RU2796863C2 - Combination therapy with a chemokine receptor 2 (ccr2) antagonist and a pd-1/pd-l1 inhibitor - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to combination therapy with a chemokine receptor 2 (CCR2) antagonist and a PD-1 and/or PD-L1 inhibitor in the treatment of colorectal cancer. A method for enhancing the therapeutic effect of a PD-1 and/or PD-L1 inhibitor in the treatment of colorectal cancer in a mammal is proposed, the said method includes administering a therapeutically effective amount of a CCR2 chemokine receptor antagonist and a therapeutically effective amount of a PD-1 and/or PD-L1 inhibitor to a mammal. Also the following is provided: the use of a therapeutically effective amount of a CCR2 chemokine receptor antagonist to enhance the therapeutic effect of a PD-1 and/or PD-L1 inhibitor in the treatment of colorectal cancer in a subject, and a kit comprising a therapeutically effective amount of a CCR2 chemokine receptor antagonist, a therapeutically effective amount of a PD-1 inhibitor, and/or PD-L1 and instructions for use.

EFFECT: provision of a significant reduction in tumor size.

25 cl, 14 dwg, 1 ex

Description

Cross-references to related applications

This application claims priority under 35 USC §119(e) on U.S. provisional application serial number 62/562,952, filed September 21, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

Federally Sponsored Research and Development Invention Claim

Not applicable.

Link to the attached "Sequence Listing", table or computer program provided on the CD

Not applicable.

Prerequisites

Malignant tumors use numerous mechanisms to avoid the body's natural cytotoxic immune response so that these tumors are not destroyed by the immune system. These mechanisms include dysfunctional T-cell signaling, suppressor regulatory cells, and immune checkpoints that are normally involved in suppressing the intensity of adaptive immune responses and protecting healthy tissues from collateral damage. For example, tumors develop immune resistance, especially against T cells that are specific for tumor antigens, by recruiting CCR2 ⁺ myeloid-derived suppressor cells (MSCs) and tumor macrophages into the tumor and their microenvironment.

CCR2 ⁺ SKMP perform immunosuppressive functions. SCMPs play a key role in the tumor's ability to suppress immune responses. Another key component of this suppression is the activation of immune checkpoints, which in turn limits the activation and entry of T cells into tumors. Immune checkpoints refer to inhibitory pathways of the immune system that are important in maintaining self-tolerance and controlling immune responses in peripheral tissues to minimize collateral tissue damage.

Programmed death-1 (PD-1) protein is one of numerous immune checkpoint receptors that are expressed by activated T cells and mediate immunosuppression. PD-1 ligands include programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), which are expressed on antigen-presenting cells as well as on many human cancer cells. PD-L1 and PD-L2 can suppress T cell activation and cytokine secretion when bound to PD-1.

Inhibitors of the PD-1/PD-L1 interaction have been shown to mediate potent antitumor activity and are effective in the treatment of certain cancers. Despite these results, there remains a need for effective treatment of cancers such as solid malignancies.

Brief summary of the invention

The present invention relates to combination therapy with a chemokine receptor 2 (CCR2) antagonist and a PD-1 and/or PD-L1 inhibitor in the treatment of cancer.

In some embodiments, the CCR2 chemokine receptor antagonist is of the formula I

where each variable is described below.

In some embodiments, the CCR2 chemokine receptor antagonist has a formula selected from the group consisting of

or their pharmaceutically acceptable salt.

In some embodiments, the CCR2 antagonist has the formula

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula

,

,

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula

or a pharmaceutically acceptable salt thereof.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is a PD-1 inhibitor.

In some embodiments, the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, IBI-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, its biosimilars, biobetters and bioequivalents.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is a PD-L1 inhibitor.

In some embodiments, the PD-L1 inhibitor is selected from the group consisting of durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, KY-1003, their biosimilars , biobetters and bioequivalents.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is a compound of formula (II)

where each variable is described below.

In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is pancreatic cancer.

Other objects, features and advantages of the present invention will be apparent to a person skilled in the art from the following detailed description and figures.

Brief description of the drawings

Fig. 1 shows immunohistochemical analysis of human normal and tumor tissue using anti-hCCR2 mAb. Panels A and B show normal colon tissue at 40X and 200X, respectively. Panels C, D, E and F show typical human colon cancer tissue at the magnifications indicated. Specific staining of CCR2 was observed in 78 malignant tumors (diagnosed at stages I-III) in patients with colon cancer, but not in normal control tissues of the colon.

Fig. 2 illustrates that CCR2 is expressed in a CD11b+ tumor-infiltrating leukocyte subset in the CT27 colorectal cancer tumor (CRC) microenvironment. Gating was used for live CD45+ cells. Panel A displays data obtained using the CCR2 marker and panel B displays data using isotype control. The circled area of each panel highlights the difference.

Fig. 3 shows that the CD11b ⁺ /CCR2 ⁺ population in CT26 tumors predominantly includes the immunophenotype of Ly6C ^hi /Ly6G ^neg - monocytic suppressor cells of myeloid origin (M-MSC). Panel A presents data obtained using CD11b ⁺ /CCR2 ⁺ markers and CD45 ⁺ gating. Panel B displays data obtained from the CD11b ⁺ /CCR2 ⁺ subset using the Ly6C/Ly6G markers.

Fig. 4 shows direct gating of M-SCMP cells isolated from CT26 tumors showing stable expression of CCR2. Panel A: Gating of live CD45 ⁺ CT26 infiltrating cells in the CD11b ⁺ population. Panel B: Gated LyC ^hi / ^Ly6G populations. Panel C: Overlay of a histogram of CCR2 staining (right) with isotype control monoclonal antibody staining (left) in a Ly6C ^hi /Ly6G ^- population.

Fig. 5 illustrates the overall study design of anti-PD-1 + Compound 1 in the CT26 model.

Fig. 6 shows that Compound 1 administered by gavage at a dose of 30 mg/kg per day provides minimal plasma levels equal to or greater than those required for complete receptor coverage. Panel A shows the plasma levels of Compound 1 on day 3 of administration. Panel B shows Compound 1 23 days after administration.

Fig. 7 shows that the combination of Compound 1 and α-PD-1 results in smaller tumor volumes. Panel A shows mice treated with 1% HPMC + isotype. Panel B shows mice treated with 1% HPMC + α-PD-1. Panel C shows mice treated with 30 mg/kg Compound 1 + isotype. Panel D shows mice treated with 30 mg/kg compound 1 + α-PD-1. The dotted line shows the largest tumor volume observed in the Compound 1 + α-PD-1 group. "1% HPMC" is the vehicle control for Compound 1, "isotype" is the identical dosed isotype control for α-PD-1.

Fig. 8 shows tetramer staining of peripheral blood lymphocytes with MHC class I molecules and associated peptides for the immunodominant CT26 antigen, demonstrating a CT26-specific CD8 T cell response in tumor bearing mice.

Fig. 9 demonstrates that CD8 T cells are required to reduce tumor size induced by Compound 1 + α-PD-1 therapy. Panel A shows tumor volume in mice treated with vehicle + α-PD-1 + α-CD8. Panel B shows tumor volume in mice treated with 30 mg/kg Compound 1 + α-PD-1 + isotype control. Panel C shows tumor volume in mice treated with 30 mg/kg Compound 1 + α-PD-1 + α-CD8.

