et al J. Immunol.151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subsets of light or heavy chain variable regions (see, e.g., carter et al Proc. Natl. Acad. Sci. USA,89:4285 (1992); and Presta et al J. Immunol.,151:2623 (1993)); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., almagro and Fransson, front. Biosci.13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., baca et al, J. Biol. Chem.272:10678-10684 (1997) and Rosok et al, J. Biol. Chem.271:22611-22618 (1996)).

Human antibodies

In certain embodiments, the antibodies described herein are human antibodies. Various techniques known in the art may be used to produce human antibodies. Human antibodies are generally described in van Dijk and van de Winkel, curr Opin Phacol.5:368-74 (2001) and Lonberg, curr Opin immunol.20:450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody having a human variable region in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci, either present extrachromosomal to the animal or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For a review of methods of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See, for example, U.S. Pat. nos. 6,075,181 and 6,150,584, describing XENOMOUSETM technology; description of the inventionTechnical U.S. patent No. 5,770,429; description of K-MTechnical U.S. Pat. No. 7,041,870 and description- >Technical U.S. patent application publication No. US2007/0061900. Human variable regions from whole antibodies produced by such animals may be further modified, for example by combining with different human constant regions.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described. (see, e.g., kozbor J.Immunol.,133:3001 (1984); brodeur et al, monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, inc., new York, 1987), and Boerner et al, J.Immunol.,147:86 (1991)) human antibodies produced via human B cell hybridoma technology are also described in Li et al, proc.Natl. Acad. Sci. USA,103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, xiandai Mianyixue,26 (4): 265-268 (2006) (describing human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, histology and Histopathology,20 (3): 927-937 (2005) and Vollmers and Brandlein, methods and Findings in Experimental and Clinical Pharmacology,27 (3): 185-91 (2005).

Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from a human phage display library. Such variable domain sequences can then be combined with the intended human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

Antibodies derived from libraries

Antibodies can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with desired binding characteristics. Such methods are reviewed in, for example, hoogenboom et al, methods in Molecular Biology 178:178:1-37 (O' Brien et al, incorporated, human Press, totowa, NJ, 2001) and further described, for example, in McCafferty et al, nature 348:552-554; clackson et al, nature 352:624-628 (1991); marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., human Press, totowa, NJ, 2003); sidhu et al, J.mol.biol.338 (2): 299-310 (2004); lee et al, J.mol.biol.340 (5): 1073-1093 (2004); felloose, proc. Natl. Acad. Sci. USA 101 (34); 12467-12472 (2004); and Lee et al, J.Immunol. Methods 284 (1-2): 119-132 (2004).

In some phage display methods, all components of the VH and VL genes are cloned individually by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library from which antigen-binding phage can then be screened as described in Winter et al, ann.rev.immunol.,12:433-455 (1994). Phage typically display antibody fragments as single chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, the initial repertoire (e.g., from humans) can be cloned to provide a single source of antibodies to a wide range of non-self and self-antigens without any immunization, as described by Griffiths et al, EMBO J,12:725-734 (1993). Finally, an initial library can also be prepared by: cloning unrearranged V gene segments from stem cells; and using PCR primers containing random sequences to encode highly variable CDR3 regions and accomplish in vitro rearrangement as described by Hoogenboom and Winter, j.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: us patent No. 5,750,373, and us publication nos. 2005/007974, 2005/019455, 2005/0266000, 2007/017126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360. Antibodies or antibody fragments isolated from a human antibody library are herein considered human antibodies or human antibody fragments.

Multispecific antibodies

In certain embodiments, the antibodies described herein are multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificities for at least two different sites. In some embodiments, the PD-1 axis component antagonist is multispecific. Wherein one of the binding specificities is for a PD-1 axis component (e.g., PD-1, PD-L1 or PD-L2) and the other is for any other antigen. In some embodiments, one of the binding specificities is for IL-17 or IL-17R, and the other is for any other antigen. In certain embodiments, the bispecific antibody can bind to two different epitopes of a PD-1 axis component (e.g., PD-1, PD-L1, or PD-L2), IL-17, or IL-17R. Bispecific antibodies can be made as full length antibodies or antibody fragments.

In some embodiments, one of the binding specificities is for a PD-1 axis component (e.g., PD-1, PD-L1, or PD-L2), and the other is for IL-17 or IL-17R. Provided herein are methods for treating or delaying progression of cancer in an individual, the methods comprising administering to the individual an effective amount of a multispecific antibody, wherein the multispecific antibody comprises a first binding specificity for a PD-1 axis component (e.g., PD-1, PD-L1, or PD-L2) and a second binding specificity for IL-17 or IL-17R. In some embodiments, the multispecific antibodies may be prepared by any of the techniques described herein and below.

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.EMBO J.10:3655 (1991)) and "pestle and mortar" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made by the following techniques: engineering electrostatic manipulation effects to produce antibody Fc-heterodimer molecules (WO 2009/089004 A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al, science 229:81 (1985)); bispecific antibodies have been generated using leucine zippers (see, e.g., kostelny et al, J.Immunol.148 (5): 1547-1553 (1992)); bispecific antibody fragments were made using the "diabody" technique (see, e.g., hollinger et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); single chain Fv (sFv) dimers (see, e.g., gruber et al, J.Immunol.,152:5368 (1994)); and the preparation of trispecific antibodies as described, for example, in Tutt et al J.Immunol.147:60 (1991).

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US2006/0025576 A1).

Antibodies or fragments herein also include "dual acting FAb" or "DAF" comprising an antigen binding site that binds to a PD-1 axis component (e.g., PD-1, PD-L1, or PD-L2), IL-17, or IL-17R, and another, different antigen (see, e.g., US 2008/0069820).