Fig. 10 demonstrates that despite the involvement of cytotoxic T cells in the reduction of tumor size, the number of tumor CD8 T cells does not change significantly with treatment. Tumor infiltrating cytotoxic T cells (Thy1 ⁺ /CD8 ⁺ ) were quantified by weighing the tumors before dissociation, which allowed the calculation of the number of cells per gram of tumor.

Fig. 11 shows that Compound 1 reduces the amount of M-SCMP in the CT26 microenvironment by day 24. M-SCMP was quantified by weighing the tumors before dissociation, which allowed the calculation of the number of cells per gram of tumor.

Fig. 12 shows that the ratio of CD8 T cells to M-SCMP is significantly increased with combination treatment. The ratio of CD8 T cells to M-SCMP was calculated from the number of cells shown in FIG. 10 and FIG. 11. The ratio in control mice (veh + iso) was 1:1, which means one M-SCMP for each CD8 T cell. The combination treatment reduced M-SCMP in favor of CD8 T cells, yielding 100 CD8 T cells for each M-SCMP. Treatment with Compound 1 alone gave 10 CD8 T cells for each M-SCMP cell. Treatment with α-PD-1 yielded a CD8 T cell for every M-SCMP cell.

Fig. 13 shows that the number of long-term survivors with CT26 in response to α-PD-1 increases with combined treatment with CCR2. On day 83, 6 survivors remained in the α-PD-1 + Compound 1 group, while only 2 survivors remained in the α-PD-1 + control solution group. Subgroups of mice taken on day 27 for cell analysis were excluded from this survival analysis. One mouse from the Iso + Veh group and one from the α-PD-1 + 598 group did not develop tumor and these two mice were excluded from this analysis. The Gehan-Breslow-Wilcoxon test was used to determine the p value between the red (middle) and blue (upper) curves.

Fig. 14 shows that long-term survivors are specifically immune to CT26 re-inoculation (panel B), but not to 4T1 breast tumor (panel A).

Detailed description of the invention

I. Abbreviations and definitions

As used in this application, the terms in the singular include not only aspects with one element, but also aspects with more than one element. For example, the singular includes the plural, unless the context clearly dictates otherwise. Thus, for example, reference to "cell" includes a plurality of such cells, and reference to "agent" includes reference to one or more agents known to those skilled in the art, and so on.

The terms "about" and "approximately" generally mean an acceptable degree of error for the quantity being measured, given the nature or accuracy of the measurements. Typical exemplary error rates are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, especially in biological systems, the terms "about" and "approximately" can mean values that are within an order of magnitude, preferably within 5 times and more preferably within 2 times the value given. The numerical values given in this document are approximate unless otherwise indicated, which means that the term "about" or "approximately" is implied unless it is explicitly stated.

The term "alkyl", by itself or as part of another substituent, means, unless otherwise indicated, a straight or branched chain hydrocarbon radical having the number of carbon atoms indicated (i.e., C _1-8 means from one to eight carbon atoms). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3 -butynyl and higher homologues and isomers. The term "cycloalkyl" refers to hydrocarbon rings having the specified number of ring atoms (eg C _3-6 cycloalkyl) and fully saturated or having no more than one double bond between the ring vertices. The term "cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. The term "heterocycloalkyl" refers to a cycloalkyl group that contains one to five heteroatoms selected from N, O and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. The heterocycloalkyl may be a monocyclic, bicyclic or polycyclic ring system. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine , pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine and the like. The heterocycloalkyl group may be attached to the rest of the molecule via a carbon or ring heteroatom. For terms such as cycloalkylalkyl and heterocycloalkylalkyl, it is understood that the cycloalkyl or heterocycloalkyl group is attached via an alkyl or alkylene linker to the rest of the molecule. For example, cyclobutylmethyl is a cyclobutyl ring that is attached by a methylene linker to the rest of the molecule.

The term "alkylene" as such or as part of another substituent means a divalent radical derived from an alkane, for example -CH ₂ CH ₂ CH ₂ CH ₂ -. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being preferred in the present invention. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having four or fewer carbon atoms. Similarly, "alkenylene" and "alkynylene" refer to unsaturated forms of "alkylene" having double or triple bonds, respectively.

», которая пересекает одинарную, двойную или тройную связь в любой химической структуре, изображенной здесь, представляет собой точечное присоединение одинарной, двойной или тройной связи к остальной части молекулы.As used herein, the wavy line "

", which crosses a single, double or triple bond in any chemical structure depicted here, is a point attachment of a single, double or triple bond to the rest of the molecule.

The term "heteroalkyl", alone or in combination with another term, means, unless otherwise indicated, a stable straight or branched chain or cyclic hydrocarbon radical, or combinations thereof, consisting of the indicated number of carbon atoms and one to three heteroatoms selected from the group consisting of of O, N, Si and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) O, N and S may be located at any internal position of the heteroalkyl group. The Si heteroatom may be located at any position on the heteroalkyl group, including the position where the alkyl group is attached to the rest of the molecule. Examples include -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N(CH ₃ )-CH ₃ , -CH ₂ -S-CH ₂ -CH ₃ , -CH ₂ -CH ₂ , -S(O)-CH ₃ , -CH ₂ -CH ₂ -S(O) ₂ -CH ₃ , -CH=CH-O-CH ₃ , -Si(CH ₃ ) ₃ , -CH ₂ -CH=N-OCH ₃ , and -CH=CH-N(CH ₃ )-CH ₃ . Up to two heteroatoms may be consecutive, such as -CH ₂ -NH-OCH ₃ and -CH ₂ -O-Si(CH ₃ ) ₃ . Similarly, the terms "heteroalkenyl" and "heteroalkynyl" alone or in combination with another term mean, unless otherwise indicated, an alkenyl group or an alkynyl group, respectively, which contains the number of carbon atoms indicated and has from one to three selected heteroatoms from a group consisting of O, N, Si and S, and where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) O, N and S may be located at any internal position of the heteroalkyl group.

The term "heteroalkylene" by itself or as part of another substituent means a divalent radical, saturated or unsaturated, or polyunsaturated, derived from heteroalkyl, exemplified by -CH ₂ -CH ₂ -S-CH ₂ CH ₂ - and -CH ₂ -S -CH ₂ -CH ₂ -NH-CH ₂ -, -O-CH ₂ -CH=CH-, -CH ₂ -CH=C(H)CH ₂ -O-CH ₂ - and -S-CH ₂ -C ≡C-. For heteroalkylene groups, heteroatoms may also occupy one or both ends of the chain (eg, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to those alkyl groups that are attached to the rest of the molecule through an oxygen atom, an amino group or a sulfur atom, respectively. In addition, for dialkylamino groups, the alkyl moieties may be the same or different, and may also combine to form a 3-7 membered ring with the nitrogen atom to which each is attached. Accordingly, a group represented as -NR ^a R ^b means the inclusion of piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like.

The terms "halo" or "halogen" by themselves or as part of another substituent mean, unless otherwise indicated, a fluorine, chlorine, bromine or iodine atom. In addition, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "C _1-4 haloalkyl" means trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term "aryl" means, unless otherwise indicated, a polyunsaturated, usually aromatic, hydrocarbon group, which may be one ring or multiple rings (up to three rings) that are fused together or covalently linked. The term "heteroaryl" refers to aryl groups (or rings) that contain one to five heteroatoms selected from N, O and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. The heteroaryl group may be attached to the rest of the molecule via a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl , benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridines, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazole yl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl and the like. Substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

For brevity, the term "aryl" when used in combination with other terms (eg, aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group that is attached to the rest of the molecule (eg, benzyl, phenethyl, pyridylmethyl, and the like).