Nucleic acid sequences, vectors and methods of production

Polynucleotides (e.g., antibodies) encoding PD1 axis binding antagonists may be used to produce the PD1 axis binding antagonists described herein. The PD1 axis binding antagonists used according to the invention may be expressed as a single polynucleotide encoding the complete bispecific antigen binding molecule, or as a plurality (e.g. two or more) of polynucleotides that are co-expressed. The polypeptides encoded by the co-expressed polynucleotides may associate, e.g., via disulfide bonds or other means, to form a functional PD1 axis binding antagonist antibody. For example, the light chain portion of a Fab fragment may be encoded by a separate polynucleotide from the portion of a bispecific antibody comprising the heavy chain portion of the Fab fragment, the Fc domain subunit and optionally (a portion of) another Fab fragment. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form a Fab fragment. In another example, a portion of a PD-1 axis binding antagonist antigen-binding portion provided herein comprising one of the two Fc domain subunits and optionally (a portion of) one or more Fab fragments can be encoded by a polynucleotide separate from a portion of a bispecific antibody provided herein comprising the other of the two Fc domain subunits and optionally (a portion of) a Fab fragment. When co-expressed, the Fc domain subunits will associate to form an Fc domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotides of the invention are RNAs, e.g., in the form of messenger RNAs (mrnas). The RNA of the present invention may be single-stranded or double-stranded.

Antibody variants

In certain embodiments, amino acid sequence variants of PD-1 axis binding antagonist antibodies are contemplated in addition to those described above. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions may be made to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

Substitution, insertion and deletion variants

In certain embodiments, variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutations include HVRs and FR. Conservative substitutions are shown under the heading "conservative substitutions" in table B. Further substantial changes are provided under the heading "exemplary substitutions" of table B, and are further described below with reference to the amino acid side chain class. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

Table B

/>

Amino acids can be grouped according to common side chain characteristics:

(1) Hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;

(3) Acid: asp, glu;

(4) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro;

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions will require exchanging members of one of these classes for the other class.

One type of substitution variant involves substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, one or more of the resulting variants selected for further investigation will have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) and/or will substantially retain certain biological properties of the parent antibody relative to the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

For example, HVRs can be altered (e.g., substituted) to improve antibody affinity. Such changes may be made in HVR "hot spots" (i.e., residues encoded by codons that undergo high frequency mutations during the somatic maturation process (see, e.g., chordhury, methods mol. Biol.207:179-196 (2008))) and/or SDR (a-CDRs), wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries has been described, for example, by Hoogenboom et al, in Methods in Molecular Biology 178:1-37 (O' Brien et al, human Press, totowa, N.J. (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation using any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis genes). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves HVR targeting methods in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such alterations do not substantially reduce the antigen binding capacity of the antibody. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the HVR that do not substantially reduce binding affinity. Such changes may be outside of HVR "hot spots" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged or comprises no more than one, two, or three amino acid substitutions.

A method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or a set of target residues (e.g., charged residues such as arg, asp, his, lys and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the point of contact between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution may be targeted or eliminated. Variants may be screened to determine if they possess the desired properties.

Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusion with an enzyme that increases the serum half-life of the antibody (e.g., for ADEPT) or the N-or C-terminus of the antibody of the polypeptide.

Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to antibodies can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

When an antibody used with the present invention contains an Fc region, the carbohydrates attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched-chain double-antenna oligosaccharides, which are typically linked by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, wright et al TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of a double-antennary oligosaccharide structure. In some embodiments, the oligosaccharides in the bispecific antibodies or antibodies that bind to DR5 of the invention may be modified to produce antibody variants with certain improved properties.

In one embodiment, bispecific antibody variants or variants of multiple antibodies are provided having a carbohydrate structure lacking fucose linked (directly or indirectly) to an Fc region. For example, the fucose content of such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e. between position 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. patent publication No. US2003/0157108 (Presta, l.); US2004/0093621 (Kyowa Hakko Kogyo Co., ltd.). The antibody variants related to "defucosylation" or "fucose deficient" include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/015614; US 2002/0164328; US 2004/0093621; US 2004/013321; US 2004/010704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); yamane-Ohnuki et al, biotech. Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al Arch. Biochem. Biophys.249:533-545 (1986), U.S. patent application Ser. No. 2003/0157108 A1,Presta,L, and WO 2004/056312A 1, adams et al, particularly example 11), and knockout cell lines such as CHO cells knocked out of the alpha-1, 6-fucosyltransferase gene (FUT 8) (see, e.g., yamane-Ohnuki et al Biotech. Bioeng.87:614 (2004); kanda, Y. Et al, biotechnol. Bioeng.,94 (4): 680-688 (2006), and WO 2003/085107).

Further provided are antibody variants comprising two typed oligosaccharides, e.g., wherein a dihedral oligosaccharide linked to the Fc region of an antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Maiset et al); U.S. Pat. No. 6,602,684 (Umana et al); and US 2005/0123946 (Umana et al). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, s.); and WO 1999/22764 (Raju, S.).

Cysteine engineered antibody variants

In certain embodiments, it may be desirable to produce a cysteine engineered antibody, such as "THIOMABS," in which one or more residues of the antibody are substituted with cysteine residues. In certain embodiments, the substituted residue is present at an accessible site of the antibody. By replacing those residues with cysteines, reactive thiol groups are thereby located at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: v205 of light chain (Kabat numbering); a118 (EU numbering) of heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, for example, in U.S. patent No. 7,521,541.

Recombinant methods and compositions

Antibodies of the invention may be obtained, for example, by solid-state peptide synthesis (e.g., merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding antibodies (or fragments), e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one embodiment, a vector, preferably an expression vector, is provided, the vector comprising one or more of the polynucleotides of the invention. Methods well known to those skilled in the art can be used to construct expression vectors containing coding sequences for antibodies and appropriate transcriptional/translational control signals. These methods include recombinant DNA technology in vitro, synthetic technology, and recombinant/genetic recombination in vivo. See, e.g., maniatis et al, molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al Current Protocols in Molecular Biology, greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector may be part of a plasmid, a virus, or may be a nucleic acid fragment. Expression vectors include expression cassettes into which polynucleotides encoding antibodies (fragments) (i.e., coding regions) are cloned in operable association with promoters and/or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid that consists of codons translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it (if present) can be considered to be part of the coding region, while any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, etc., are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs (e.g., on separate (different) vectors). In addition, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides that are separated into the final proteins by proteolytic cleavage after or at the time of translation. Furthermore, the vectors, polynucleotides or nucleic acids of the invention may encode heterologous coding regions, fused or unfused to polynucleotides encoding antibodies or variants or derivatives thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretion signal peptides or heterologous functional domains. An operable association is when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in a manner such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequence to direct expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, if a promoter is capable of affecting transcription of the nucleic acid, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs substantial transcription of DNA in only a predetermined cell.

In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcriptional termination signals, may be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. A variety of transcriptional control regions are known to those skilled in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter binding intron-a), simian virus 40 (e.g., early promoter), and retroviruses (such as, for example, rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock proteins, bovine growth hormone, and rabbitGlobin) and other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetradA cyclic-element inducible promoter). Similarly, various translational control elements are known to those of ordinary skill in the art. These translational control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from the viral system (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also include other features, such as an origin of replication, and/or chromosomal integration elements, such as retroviral Long Terminal Repeats (LTRs), or adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs).

The polynucleotides and nucleic acid coding regions of the invention may be associated with additional coding regions encoding a secretory peptide or signal peptide which direct secretion of the polypeptide encoded by the polynucleotides of the invention. For example, if secretion of an antibody is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid of the antibody or fragment thereof of the invention. Based on the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretion leader that is cleaved from the mature protein once the growing protein chain has been initiated to export across the rough endoplasmic reticulum. One of ordinary skill in the art knows that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a natural signal peptide (e.g., an immunoglobulin heavy chain or light chain signal peptide), or a functional derivative of such a sequence that retains the ability to direct secretion of a polypeptide with which it is operably associated, is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced by a human Tissue Plasminogen Activator (TPA) or a mouse β -glucuronidase leader sequence.

DNA encoding short protein sequences (which may be used to facilitate subsequent purification (e.g., histidine tags) or to aid in labeling the antibody) may be contained within or at the ends of the antibody (fragment) encoding polynucleotide.

In another embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments, host cells comprising one or more vectors of the invention are provided. The polynucleotide and vector may be infiltrated with any of the features described herein with respect to the polynucleotide and vector, respectively, alone or in combination. In one such embodiment, the host cell comprises (e.g., has been transformed or transfected with) a vector comprising a polynucleotide encoding an antibody or portion thereof of the invention. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce antibodies of the invention (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies) or fragments thereof. Host cells suitable for replication and supporting expression of the antibodies of the invention are well known in the art. Such cells can be appropriately transfected or transduced with a particular expression vector, and a large number of vector-containing cells can be cultured for inoculation of a large-scale fermenter to obtain a sufficient amount of antibody for clinical use. Suitable host cells include prokaryotic microorganisms, such as E.coli, or various eukaryotic cells, such as Chinese hamster ovary Cells (CHO), insect cells, and the like. For example, polypeptides may be produced in bacteria, particularly when glycosylation is not required. The polypeptide may be isolated from the bacterial cell paste in a soluble fraction after expression and may be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of polypeptides having a partially or fully human glycosylation pattern. See Gerngross, nat Biotech 22,1409-1414 (2004) and Li et al, nat Biotech 24,210-215 (2006). Suitable host cells for expressing (glycosylating) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures may also be used as hosts. See, for example, U.S. Pat. nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (described for use in Antibody production in transgenic plants ^TM Technology). Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells as for example described in Graham et al, J Gen Virol 36,59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM 4 cells as for example described in Mather, biol Reprod 23,243-251 (1980)), monkey kidney cells (CV 1), african green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as for example described in Mather et al, annals N.Y. Acad Sci 383,44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including dhfr ^- CHO cells (Urlaub et al Proc Natl Acad Sci USA, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., yazaki and Wu, methods in Molecular Biology, vol.248 (B.K.C.Lo. Editors, humana Press, totowa, N.J.), pages 255-268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, to name a few, as well as transgenic animals, transgenic plants, or cells contained in cultured plants or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a lymphocyte (e.g., Y0, NS0, sp20 cell).

Standard techniques for expressing exogenous genes in these systems are known in the art. Cells expressing polypeptides comprising antigen binding domains, such as the heavy or light chains of an antibody, can be engineered to also express another antibody chain, such that the expressed product is an antibody having a heavy chain and a light chain.