The above terms (eg, "alkyl", "aryl" and "heteroaryl") in some embodiments, the implementation will include both substituted and unsubstituted forms of the specified radical. Preferred substituents for each type of radical are listed below. For brevity, the terms "aryl" and "heteroaryl" will refer to the substituted or unsubstituted variants below, while the term "alkyl" and related aliphatic radicals refer to an unsubstituted variant unless indicated to be substituted.

Substituents for alkyl radicals (including groups often referred to as alkylene, alkenyl, alkynyl and cycloalkyl) can be various groups selected from: -halo, -OR', -NR'R", -SR', -SiR'R"R"',-OC(O)R',-C(O)R', -CO ₂ R', -CONR'R”, -OC(O)NR'R”, -NR”C(O)R', -NR'-C(O)NR”R”', -NR”C(O) ₂ R', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH- C(NH ₂ )=NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R", -NR'S(O) ₂ R", -CN and - NO ₂ in an amount from zero to (2m' + 1), where m' is the total number of carbon atoms in such a radical. R', R" and R"' are each independently hydrogen, unsubstituted C _1-8 alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halo, unsubstituted C _1-8 alkyl, C _1-8 alkoxy, or C _1-8 thioalkoxy group or unsubstituted aryl-C _1-4 alkyl groups. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" means 1 -pyrrolidinyl and 4-morpholinyl The term "acyl", whether used alone or as part of another group, refers to an alkyl radical in which the two substituents on the carbon atom closest to the point of attachment of the radical are replaced by a =O substituent (e.g. , -C(O)CH ₃ , -C(O)CH ₂ CH ₂ OR' and the like).

Likewise, substituents for aryl and heteroaryl groups may vary and are typically selected from: -halo, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO ₂ , -CO ₂ R', -CONR'R”, -C(O)R', -OC(O)NR'R”, -NR”C(O)R', -NR”C(O) ₂ R', ,-NR'-C(O)NR”R”', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR ', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R”, -NR'S(O) ₂ R”, -N ₃ , perfluoro(C ₁ -C ₄ )alkoxy and perfluoro(C ₁ -C ₄ )alkyl, in an amount from zero to the total number of open valences in the aromatic ring system; and where R', R" and R"' are independently selected from hydrogen, C _1-8 alkyl, C _3-6 cycloalkyl, C _2-8 alkenyl, C _2-8 alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)- C _1-4 alkyl and unsubstituted aryloxy-C _1-4 alkyl. Other suitable substituents include each of the above aryl substituents attached to the ring atom by an alkylene unit of 1-4 carbon atoms.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted with a substituent of the formula -TC(O)-(CH ₂ ) _q -U-, where T and U are independently -NH-, -O-, -CH ₂ - or a single bond, and q is an integer from 0 to 2. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -A-(CH ₂ ) _r -B-, where A and B are independently represent -CH ₂ -, -O-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -S(O) ₂ NR'- or a single bond, and r represents is an integer from 1 to 3. One of the single bonds of the new ring thus obtained may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may be optionally substituted with a substituent of the formula -(CH _2)s -X-(CH ₂ ) _t - where s and t are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O) ₂ - or -S(O) ₂ NR'-. The substituent R' in -NR'- and -S(O) ₂ NR'- is selected from hydrogen or unsubstituted C _1-6 alkyl.

Used in this application, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

For the compounds provided herein, a bond that is directed from a substituent (usually an R-group) to the center of an aromatic ring (e.g., benzene, pyridine, and the like) should be understood to refer to a bond providing a bond at any of the available vertices. aromatic ring. In some embodiments, the image will also include a ring compound that is fused to an aromatic ring. For example, a bond directed towards the center of the benzene moiety of an indole would indicate a bond to any available vertex of the six- or five-membered ring moieties of the indole.

The term "pharmaceutically acceptable salts" is intended to include salts of active compounds that are prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the compounds described in this application. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be prepared by contacting the neutral form of such compounds with a sufficient amount of the required base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include salts of aluminum, ammonium, calcium, copper, ferric iron, ferrous iron, lithium, magnesium, ferric manganese, ferrous manganese, potassium, sodium, zinc and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, natural amines, and the like, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine , trimethylamine, tripropylamine, tromethamine and the like. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be prepared by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrocarboxylic, phosphoric, monohydrophosphoric, dihydrophosphoric, sulfuric, monohydrosulfuric, hydroiodic or phosphorous acids and the like, as well as salts derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids such as glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Some specific compounds of the present invention contain both basic and acidic functional groups which allow the compounds to be converted into base or acid addition salts.

The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the usual manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but the salts are otherwise equivalent to the parent form of the compound for purposes of the present invention.

In addition to salt forms, the present invention provides compounds that are in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to give the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods under ex vivo conditions. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical.

Some compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are intended to be included within the scope of the present invention. Some compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for use contemplated by the present invention and are intended to be within the scope of the present invention.

Some compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers, and individual isomers (eg, individual enantiomers) are intended to be included within the scope of the present invention. When compounds are presented herein with identified stereochemistry (denoted as R or S, or indicated by dotted lines or wedge-bond signs), those skilled in the art will appreciate that these compounds are substantially free of other isomers (e.g., at least 80%, 90%, 95%, 98%, 99% and up to 100% free from other isomer).

The compounds of the present invention may also contain non-natural proportions of atomic isotopes on one or more of the atoms constituting such compounds. The non-natural proportions of an isotope can be defined as ranging from the naturally occurring amount to the amount consisting of 100% of the atom in question. For example, compounds may include radioactive isotopes such as, for example, tritium ( ³ H), iodine-125 ( ¹²⁵ I) or carbon-14 ( ¹⁴ C), or non-radioactive isotopes such as deuterium ( ² H) or carbon-13 ( ¹³ C). Such isotopic variations may provide additional benefit to those compounds described elsewhere in this application. For example, isotopic variants of the compounds of the invention may find additional uses, including but not limited to use as diagnostic and/or imaging reagents or as cytotoxic/radiotoxic therapeutic agents. In addition, isotopic variants of the compounds of the invention may have altered pharmacokinetic and pharmacodynamic characteristics that may improve safety, tolerability, or efficacy during treatment. All isotopic variants of the compounds of the present invention, whether radioactive or not, are intended to be included within the scope of the present invention.

The term "cancer" refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, glioblastoma, and the like, but are not limited to. The terms "tumor" and "cancer" are used interchangeably in this application, for example, both terms cover solid and liquid, for example, diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous as well as malignant neoplasms and tumors.

The term "PD-1" or "PD-1 receptor" refers to the programmed death protein-1, a co-inhibitor of T cells, also known as CD279. The amino acid sequence of the full-length human PD-1 protein is given, for example, in GenBank accession number NP_005009.2. PD-1 is a 288 amino acid protein with an extracellular N-terminal domain, which is an IgV-like transmembrane domain, and an intracellular domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switching motif (ITSM) (Chattopadhyay et al., ImmunolRev, 2009, 229 (1): 356-386). The term "PD-1" includes recombinant PD-1, or a fragment thereof, or variants thereof. The PD-1 receptor has two ligands, PD ligand-1 (PD-L1) and PD ligand-2 (PD-L2).