Antibodies, antibody fragments, antigen binding domains or variable regions of any animal species may be used in the antibodies used according to the invention. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the present invention may be of murine, primate or human origin. If the antibody is intended for human use, chimeric forms of the antibody may be used, wherein the constant regions of the antibody are from human. Humanized or fully human forms of antibodies can also be prepared according to methods well known in the art (see, e.g., winter, U.S. Pat. No. 5,565,332). Humanization can be achieved by a variety of methods including, but not limited to, (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) framework and constant regions with or without the retention of critical framework residues (e.g., critical framework residues important for maintaining good antigen binding affinity or antibody function), (b) grafting only non-human specific determinant regions (SDR or a-CDRs; residues critical for antibody-antigen interactions) onto human framework and constant regions, or (c) grafting the entire non-human variable domains, but "hiding" them with human-like segments by replacing surface residues. Humanized antibodies and methods of making them are reviewed in, for example, almagro and Franson, front Biosci 13,1619-1633 (2008), and further described, for example, in Riechmann et al, nature 332,323-329 (1988); queen et al, proc Natl Acad Sci USA, 86,10029-10033 (1989); U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; jones et al, nature321,522-525 (1986); morrison et al Proc Natl Acad Sci, 81,6851-6855 (1984); morrison and Oi, adv Immunol 44,65-92 (1988); verhoeyen et al, science 239,1534-1536 (1988); padlan, molecular Immun 31 (3), 169-217 (1994); kashmiri et al Methods 36,25-34 (2005) (describing SDR (a-CDR) porting); padlan, mol Immunol 28,489-498 (1991) (describing "surface reshaping"); dall' Acqua et al, methods 36,43-60 (2005) (describing "FR shuffling"); and Osbourn et al, methods 36,61-68 (2005) and Klimka et al, br J Cancer 83,252-260 (2000) (describing "guide selection" Methods for FR shuffling). Various techniques known in the art can be used to produce human antibodies and human variable regions. Human antibodies are generally described in van Dijk and van de Winkel, curr Opin Pharmacol, 368-74 (2001) and Lonberg, curr Opin Immunol, 20,450-459 (2008). The human variable region may form part of and be derived from a human monoclonal antibody prepared by the hybridoma method (see, e.g., monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, inc., new York, 1987)). Human antibodies and human variable regions can also be prepared by: the immunogen is administered to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody having a human variable region responsive to antigen challenge (see, e.g., lonberg, nat Biotech 23,1117-1125 (2005)). Human antibodies and human variable regions can also be produced by: fv clone variable region sequences selected from phage display libraries of Human origin were isolated (see, e.g., hoogenboom et al Methods in Molecular Biology 178,1-37 (O' Brien et al ed., human Press, totowa, N.J., 2001), and McCafferty et al Nature 348,552-554; clackson et al Nature 352,624-628 (1991)). Phage typically display antibody fragments as single chain Fv (scFv) fragments or Fab fragments.

In certain embodiments, antigen binding portions useful in the present invention are engineered to have enhanced binding affinity according to methods disclosed, for example, in U.S. patent application publication No. 2004/013066, the entire disclosure of which is incorporated herein by reference. The ability of the antibodies of the invention to bind to a particular epitope can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance techniques (analysis on BIACORE T100 system) (Liljeblad, et al, glyco J17, 323-329 (2000)) as well as conventional binding assays (Heeley, endocr Res 28,217-229 (2002)), competition assays can be used to identify antibodies, antibody fragments, antigen binding domains or variable domains that bind to a reference antibody, in certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) to which the reference antibody binds, detailed exemplary methods for mapping the epitope bound by the antibody are provided in volume Methods in Molecular Biology (Humana Press, totowa, NJ) 'Epitope Mapping Protocols', in an exemplary competition assay, an immobilized antigen (e.g., PD-1) is incubated in a first antibody (e.g., 6, 4) comprising binding to the antigen, and a second antibody (e.g., 4, 297) in a second antibody) in a non-binding format, and a non-binding antibody is not allowed to bind to the antigen in a control antibody in the first antibody, and a non-binding antibody is removed in a non-binding format to the second antibody in a non-binding antibody to the second antibody, and a non-binding antibody is detected in a non-binding antibody to the second antibody is detected in a contrast to the first antibody and a detection antibody is not allowed to bind to the antigen in a sample, a substantial decrease in the amount of label associated with the immobilized antigen in the test sample indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies, A Laboratory Manual chapter 14 (Cold Spring Harbor Laboratory, cold Spring Harbor, N.Y.).

In certain embodiments, antigen binding portions useful in the present invention are engineered to have enhanced binding affinity according to methods disclosed, for example, in U.S. patent application publication No. 2004/013066, the entire disclosure of which is incorporated herein by reference. The ability of the antibodies of the invention to bind to a particular epitope can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance techniques (analyzed on the BIACORE T100 system) (Liljeblad, et al, glyco J17, 323-329 (2000)) as well as conventional binding assays (Heeley, endocr Res 28,217-229 (2002)), competition assays can be used to identify antibodies, antibody fragments, antigen binding domains or variable domains that bind to a reference antibody, in certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) as the reference antibody binding to the epitope provided in the volume Methods in Molecular Biology (Humana Press, totowa, NJ) 'Epitope Mapping Protocols'), in an exemplary competition assay, incubating the immobilized antigen in a sample solution comprising a first labeled antibody that binds to the antigen and a test antibody that is not capable of binding to the first labeled antibody to the antigen, and removing the amount of binding to the immobilized antigen from a sample in a sample comprising the immobilized antigen in a contrast to which the immobilized antigen is not bound to the immobilized antigen is substantially reduced in an amount of the sample, and in the sample is not allowed to be hybridized to the immobilized antigen in a sample, the second antibody is shown competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies, A Laboratory Manual chapter 14 (Cold Spring Harbor Laboratory, cold Spring Harbor, N.Y.).

Antibodies prepared as described herein can be purified by techniques known in the art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, bispecific antibodies or antibodies, ligands, receptors or antigens to which antibodies that bind DR5 can be used. For example, for affinity chromatography purification of the bispecific antibodies of the invention, a matrix with protein a or protein G may be used. Bispecific antibodies can be isolated using sequential protein a or G affinity chromatography and size exclusion chromatography, substantially as described in the examples. The purity of the bispecific antibody or antibody that binds to DR5 can be determined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.

Measurement

The physical/chemical properties and/or biological activity of the antibodies provided herein (e.g., anti-PD-1 axis binding antagonist antibodies) can be identified, screened, or characterized by various assays known in the art.