The term "PD-L1" or "programmed death ligand 1" refers to the PD-1 receptor ligand, also known as CD274 and B7H1. The amino acid sequence of the full-length human PD-L1 protein is listed, for example, in GenBank accession number NP_054862.1. PD-L1 is a 290 amino acid protein with an extracellular IgV-like domain, a transmembrane domain, and a highly conserved intracellular domain of approximately 30 amino acids. PD-L1 is constitutively expressed on many cells, such as antigen-presenting cells (eg, dendritic cells, macrophages, and B cells), as well as hematopoietic and non-hematopoietic cells (eg, vascular endothelial cells, pancreatic islets, and sites of immune privilege) . PD-L1 is also expressed in a wide variety of tumors, virally infected cells, and autoimmune tissue.

Programmed death pathway 1 (PD-1/PD-L1) acts as a checkpoint to limit T-cell mediated immune responses. Both PD-1 ligands, PD-L1 and PD-L2, can interact with the PD-1 receptor and induce PD-1 signaling and reversible inhibition of T cell activation and proliferation. When PD-1 ligands are on the surface of cancer cells or neighboring cells, these ligands bind to PD-1 receptor positive immune effector cells and use the PD-1 pathway to evade the immune response.

The term "immune checkpoint inhibitor" or "immune checkpoint blockade" refers to any agent, molecule, compound, chemical, protein, polypeptide, macromolecule, etc. that blocks or inhibits inhibitory pathways in a statistically, clinically or biologically significant manner. immune system. Such inhibitors may include small molecule inhibitors or may include antibodies or antigen-binding fragments thereof that bind and block or inhibit immune checkpoint receptors, or antibodies that bind and block or inhibit immune checkpoint receptor ligands. Exemplary immune checkpoint molecules that can be targeted to block or inhibit include CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γδ and CD8+(αβ) memory T cells), CD160 ( also referred to as BY55) and CGEN-15049, but are not limited to. Exemplary immune checkpoint inhibitors include durvalumab (anti-PD-L1 antibody; MEDI4736), pembrolizumab (anti-PD-1 monoclonal antibody), nivolumab (anti-PD-1 antibody), pidilizumab (CT-011; humanized anti-PD-1 monoclonal antibody ), AMP224 (recombinant B7-DC-Fc fusion protein), BMS-936559 (anti-PD-L1 antibody), atezolizumab (MPLDL3280A; Fc-optimized human anti-PD-L1 monoclonal antibody), avelumab (MSB0010718C; human anti-PD- L1), ipilimumab (anti-CTLA-4 checkpoint inhibitor), tremelimumab (anti-CTLA-4 blocking antibody), and anti-OX40.

The terms "CCR2 antagonist" and "CCR2 chemokine receptor antagonist" are used interchangeably and refer to a small molecule that antagonizes the interaction of the CCR2 chemokine receptor and any of its ligands. Such a compound can inhibit the processes usually triggered by the interaction of the receptor with the ligand.

Used in this application, the term "complete response" or "CR" refers to the disappearance of all target lesions; "partial response" or "PR" refers to at least a 30% reduction in the sum of longest diameters (SLD) of the target lesions, based on a baseline SLD; and "stable disease" or "SD" refers to a case where there is neither enough reduction in target lesions to be defined as a partial response, nor enough increase to be defined as disease progression, with the lowest SLD score since the start of treatment being taken as a benchmark .

As used herein, the term "progressive disease" or "PD" refers to an increase in the SLD of target lesions of at least 20%, taking into account the lowest SLD value recorded since the start of treatment, or the presence of one or more new lesions.

As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the disease being treated (eg, cancer) does not worsen. Progression-free survival may include the amount of time patients have a complete or partial response, as well as the amount of time patients have stable disease.

As used herein, "overall response rate" (ORR) refers to the sum of the complete response rate (CR) and the partial response rate (PR).

Used in this application, the term "overall survival" refers to the percentage of people in a group who can be alive after a certain period of time.

As used herein, the term "mammal" is defined as humans, other primates, cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. The compounds, agents and compositions described herein are useful in the treatment of a wide range of cancers, including solid cancers.

The term "therapeutically effective amount" means the amount of the compound in question that will elicit a biological or medical response in a cell, tissue, system, or animal, such as a human, that is necessary for the researcher, veterinarian, physician, or other person providing treatment.

II. general information

The present invention addresses the surprising and unexpected fact that combination therapy using a CCR2 antagonist and a PD-1 and/or PD-L1 inhibitor significantly improves cancer treatment compared to PD-1 and/or PD-L1 inhibition alone.

III. Combination therapy with a CCR2 antagonist and a PD-1 and/or PD-L1 inhibitor

Provided herein are methods, compositions and kits that utilize the synergistic effect of CCR2 antagonists and PD-1 and/or PD-L1 inhibitors in the treatment of cancer. A combination treatment that includes both a CCR2 antagonist and a PD-1 and/or PD-L1 inhibitor is more effective in treating cancer than compound/antibody alone.

In one aspect, provided herein are methods for treating cancer in a mammal. The method includes administering to a subject in need thereof a therapeutically effective amount of a CCR2 chemokine receptor antagonist and a therapeutically effective amount of a PD-1 and/or PD-L1 inhibitor.

In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a CCR2 chemokine receptor antagonist and a therapeutically effective amount of a PD-1 inhibitor.

In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a CCR2 chemokine receptor antagonist and a therapeutically effective amount of a PD-L1 inhibitor.

In some embodiments, the CCR2 chemokine receptor antagonist is a compound of formula I of its subformulas below. In some embodiments, the CCR2 chemokine receptor antagonist is selected from the group consisting of:

,

,

and their pharmaceutically acceptable salt.

In some embodiments, the CCR2 chemokine receptor antagonist is selected from the group consisting of AZ889, AZD2423, INCB-8761, MK-0812, BMS-813160, INCB-003284, PF-04634817, BMS-741672, cenicriviroc, CCX-140.

In some embodiments, the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, IBI-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001 GB-226, STI -1110, their biosimilars, biobetters and bioequivalents.

In some embodiments, the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, IBI-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001 GB-226, and STI -1110.

In some embodiments, the PD-1 inhibitor is RPM1-14.

In some embodiments, the PD-L1 inhibitor is selected from the group consisting of durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, KY-1003, their biosimilars , biobetters and bioequivalents.

In some embodiments, the PD-L1 inhibitor is selected from the group consisting of durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, CA-327, STI-1014, KY- 1003, their biosimilars, biobetters and bioequivalents.

In some embodiments, the PD-L1 inhibitor is selected from the group consisting of durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-327, STI-1014, KY-1003, their biosimilars , biobetters and bioequivalents.

In some embodiments, the PD-L1 inhibitor is selected from the group consisting of durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, and KY-1003.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is selected from the compounds disclosed in US2015291549, WO16039749, WO15034820 and US2014294898 (BRISTOL MYERS SQUIBB CO), which are hereby incorporated by reference.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is selected from compounds disclosed in WO14151634, WO15160641, WO16039749, WO16077518, WO16100608, WO16149351, WO2016057624, WO2016100285, US2016 194307, US2016222060 and US2014294898 (BRISTOL MYERS SQUIBB CO), which most incorporated by reference.

In some embodiments, the small molecule PD-1/PD-L1 inhibitor is selected from the compounds or pharmaceutical compositions disclosed in WO 2018/005374 filed by ChemoCentryx June 26, 2017, the contents of which are incorporated herein for all purposes.

In some embodiments, a CCR2 chemokine receptor antagonist and a PD-1 inhibitor or PD-L1 inhibitor are co-administered.