Affinity assay

The affinity of an antibody provided herein (e.g., an anti-PD-1 axis binding antagonist antibody) for its respective antigen (e.g., PD-1, PD-L1) can be determined by Surface Plasmon Resonance (SPR) according to the methods set forth in the examples using standard instruments such as BIAcore instrument (GE Healthcare) and receptors or target proteins such as can be obtained by recombinant expression. Alternatively, cell lines expressing a particular receptor or target antigen may be used, for example, by flow cytometry (FACS) to assess the binding of antibodies provided herein to their respective antigens.

At 25℃it is possible to useT100 instrument (GE Healthcare) measures K by surface plasmon resonance _D . To analyze the interaction between the Fc portion and Fc receptor, his-tagged recombinant Fc receptor was captured by anti-pentahistidine antibody (Qiagen) immobilized on CM5 chip ("Penta His"), a bispecific construct was used as the analyte. Briefly, according to the supplier's instructions, carboxymethylated dextran biosensor chips (CM 5, GE Healthcare) were activated with N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Anti-pentahistidine antibody ("Penta His") was diluted to 40 μg/ml with 10mM sodium acetate pH 5.0, followed by injection at a flow rate of 5 μl/min to obtain about 6500 Response Units (RU) conjugated protein. After injection of the ligand, 1M ethanolamine was injected to block unreacted groups. The Fc receptor was then captured at 4 or 10nM for 60s. For kinetic measurements, four-fold serial dilutions of bispecific constructs (ranging between 500nM and 4000 nM) were injected into HBS-EP (GE Healthcare,10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) at 25℃at a flow rate of 30 μl/min for 120 s.

To determine affinity to the target antigen, bispecific constructs were captured by anti-human Fab specific antibodies (GE Healthcare) immobilized on the surface of activated CM5 sensor chip as described for anti-Penta histidine antibodies ("Penta His"). The final amount of coupled protein was about 12000RU. Bispecific constructs were captured for 90s at 300 nM. The target antigen was passed through the flow cell at a flow rate of 30. Mu.l/min for 180s at a concentration range of 250 to 1000 nM. Dissociation was monitored for 180s.

The bulk refractive index difference is corrected by subtracting the response obtained at the reference flow cell. Steady state response for deriving dissociation constant K by nonlinear curve fitting of langmuir binding isotherms _D . Using a simple one-to-one Langmuir binding modelT100Evaluation Software version 1.1.1) the association rate (k) was calculated by fitting the association and dissociation sensor maps simultaneously _on ) And dissociation rate (k) _off ). Equilibrium dissociation constant (K) _D ) Calculated as the ratio k _off /k _on . See, e.g., chen et al, J Mol Biol 293,865-881 (1999).

Binding assays and other assays

In one aspect, the antibodies of the invention (e.g., anti-PD-1 axis binding antagonist antibodies) are tested for antigen binding activity, e.g., by known methods such as ELISA, western blot, and the like.

In another aspect, competition assays can be used to identify antibodies or fragments that compete with a specific reference antibody for binding to the respective antigen. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) to which a particular reference antibody binds. A detailed exemplary method for mapping epitopes to which antibodies bind is provided in Morris (1996) "Epitope Mapping Protocols" in volume Methods in Molecular Biology, 66 (Humana Press, totowa, N.J.). Other methods are described in the examples section.

Activity determination

In one aspect, assays for identifying antibodies (e.g., the bioactive anti-PD-1 axis binding antagonist antibodies provided herein) are provided. Biological activity may include, for example, induction of DNA fragmentation, induction of apoptosis, and lysis of target cells. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, antibodies of the invention are tested for such biological activity. Assays for detecting cell lysis (e.g., by measuring LDH release) or apoptosis (e.g., using TUNEL assays) are well known in the art. Assays for measuring ADCC or CDC are also described in WO 2004/065540 (see example 1 therein), the entire contents of which are incorporated herein by reference.

Pharmaceutical preparation

Antibodies as described herein (e.g., anti-PD-1 axis binding antagonist antibodies) are prepared in the form of a lyophilized formulation or an aqueous solution by mixing such antibodies of desired purity with one or more optional pharmaceutically acceptable carriers (Remington' sPharmaceutical Sciences, 16 th edition, osol, a.ed. (1980)). Pharmaceutically acceptable 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., octadecyldimethylbenzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates, such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming 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 rHuPH 20) descriptionsIn U.S. patent publication Nos. 2005/026086 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases (such as chondroitinase).

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations comprising histidine-acetate buffer.

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.

The active ingredient may be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively); embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or embedded in a macroemulsion. Such techniques are disclosed in Remington' sPharmaceutical Sciences, 16 th edition, osol, a. Ed., 1980.

A slow release preparation may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations for in vivo administration are typically sterile. For example, sterility can be readily achieved by filtration through sterile filtration membranes.

Antibodies may be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and may be administered intralesionally if local treatment is desired. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various points in time, bolus administrations, and pulse infusion.

Another embodiment of the invention provides pharmaceutical compositions comprising one or more compositions, wherein each composition comprises one or more compounds for use according to the invention and one or more therapeutically inert carriers, diluents or excipients, and methods of preparing such pharmaceutical compositions. In one example, the compound of formula (I) may be formulated in a galenical administration form by mixing with a physiologically acceptable carrier (i.e., a carrier that is non-toxic to the recipient at the dosage and concentration used) at an ambient temperature at an appropriate pH and desired purity. The pH of the formulation will depend primarily on the particular 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 acetate buffer at pH 5. In another embodiment, the compound of formula (I) is sterile. The compounds may be stored, for example, as solid or amorphous compositions, as lyophilized formulations, or as aqueous solutions.