In other embodiments, the CCR2 chemokine receptor antagonist and the PD-1 inhibitor or PD-L1 inhibitor are formulated for sequential use.

In some embodiments, a tumor may be a malignant or potentially malignant neoplasm or mass of tissue of any size, and includes primary tumors and secondary neoplasms. A solid tumor may be an abnormal growth or mass of tissue that does not contain cysts or areas of fluid.

In some embodiments, the use of the compounds, agents, and compositions of the present invention may reduce or reduce tumor burden, tumor mass, tumor size, and/or number of tumors in a subject. In some instances, the compounds, agents, and compositions may prevent or minimize tumor metastasis. In other instances, the compounds, agents, and compositions may cause or enhance tumor necrosis.

In some embodiments, the use of the compounds, agents, and compositions of the present invention may result in partial response or complete response (progression-free survival), delay disease progression, and/or improve overall survival. In some instances, the compounds, agents, and compositions can increase the duration of overall response to treatment, promote tumor regression, cancer regression, or disease stabilization, and/or provide clinical benefit. In other cases, the compounds, agents and compositions can reduce the severity of at least one symptom of the disease, increase the frequency and duration of asymptomatic periods of the disease, or prevent impairment or disability due to cancer. In some cases, cancer development or cancer recurrence can be reduced.

Cancer typically includes any of a variety of malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissues and metastasize to new areas of the body. Non-limiting examples of various types of cancer suitable for treatment using the compositions of the present invention include ovarian cancer, breast cancer, lung cancer (such as non-small cell lung cancer), bladder cancer, thyroid cancer, liver cancer, pleural cancer, pancreatic cancer. cancer of the cervix, prostate cancer, testicular cancer, colon cancer, anal cancer, colorectal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gallbladder cancer, rectal cancer, appendix cancer, small intestine cancer colon, stomach cancer, kidney cancer (i.e. renal cell carcinoma), central nervous system malignancies, skin cancer, choriocarcinomas, head and neck cancer, bone cancer, osteosarcoma, fibrosarcoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma , neuroblastoma, glioma, melanoma, leukemia (eg, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, or hairy cell leukemia), lymphoma (eg, non-Hodgkin's lymphoma, Hodgkin's lymphoma, B-cell lymphoma, or Burkitt's lymphoma), and multiple myeloma.

Additional examples of cancers include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, squamous cell carcinoma, base all-cell carcinoma, sweat gland carcinoma , sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, biliary tract carcinoma, choriocarcinoma, seminoma, fetal carcinoma, Wilms tumor, testicular tumor, lung cancer, small cell lung cancer, but not limited to bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

In some embodiments, the cancer is lung cancer (eg, non-small cell lung cancer), melanoma, epithelial cancer (eg, prostate cancer, ovarian cancer, breast cancer), or hemoblastosis (eg, leukemia, lymphoma, multiple myeloma).

In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is pancreatic cancer.

A. CCR2 antagonists

In some embodiments, the CCR2 antagonists are a small molecule inhibitor of CCR2 having formula (I):

or a pharmaceutically acceptable salt, hydrate, stereoisomer or rotamer thereof; where A represents C(R ⁵ )(R ⁶ ) or N(R ⁵ );

each index m and n is independently an integer from 0 to 2, and m + n is ≤ 3;

R ¹ is selected from the group consisting of aryl, aryl-C _1-4 -alkyl, heteroaryl and heteroaryl-C _1-4 -alkyl, where the heteroaryl moiety has 1-3 heteroatoms as ring members selected from N, O and S ; and where said aryl and heteroaryl groups or moieties are optionally substituted with 1 to 5 R ^x substituents;

R ² is selected from the group consisting of H, C _1-8 alkyl, C _3-8 cycloalkyl, C _3-8 cycloalkyl-C _1-4 alkyl, aryl, aryl-C _1-4 alkyl, heteroaryl and heteroaryl-C _{1 -4} alkyl, where the heteroaryl portion has 1-3 heteroatoms as ring members selected from N, O and S; and where said aryl and heteroaryl groups or moieties are optionally substituted with 1 to 4 R ^x substituents;

or, optionally, R ¹ and R ² are combined with the nitrogen atom to which they are each attached to form a 6-11 membered monocyclic or fused bicyclic-heterocyclic or heteroaryl ring, where -NR ¹ R ² is optionally further substituted from 1 to 4 R ^x substituents;

R ³ is selected from the group consisting of H, C _1-8 -alkyl, C _3-8 -cycloalkyl and C _3-8 -cycloalkyl-C _1-4 alkyl, each of which is optionally substituted with 1-3 substituents R ^y ;

R ⁴ is selected from the group consisting of H, C _1-8 alkyl optionally substituted with 1 to 2 R ^y , and —CO ₂ H;

R ⁵ is selected from the group consisting of C _1-8 alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, C _3-8 cycloalkyloxy, C _3-8 cycloalkyl-C _1-4 alkyl, C _1-8 alkylamino, di-C _1-8 alkylamino, aryl, aryloxy, arylamino, aryl-C _1-4 alkyl, heteroaryl, heteroaryloxy, heteroarylamino and heteroaryl-C _1-4 alkyl, each optionally substituted with 1 to 5 R ^z substituents;

R ⁶ is selected from the group consisting of H, F, OH, C _1-8 alkyl and C _1-8 alkoxy, where the C _1-8 alkyl and C _1-8 alkoxy groups are optionally substituted with 1-3 R ^z substituents;

or, optionally, R ⁵ and R ⁶ are connected to form a spirocyclic 5- or 6-membered cycloalkyl ring which is optionally unsaturated and has a fused aryl group which is optionally substituted with 1-4 R ^z substituents;

each R ^x is independently selected from the group consisting of halogen, -CN, -R ^c , -CO ₂ R ^a , -CONR ^a R ^b , -C(O)R ^a , -OC(O)NR ^a R ^b , - NR ^b C(O)R ^a , -NR ^b C(O) ₂ R ^c , -NR ^a -C(O)NR ^a R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a R ^b , -OR ^a , -OX ¹ -OR ^a , -OX ¹ -NR ^a R ^b , -OX ¹ -CO ₂ R ^a , -OX ¹ -CONR ^a R ^b , -X ¹ -OR ^a , -X ¹ - NR ^a R ^b , -X ¹ -CO ₂ R ^a , -X ¹ -CONR ^a R ^b , -SF ₅ , -S(O) ₂ NR ^a R ^b , and 5- or 6-membered aryl or heteroaryl, where each X ¹ is C _1-4 alkylene; each R ^a and R ^b is independently selected from hydrogen, C _1-8 alkyl and C _1-8 haloalkyl or, when attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a five or six membered ring having 0 to 2 additional heteroatoms as ring members selected from N, O or S and optionally substituted with an oxo group; each R ^c is independently selected from the group consisting of C _1-8 alkyl, C _1-8 haloalkyl and C _3-6 cycloalkyl; and optionally, when two R ^x substituents are on adjacent atoms, combine to form a fused five- or six-membered carbocyclic ring, and wherein the aryl or heteroaryl groups are optionally substituted with 1-3 members selected from halogen, hydroxyl, C _1-4 alkyl C _{1 -4} alkoxy, C _1-4 haloalkyl and C _1-4 haloalkoxy;