The compositions are formulated, metered and administered in a manner consistent with good medical practice. Factors to be considered in this case include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the practitioner.

As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all materials compatible with pharmaceutical administration, including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other materials and compounds compatible with pharmaceutical administration. Except insofar as any conventional medium or agent is incompatible with the active compound, it is contemplated that it will be used in the compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.

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

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

Pharmaceutical compositions of BRAF inhibitors (alone or in combination) can be prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the active ingredient of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington' sPharmaceutical Sciences, 16 th edition, osol, a. (edit) (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethyldiammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or nonionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).

Pharmaceutical compositions of BRAF inhibitors include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The composition may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will generally be that amount treated by the BRAF inhibitor or PD-1 axis binding antagonist that produces a therapeutic effect. Typically, this amount will range from about 1% to about 90%, preferably from about 5% to about 70%, most preferably from about 10% to about 30%, of the active ingredient in percent. Methods of making these compositions include the step of associating a BRAF inhibitor with a carrier and optionally one or more accessory ingredients. In general, the pharmaceutical compositions may be prepared by uniformly and intimately bringing into association the BRAF inhibitor with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, sachets, pills, tablets, lozenges (using a flavored basis, typically 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 a lozenge (using an inert basis such as gelatin and glycerin, or sucrose and acacia) and/or as a mouthwash, and the like, each containing a predetermined amount of a BRAF inhibitor as the active ingredient. The BRAF inhibitor may also be administered as a bolus, electuary or paste.

In one embodiment of the invention, the BRAF inhibitor and PD-1 axis binding antagonist treatment are formulated as two separate pharmaceutical compositions.

The active ingredient may be embedded in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, 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, 16 th edition, osol, a. (edit) (1980).

The formulation to be used for in vivo administration must be sterile. This can be easily achieved by filtration through sterile filtration membranes.

The dosage can vary within a wide range but will of course have to be adjusted according to the individual requirements of each particular situation. In the case of oral administration, the dosage for adults can vary from about 0.01mg to about 1000mg of a compound of formula (I) or a corresponding amount of a pharmaceutically acceptable solvate thereof per day. The daily dose may be administered as a single dose or in multiple divided doses, and furthermore, when an indication is found that the upper limit may be exceeded, the upper limit may also be exceeded.

The following examples illustrate, but do not limit, the invention, but are merely representative of the invention. The pharmaceutical compositions conveniently contain from about 1mg to 500mg, especially from 1mg to 100mg, of a compound of formula (I).

Examples of compositions according to the invention are:

example A

Tablets of the following composition were produced in a conventional manner:

/>

table 3: possible tablet composition

Production program

1. Ingredients 1, 2, 3 and 4 were mixed and granulated with purified water.

2. The granules were dried at 50 ℃.

3. The particles are passed through a suitable milling apparatus.

4. Adding the component 5, and mixing for three minutes; pressing on a suitable press.

Example B-1

Capsules were produced of the following composition:

table 4: possible composition of the capsule components

Production program

1. Ingredients 1, 2 and 3 were mixed in a suitable mixer for 30 minutes.

2. Ingredients 4 and 5 were added and mixed for 3 minutes.

3. Filled into suitable capsules.

The compound of formula (I), lactose and corn starch are first mixed in a mixer and then mixed in a pulverizer. Returning the mixture to the mixer; talc powder was added thereto and mixed well. The mixture is filled with a machine into suitable capsules, such as hard gelatin capsules.

Example B-2

Soft gelatin capsules were manufactured having the following composition:

Composition of the components	mg/capsule
		A compound of formula (I)	5
Yellow wax	8
		Hydrogenated soybean oil	8
Partially hydrogenated vegetable oil	34
		Soybean oil	110
Totals to	165

Table 5: possible soft gelatin capsule ingredient compositions

Composition of the components	mg/capsule
		Gelatin	75
Glycerin 85%	32
		Karion 83	8 (Dry matter)
Titanium dioxide	0.4
		Iron oxide yellow	1.1
Totals to	116.5

Table 6: possible soft gelatin capsule compositions

Production program

The compound of formula (I) is dissolved in a warm melt of the other ingredients and the mixture is filled into soft gelatin capsules of suitable size. Filled soft gelatin capsules are processed according to general procedures.

Example C

Suppositories were manufactured with the following composition:

composition of the components	mg/suppository
		A compound of formula (I)	15
Suppository matrix	1285
		Totals to	1300

Table 7: possible suppository compositions

Production program

The plug matrix was melted in a glass or steel vessel, thoroughly mixed and cooled to 45 ℃. At this time, fine powder of the compound of formula (I) was added thereto and stirred until the compound was completely dispersed. Pouring the mixture into a bolt mold with proper size, standing and cooling; the suppositories are then removed from the mold and individually packaged in waxed paper or foil.

Example D

An injection of the following composition was produced:

composition of the components	mg/injection.
		A compound of formula (I)	3
Polyethylene glycol 400	150
		Acetic acid	Proper amount, the pH is adjusted to 5.0
Water for injection	To 1.0ml

Table 8: possible injection solution composition

Production program

The compound of formula (I) is dissolved in a mixture of polyethylene glycol 400 and water for injection (part of). The pH was adjusted to 5.0 by acetic acid. The volume was adjusted to 1.0ml by adding the balance water. The solution is filtered, filled into vials using a suitable overfill and sterilized.

Example E

A bagging agent having the following composition was produced:

composition of the components	mg/bag
		A compound of formula (I)	50
Lactose, fine powder	1015
		Microcrystalline cellulose (AVICEL PH 102)	1400
Sodium carboxymethyl cellulose	14
		Polyvinylpyrrolidone K30	10
Magnesium stearate	10
		Flavoring additive	1
Totals to	2500

Table 9: possible compositions of sachets

Production program

The compound of formula (I) is mixed with lactose, microcrystalline cellulose and sodium carboxymethyl cellulose and granulated with a mixture of polyvinylpyrrolidone in water. The granules were mixed with magnesium stearate and flavouring additives and filled into bags.