each R ^y is independently selected from the group consisting of halogen, -CN, -R ^f , -CO ₂ R ^d , -CONR ^d R ^e , -C(O)R ^d , -OC(O)NR ^d R ^e , - NR ^e C(O)R ^d , -NR ^e C(O) ₂ R ^f , -NR ^d C(O)NR ^d R ^e , -NR ^d C(O)NR ^d R ^e , -NR ^d R ^e , -OR ^d , and -S(O) ₂ NR ^d R ^e ; where each R ^d and R ^e is independently selected from hydrogen, C _1-8 alkyl and C _1-8 haloalkyl, or when attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a five- or six-membered ring , having from 0 to 2 additional heteroatoms as ring members selected from N, O or S; each R ^f is independently selected from the group consisting of C _1-8 alkyl, C _1-8 haloalkyl and C _3-6 cycloalkyl;

each R ^z is independently selected from the group consisting of halogen, -CN, -R ⁱ , -CO ₂ R ^g , -CONR ^g R ^h , -C(O)R ^g , -OC(O)NR ^g R ^h , - NR ^h C(O)R ^g , -NR ^h C(O) ₂ R ⁱ , -NR ^g C(O)NR ^g R ^h , -NR ^g R ^h , -OR ^g , -S(O) ₂ NR ^g R ^h , -X ¹ -R ^j , -X ¹ -NR ^g R ^h , -X ¹ -CONR ^g R ^h , -X ¹ -NR ^h C(O)R ^g , -NHR ^j , -NHCH ₂ R ^j and tetrazole; where each R ^g and R ^h are independently selected from hydrogen, C _1-8 alkyl, C _3-6 cycloalkyl and C _1-8 haloalkyl, or when they are attached to the same nitrogen atom, they can be combined with a nitrogen atom with forming a five or six membered ring having 0 to 2 additional heteroatoms as ring members selected from N, O or S and optionally substituted with one or two oxo groups; each R ⁱ is independently selected from the group consisting of C _1-8 alkyl, C _1-8 haloalkyl and C _3-6 cycloalkyl; and each R ^j is selected from the group consisting of C _3-6 cycloalkyl, pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuranyl and tetrahydropyranyl.

It should be understood that when R ¹ and R ² are combined with the nitrogen atom to which each is attached to form a 6-11 membered monocyclic or fused bicyclic-heterocyclic ring, the 6-11 membered monocyclic or fused bicyclic-heterocyclic ring includes monocyclic heterocyclic rings fused to an aryl or heteroaryl ring.

In Formula I, R ³ is in one embodiment selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, and cyclobutylmethyl.

In the descriptions given in this application, a person skilled in the art will understand that the wavy line crossing the bond is intended to identify the point of attachment of a given substituent or group to the rest of the molecule.

As noted above, the subscript m and n are each integers selected from 0, 1, and 2, and m + n is ≤ 3. When the subscript is 0, one skilled in the art will understand that a vertex loop is meant ring A, but neighboring vertices of the ring on both sides of the brackets are connected by a link. Accordingly, the present invention includes structures in which a ring having A as a vertex should include:

In one selected group of embodiments, m and n are both 0. In another selected group of embodiments, m and n are both 1. In another group of selected embodiments, m is 1 and n is 0. In another group of embodiments, m is 1 , and n is 2.

In other selected embodiments, the ring having vertex A is represented by a formula selected from:

In one subset of embodiments, the compounds of formula (I) are represented as:

In formula (Ia), a number of selected embodiments are represented as formulas Ia1, Ia2, Ia3, Ia4 and Ia5.

In each of the formulas Ia, Ia1, Ia2, Ia3, Ia4 and Ia5, the marked substituents (R ¹ -R ⁶ , R ^x and R ^z ) and the indices m and n have the meanings indicated above with respect to formula I. The indices p and q have the following meanings: for Ia1, Ia4 and Ia5 index q is an integer from 0 to 5; for Ia2 and Ia4, the index p is an integer from 0 to 4; and for Ia3 and Ia5, the index p is an integer from 0 to 5.

In other selected embodiments, the compounds provided herein are represented by formulas selected from:

where each compound essentially does not contain other stereoisomers, and where the substituents (R ¹ -R ⁶ , R ^x and R ^z ) and the indices m and n have the meanings indicated above with respect to formula I. The indices p and q have the following meanings: for Ia1', Ia4' and Ia5' index q is an integer from 0 to 5; for Ia2' and Ia4' index p is an integer from 0 to 4; and for Ia3' and Ia5' the index p is an integer from 0 to 5.

In another group of embodiments of formula IA is C(R ⁵ )(R ⁶ ), where R ⁵ and R ⁶ are combined to form a ring. Selected embodiments are presented as follows:

In each of the formulas Ib, Ib1 and Ib2, the marked substituents (R ¹ -R ⁶ , R ^x and R ^z ) and the subscripts m and n have the meanings given above with respect to formula I. The subscripts p and q have the following meanings: for Ib, Ib1 and Ib2 index q is an integer from 0 to 5; for Ib1, index p is an integer from 0 to 4; and for Ib2, index p is an integer from 0 to 5.

In another group of formula embodiments, IA is NR ⁵ (see Formula Ic). Selected embodiments are presented as follows:

In each of the formulas Ic, Ic1, Ic2, Ic3, Ic4 and Ic5, the marked substituents (R ¹ -R ⁶ , R ^x and R ^z ) and the indices m and n have the meanings given above with respect to formula I. The indices p and q have the following values: for Ic1, Ic4 and Ic5 index q is an integer from 0 to 5; for Ic2 and Ic4 index p is an integer from 0 to 4; and for Ic3 and Ic5, the index p is an integer from 0 to 5.

In still other selected embodiments, the compounds provided herein are represented by formulas selected from:

where each compound essentially does not contain other stereoisomers, and where the substituents (R ¹ -R ⁶ , R ^x and R ^z ) and the indices m and n have the meanings indicated above with respect to formula I. The indices p and q have the following meanings: for Ic1', Ic4' and Ic5' index q is an integer from 0 to 5; for Ic2' and Ic4' index p is an integer from 0 to 4; and for Ic3' and Ic5' index p is an integer from 0 to 5.

In other selected embodiments, the compounds are each of I, Ia, Ia1, Ia1', Ib, Ic, Ic1 and Ic1' described above, wherein -N(R ¹ )(R ² ) is selected from:

Still other selected embodiments are provided in each of I, Ia, Ia1, Ia1', Ib, Ic, Ic1 and Ic1' described above, where -N(R1)(R2) is selected from:

Still other selected embodiments are presented in each of I, Ia, Ia1, Ia1', Ib, Ic, Ic1 and Ic1' described above, where -N(R1)(R2) is selected from:

In some embodiments, the invention provides compounds of formulas I, Ia, Ia2, Ia3, Ia2' and Ia3', where A is C(R5)(R6) or shown in the formula as C(R5)(R6), where R5 is selected from aryl, aryloxy, arylamino, aryl-C1-4 alkyl, heteroaryl, heteroaryloxy, heteroarylamino and heteroaryl-C _1-4 alkyl, wherein the aryl or heteroaryl groups or moieties are selected from:

In other selected embodiments, the invention provides compounds of formulas I, Ia, Ia2, Ia3, Ia2' and Ia3', where A is C(R5)(R6) or shown in the formula as C(R ⁵ )(R ⁶ ), where R ⁵ is selected from aryl, aryloxy, arylamino and aryl-C _1-4 alkyl, where the aryl group or moiety is selected from:

In other selected embodiments, the invention provides compounds of formulas I, Ia, Ia2, Ia3, Ia2' and Ia3', where A is C(R5)(R6) or shown in the formula as C(R5)(R6), where R5 is selected from heteroaryl, heteroaryloxy, heteroarylamino and heteroaryl-C _1-4 alkyl, wherein the heteroaryl group or moiety is selected from:

In some embodiments, compounds of formulas I, Ic, Ic2, Ic3, Ic2', and Ic3' are provided, where A is N(R5) or shown in the formula as N(R5), where R5 is selected from aryl, aryl-C1-4 alkyl, heteroaryl and heteroaryl-C1-4 alkyl, wherein the aryl or heteroaryl groups or moieties are selected from Group 1 above. In some selected embodiments, compounds of formulas I, Ic, Ic2, Ic3, Ic2', and Ic3' are provided, where A is N(R ⁵ ) or denoted in the formula as N(R ⁵ ), where R ⁵ is selected from aryl and aryl -C _1-4 alkyl, where the aryl group or part is selected from Subgroup 1a above. In other selected embodiments, the invention provides compounds of formulas I, Ic, Ic2, Ic3, Ic2' and Ic3', where A is N(R ⁵ ) or shown in the formula as N(R ⁵ ), where R ⁵ is selected from heteroaryl and heteroaryl-C _1-4 alkyl, where the heteroaryl group or part is selected from Subgroup 1b above.

In some embodiments, the CCR2 antagonist has a formula selected from the group consisting of:

or their pharmaceutically acceptable salt.

In some embodiments, the CCR2 antagonist has the formula

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist has the formula

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR2 antagonist is selected from the compounds or pharmaceutical compositions disclosed in US2016/0340356, derived from Application No. 15/158,713, filed May 19, 2016 by ChemoCentryx, the contents of which are incorporated herein for any purpose.

In some embodiments, the CCR2 chemokine receptor antagonist is selected from the group consisting of AZ889, AZD2423, INCB-8761, MK-0812, BMS-813160, INCB-003284, PF-04634817, BMS-741672, cenicriviroc, CCX-140.

B. PD-1 inhibitors and PD-L1 inhibitors

The methods, compositions, and kits provided herein include immune checkpoint inhibitors, such as inhibitors (agents) of the PD-1/PD-L1 pathway. The PD-1 and/or PD-L1 inhibitors of the present invention include small molecules and antibodies.

In some embodiments, the PD-L1 inhibitor may be durvalumab or atezolizumab or avelumab or BMS-936559 (MDX-1105) or ALN-PDL or TSR-042 or KD-033 or CA-170 or CA -327 or STI-1014 or MEDI-0680 or KY-1003.

In some embodiments, the PD-L1 inhibitor may be durvalumab or atezolizumab or avelumab or BMS-936559 (MDX-1105) or ALN-PDL or TSR-042 or KD-033 or CA-170 or STI -1014 or MEDI-0680 or KY-1003. Durvalmab (MEDI4736) is a human monoclonal antibody directed against PD-L1. Atrexolizumab (MPDL3280A) is a fully humanized, engineered anti-PD-L1 IgG1 monoclonal antibody. Avelumab (MSB0010718C) is a fully humanized, engineered anti-PD-L1 IgG1 monoclonal antibody. BMS-936559 (MDX-1105) is a fully human anti-PD-L1 IgG4 monoclonal antibody. ALN-PDL is an inhibitory RNA (RNAi) targeted to PD-L1. TSR-042 refers to an engineered chimeric antibody that is directed against the PD-1/PD-L1 pathway. KD-033 refers to a bifunctional anti-PD-L1/IL-15 fusion protein in which an anti-PD-L1 antibody is linked in its tail to an IL-15 cytokine via the sushi domain of the IL-15 receptor. CA-170 refers to a small molecule antagonist of PD-L1 and VISTA. STI-1014 refers to an antibody against PD-L1. KY-1003 is a monoclonal antibody against PD-L1. CA-327 refers to a small molecule PD-L1 and TIM3 antagonist.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is selected from the group consisting of durvalumab, atezolizumab, pembrolizumab, nivolumab, AP-106, AP-105, MSB-2311, CBT-501, avelumab, AK-105, IO-102, IO-103, PDR-001, CX-072, SHR-1316, JTX-4014, GNS-1480, recombinant humanized anti-PD1 mAb (Shanghai Junshi Biosciences), REGN-2810, pelareorepa, SHR-1210, PD1/PDL1 inhibitor vaccine (THERAVECTYS), BGB-A317, recombinant humanized anti-PD-1 mAb (Bio-Thera Solutions); Probody™ targeting PD-1 (CytomX); XmAb-20717, FS-118, PSI-001, SN-PDL01, SN-PD07, PD-1-modified antitumor effector lymphocytes (Sangamo Therapeutics), PRS-332, FPT-155, jienuo mAb (Genor Biopharma), TSR- 042, REGN-1979, REGN-2810, resminostat, FAZ-053, PD-1/CTLA-4 bispecific antibody (MacroGenics), MGA-012, MGD-013, M-7824, PD-1-based bispecific antibody (Beijing Hanmi Pharmaceutical), AK-112, AK-106, AK-104, AK-103, BI-754091, ENUM-244C8, MCLA-145, MCLA-134, anti-PD1 oncolytic monoclonal antibody (Transgene SA), AGEN-2034, IBI-308, WBP-3155, JNJ-63723283, MEDI-0680, SSI-361, CBT-502, anti-PD-1 bispecific antibody, anti-PD-1/LAG-3 (TESARO) dual targeting monoclonal antibody, dual targeting monoclonal antibody targeting against PD-1/TIM-3 (TESARO), PF-06801591, LY-3300054, BCD-100, STI-1110, pembrolizumab biosimilar, nivolumab biosimilar, PD-L1-TGF-beta therapy, KY-1003, STI -1014, GLS-010, AM-0001, GX-P2, KD-033, PD-L1/BCMA bispecific antibody (Immune Pharmaceuticals), anti-PD-1/Ox40 bispecific antibody (Immune Pharmaceuticals), BMS-936559, anti- PD-1/VEGF-A DARPins (Molecular Partners), mDX-400, ALN-PDL, PD-1 inhibitory peptide (Aurigene), siRNA-loaded dendritic cell vaccine (Alnylam Pharmaceuticals), GB-226, PD-L1- targeted immunotherapy based on CAR-TNK (TNK Therapeutics/NantKwest), INSIX RA, INDUS-903, AMP-224, anti-CTLA-4/anti-PD-1 bispecific humanized antibody (Akeso Biopharma), B7-H1 vaccine (State Cancer Biology Key Laboratory/Fourth Military Medical University) and GX-D1.

In some embodiments, the PD-1 inhibitor may be pembrolizumab, or nivolumab, or IBI-308, or mDX-400, or BGB-108, or MEDI-0680, or SHR-1210, or PF-06801591, or PDR-001, or GB-226 or STI-1110. Nivolumab (also known as OPDIVO™, MDX-1106, BMS-936558 and ONO-4538) is a human anti-PD-1 IgG4 monoclonal antibody. Pembrolizumab (also known as KEYTRUDA®, lambrolizumab, and MK-34) is a humanized monoclonal antibody of the kappa IgG4 isotype against PD-1. IBI-308 refers to a monoclonal antibody directed to PD-1. mDX-400 refers to a mouse anti-PD-1 antibody. BGB-108 is a humanized anti-PD-1 monoclonal antibody. MEDI-0680 (AMP-514) is a humanized anti-PD-1 IgG4 monoclonal antibody. SHR-1210 refers to a monoclonal antibody against PD-1. PF-06801591 is a monoclonal antibody against PD-1. PDR-001 refers to a monoclonal antibody against PD-1. GB-226 refers to a monoclonal antibody against PD-1. STI-1110 refers to a monoclonal antibody against PD-1.