Examples

Abbreviations (abbreviations)

CAS = american chemical abstracts; DCM = dichloromethane; dipea=n, N-diisopropylethylamine; DMF = dimethylformamide; DMSO = dimethylsulfoxide; DNA = deoxyribonucleic acid; edc·hcl=1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; ESI = electrospray ionization; etOAc = ethyl acetate; hoobt=3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine; LC-MS/ms=liquid chromatography-MS/MS; meoh=methanol; ms=mass spectrum; NMP = N-methyl-2-pyrrolidone; PCR = polymerase chain reaction; rt=room temperature; SFC = supercritical fluid chromatography; THF = tetrahydrofuran.

The following examples and figures are provided to illustrate the invention and are not limiting in nature.

Measuring reagent

(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 compound Ia) was provided as a powder from basil, basel, switzerland and resuspended before use. Anti-mouse PD-1 (CD 279; referred to herein as anti-mPD-1) was purchased from BioXcell (clone RMP1-14, catalog number: BP 0146).

Cell lines and culture conditions

Cell lines were obtained from ATCC and stored under standard conditions in humidified incubator with 5% CO2 and passaged twice a week. Culture conditions are reported in the following table:

the YUMM1.7 cell line was originally derived from a genetically engineered mouse melanoma model, with the following alleles: tyrosinase: creet 2 (activation is limited to CRE recombinase expression by cells expressing tyrosinase genes; expression is limited to melanocyte compartments), BRAF V600E/WT (BRAF 600E is conditionally active only in cells expressing CRE recombinase), PTEN-/-, CDKN2A-/- (PTEN and CDKN2A are conditionally inactive only in cells expressing CRE recombinase). Murine cells derived from these tumors were transplanted into immunocompetent CBL/6 mice, allowing for studies of interactions with immune compartments. The model is initially described in the following: meeth K et al, the YemM lines a series of congenic mouse melanoma cell lines with defined genetic alterations. Pigment Cell Melanoma Res (5): 590-597,2016.PubMed 27287723.

Animals:

in vivo studies, female mice 7-9 weeks old (at the beginning of the experiment) were purchased from Charles river laboratory (Charles River Laboratories). The strain used in the experiment was C57BL/6.

For xenograft establishment, cells were suspended in a medium consisting of 50% matrigel and 50% Hank balanced salt solution and subcutaneously injected to the right. When the tumor volume reaches about 100mm ³ At this time, mice were randomized prior to treatment.

In vivo studies, female mice 7-9 weeks old (at the beginning of the experiment) of immunocompetent strain C57BL/6 were used. The experiment was performed at the trusted research institute (CRO) covancer, who purchased mice for tumor establishment, drug treatment, and tumor monitoring.

For xenograft establishment, YUMM1.7 cells were suspended in a medium consisting of 50% matrigel and 50% hank balanced salt solution and subcutaneously injected to the right. When the tumor volume reaches about 100mm ³ At this time, mice were randomized prior to treatment. Mice were treated according to the protocol reported in fig. 2.

For immunohistochemical analysis, female C57/BL6 mice were subQ injected with YUMM1.7 cells. When the tumor reached about 250mm3 (day 14), the mice were treated with 1 or 5mg/kg of compound Ia per day (oral administration), 12.5mg/kg of anti-mouse PD1 once (single intravenous administration) or a combination of the following: compound Ia and anti-mPD 1. Mice were treated according to the protocol reported in fig. 5. Mice were sacrificed 3 or 5 days from the start of treatment and tumors were collected, digested and used for fixation and paraffin-embedded cytofluorescence analysis. The cell suspension was mixed with Zombie Aqua (1:1000) ^TM Dyes (biologid) were incubated together to distinguish between living and dead cells and by anti-mouse labeled with PECells were stained for either CD45 or anti-mouse CD3 (clone SP-7 Abcam) for cell fluorescence analysis. For paraffin-embedded tumors, samples were stained with anti-mouse CD45 (clone 30-F11 Biolegend USA) according to standard procedures (Cancer Res.2015Mar 15;75 (6): 1091-101).

Examples

Compound Ia is a novel paradox inhibitor BRAFi, as shown in figure 1, and uses cell line a375 containing the mutations BRAF V600E and NRAS Q61K; the cell line served as a resistance model for the first generation of BRAFi and BRAF/MEKi combinations.

The results demonstrate that compound Ia elicits potent P-ERK inhibition in this cell line model, which is superior to the effect achieved by Kang Naifei ni as a single agent. Furthermore, the combination with a fixed dose of MEKi cobicitinib of 10nM resulted in a better P-ERK reduction than the activity achieved by Kang Naifei Ni/Bimetinib.

Example 1

Mice were implanted with the BRAF V600E mutant cell line YUMM1.7. After tumor establishment (100 mm 3), mice were randomized and orally administered (PO) once daily (QD) compound Ia (1 mg/kg or 5 mg/kg), mouse anti-PD-1 antibodies once weekly by intravenous injection, or respective combinations of PD-1 and 1 or 5mg/kg compound Ia. For the group with 5mg/kg of compound Ia and 5mg/kg of combined PD-1/compound Ia, treatment was performed until day 11 (treatment started after random grouping) to day 31, or for the group with 1mg/kg of compound Ia and 1mg/kg of combined PD-1/compound Ia, treatment was performed until day 11 to day 45. After the end of treatment, mice were monitored for signs of tumor recurrence until day 58.

The results reported in fig. 3 and 4 demonstrate that the combination of compounds Ia and PD-1 shows a strong effect on the number of mice exhibiting disease recurrence.

Example 2

The results reported in fig. 6 and 7 clearly support the efficacy of the combination of compound Ia with a PD-1 axis binding antagonist. In these experiments, as observed in flow cytometry and IHC data, treatment of compound Ia is driving a powerful anti-tumor response and thereby triggering recruitment of inflammatory infiltrates. When compound Ia is combined with a PD-1 axis binding antagonist, the activation of the immune system by compound Ia supports a powerful effect. Furthermore, an increase in tumor antigen release may promote additional efficacy of PD-1 axis blocking.

Article of manufacture

In another aspect of the invention, an article of manufacture is provided that contains a substance useful for treating, preventing and/or diagnosing the above-mentioned disorders. The article includes a container and a label or package insert (package insert) on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition that is effective in treating, preventing, and/or diagnosing a condition, either by itself or in combination with another composition, and the container may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a bispecific antibody and the additional active agent is an additional chemotherapeutic agent as described herein. The label or package insert indicates that the composition is to be used to treat the selected condition. Furthermore, the article of manufacture may comprise (a) a first container comprising a composition therein, wherein the composition comprises a bispecific antibody; and (b) a second container containing a composition therein, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition is useful for treating a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. The article of manufacture may also include other substances desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Amino acid sequences of exemplary embodiments

Exemplary anti-PD-1 and anti-PD-L1 antagonist sequences

/>

Although the invention has been described in considerable detail by way of illustration and example for the purpose of clarity of understanding, such illustration and example should not be construed to limit the scope of the invention. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

Claims

1. A combination of a BRAF inhibitor and a PD-1 axis binding antagonist, wherein the BRAF inhibitor is a compound of formula (I)

Or a pharmaceutically acceptable salt or solvate thereof.

2. The combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to claim 1, 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.

3. The combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to claim 1 or 2, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.

4. The combination of a BRAF inhibitor of any one of claims 1-3 and a PD-1 axis binding antagonist, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

5. The combination of a BRAF inhibitor according to any one of claims 1-3 and a PD-1 axis binding antagonist, wherein the PD-1 axis binding antagonist is selected from the group consisting of a cimrpe Li Shan antagonistPembrolizumab +.>Nawu monoclonal antibodyRN888 (PF-06801591), alemtuzumab +.>Avermectin->And Devaluzumab (ImfinziTM).

6. The combination of a BRAF inhibitor according to any one of claims 1-4 and a PD-1 axis binding antagonist, wherein the PD-1 axis binding antagonist is alemtuzumab

7. The combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to any one of claims 1-6 for use as a medicament.

8. The combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to any one of claims 1-6 for use in the therapeutic and/or prophylactic treatment of cancer.

9. Use of a combination of a BRAF inhibitor and a PD-1 axis binding antagonist according to any one of claims 1-6 for the manufacture of a medicament for the treatment or prevention of cancer.

10. A method for treating or preventing cancer, in particular melanoma, non-small cell lung cancer or leptomeningeal cancer, comprising administering to a patient in need thereof an effective amount of a combination of a BRAF inhibitor according to any one of claims 1 to 6 and a PD-1 axis binding antagonist.

11. A pharmaceutical composition comprising the combination of a BRAF inhibitor according to any one of claims 1 to 6 and a PD-1 axis binding antagonist and one or more pharmaceutically acceptable excipients.

12. The combination, use, method or pharmaceutical composition according to any one of claims 7 to 11, wherein the BRAF inhibitor is administered orally and the PD-1 axis binding antagonist is administered by injection, such as intravenous or subcutaneous injection.

13. The combination, use, method or pharmaceutical composition of any one of claims 7-11, wherein the BRAF inhibitor is administered concurrently with the PD-1 axis binding antagonist.

14. The combination, use, method or pharmaceutical composition according to any one of claims 7 to 11, wherein the BRAF inhibitor and the PD-1 axis binding antagonist are administered sequentially.

15. The combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to any one of claims 7-9, wherein the cancer is thyroid cancer, colorectal cancer, melanoma, brain cancer, pia mater cancer or non-small cell lung cancer.

16. BRAF inhibition for use according to any one of claims 7 to 10Combination of an agent and a PD-1 axis binding antagonist, wherein the cancer is associated with BRAF ^V600X Mutations are associated.

17. The combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to any one of claims 7-10, wherein the cancer is BRAF ^V600X Mutation-positive unresectable or metastatic cancer.

18. The combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to any one of claims 7-10, wherein BRAF is determined using a method comprising ^V600X Mutation: (a) PCR or sequencing nucleic acids (e.g., DNA) extracted from a sample of tumor tissue and/or body fluid of the patient; and (b) determining BRAF in the sample ^V600 Is expressed by (a).

19. The combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to any one of claims 7-10, comprising one or more additional anticancer agents selected from the group consisting of MEK inhibitors, MEK degradants, EGFR inhibitors, EGFR degradants, inhibitors of HER2 and/or HER3, degradants of HER2 and/or HER3, SHP2 inhibitors, SHP2 degradants, axl inhibitors, axl degradants, ALK inhibitors, ALK degradants, PI3K inhibitors, PI3K degradants, SOS1 inhibitors, SOS1 degradants, signal transduction pathway inhibitors, checkpoint inhibitors, modulators of the apoptotic pathway, cytotoxic chemotherapeutic agents, angiogenesis targeting therapies, immune targeting agents, and antibody drug conjugates.

20. The combination of a BRAF inhibitor and a PD-1 axis binding antagonist for use according to any one of claims 7-10, further comprising a MEK inhibitor.

21. The invention as hereinbefore described.