In some embodiments, the PD-1 inhibitor is RPM1-14.

In some embodiments, the PD-1 inhibitor is an antibody selected from nivolumab, pembrolizumab, and pidilizumab.

The anti-PD-1 antibodies and antibody fragments described herein encompass proteins having amino acid sequences that differ from those of the antibodies described, but which retain the ability to bind PD-1.

In some embodiments, anti-PD-1 antibodies include bispecific antibodies and antibody-like therapeutic proteins, including DARTs®, DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives, and the like, that bind to PD-1.

The anti-PD-L1 antibodies and antibody fragments described herein encompass proteins having amino acid sequences that differ from those of the antibodies described, but which retain the ability to bind PD-L1. Such antibody variants and fragments may contain one or more amino acid insertions, deletions, or substitutions from the original sequence, but exhibit biological activity that is substantially equivalent or substantially bioequivalent to that of the described antibodies.

In some embodiments, anti-PD-L1 antibodies include bispecific antibodies and antibody-like therapeutic proteins, including DARTs®, DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives, and the like, that bind to PD-L1.

Non-limiting examples of additional inhibitors of the PD-1/PD-L1 pathway are described, for example, in Chen and Han, Jour Clin Invest, 2015, 125 (9): 3384-3391, US Pat. U.S. Patent Application Publication No. 2014/0341917; 2015/0203580 and 2015/0320859; International Patent Application Publication No. WO2015/026634.

A biological product, such as an antibody or fragment thereof, is considered a biosimilar if, for example, the biological product has a high similarity to an already FDA-approved biological product, known as a reference product. The biosimilar has no clinically significant differences in terms of safety and efficacy from the reference product. A biosimilar may also have the same mode of action, route of administration, dosage form, and dosage size as its reference product.

Two biological products, such as antibodies or fragments thereof, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives, the rate and extent of absorption of which do not show a significant difference when administered at the same molar dose under similar experimental conditions, in single or multiple dosage. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and yet they can be considered bioequivalent, since such differences in absorption rate are intentional and reflected in the labeling, are not significant for to achieve effective concentrations of drugs in the body, such as chronic use, and are considered medically negligible for a particular investigational drug.

In some embodiments, two biological products (eg, two antibodies or fragments thereof) are bioequivalent if there are no clinically significant differences in their safety, purity, or potency.

In other embodiments, two biological products (e.g., two antibodies or fragments thereof) are bioequivalent if the patient can switch one or more times between the reference product and the biological product without an expected increase in the risk of side effects, including a clinically significant change in immunogenicity or a decrease in potency. compared with continuing therapy without such a switch.

In other embodiments, two biological products (eg, two antibodies or fragments thereof) are bioequivalent if they both act through a common mechanism of action for the conditions of use, to the extent such mechanisms are known.

Bioequivalence can be demonstrated by in vivo and/or in vitro methods. Bioequivalence measurements include, for example, (a) an in vivo test in humans or other mammals, in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum, or other body fluid as a function of time; (b) an in vitro test that is correlated with in vivo human bioavailability data and is reasonably predictive; (c) an in vivo test in humans or other mammals, in which the corresponding acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of the antibody.

Biobetter variants of the antibodies described herein may be based on an existing reference antibody specific for the target antigen, e.g. its target antigen and/or has become associated with a different epitope than the reference antibody, or has received more desirable therapeutic efficacy, expression and/or biophysical characteristics.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is a small molecule PD-1/PD-L1 inhibitor having the formula:

или

.

or

.

In some embodiments, the PD-1 and/or PD-L1 inhibitor is a small molecule PD-1/PD-L1 inhibitor having formula (II)

or a pharmaceutically acceptable salt thereof; wherein:

R ¹ is selected from the group consisting of halogen, C _5-8 cycloalkyl, C _6-10 aryl and thienyl,

where C _6-10 aryl and thienyl are optionally substituted with 1-5 substituents R ^x ;

each R ^x is independently selected from the group consisting of halogen, -CN, -R ^c , -CO ₂ R ^a , -CONR ^a R ^b , -C(O)R ^a , -OC(O)NR ^a R ^b , - NR ^b C(O)R ^a , -NR ^b C(O) ₂ R ^c , -NR ^a -C(O)NR ^a R ^b , -NR ^a R ^b , -OR ^a , -OX ¹ -OR ^a , -OX ¹ -CO ₂ R ^a , -OX ¹ -CONR ^a R ^b , -X ¹ -OR ^a , -X ¹ -NR ^a R ^b , -X ¹ -CO ₂ R ^a , -X ¹ -CONR ^a R ^b , -SF ₅ , and -S(O) ₂ NR ^a R ^b , where each X ¹ is C _1-4 alkylene;

each R ^a and R ^b is independently selected from hydrogen, C _1-8 alkyl and C _1-8 haloalkyl or, when attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a five or six membered ring having 0 to 2 additional heteroatoms as ring members selected from N, O or S, where the five- or six-membered ring is optionally substituted with an oxo group; each R ^c is independently selected from the group consisting of C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, and C _1-8 haloalkyl; and optionally, when two R ^x substituents are on adjacent atoms, they combine to form a fused five-, six-, or seven-membered carbocyclic or heterocyclic ring optionally substituted with 1-3 substituents independently selected from halogen, oxo, C _1-8 haloalkyl and C _1-8 alkyl;

each R ^2a , R ^2b and R ^2c is independently selected from the group consisting of H, halogen, -CN, -R ^d , -CO ₂ R ^e , -CONR ^e R ^f , -C(O)R ^e , -OC( O)NR ^e R ^f , -NR ^f C(O)R ^e , -NR ^f C(O) ₂ R ^d , -NR ^e -C(O)NR ^e R ^f , -NR ^e R ^f , -OR ^e , -OX ² -OR ^e , -OX ² -NR ^e R ^f , -OX ² -CO ₂ R ^e , -OX ² -CONR ^e R ^f , -X ² -OR ^e , -X ² -NR ^e R ^f , -X ² -CO ₂ R ^e , -X ² -CONR ^e R ^f , -SF ₅ , -S(O) ₂ NR ^e R ^f , C _6-10 aryl and C _5-10 heteroaryl, where each X ² is C _1-4 alkylene; each R ^e and R ^f is independently selected from hydrogen, C _1-8 alkyl and C _1-8 haloalkyl, or when attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a five- or six-membered ring, having 0 to 2 additional heteroatoms as ring members selected from N, O and S and optionally substituted with an oxo group; each R ^d is independently selected from the group consisting of C _1-8 alkyl, C _2-8 alkenyl and C _1-8 haloalkyl;

R ³ is selected from the group consisting of -NR ^g R ^h and C _4-12 heterocyclyl, where C _4-12 heterocyclyl is optionally substituted with 1-6 R ^y ;

each R ^y is independently selected from the group consisting of halogen, -CN, -R ⁱ , -CO ₂ R ^j , -CONR ^j R ^k , -CONHC _1-6 alkyl-OH, -C(O)R ^j , -OC (O)NR ^j R ^k , -NR ^j C(O)R ^k , -NR ^j C(O) ₂ R ^k , CONOH, PO ₃ H ₂ , -NR ^j -C _1-6 alkyl-C(O) ₂ R ^k , -NR ^j C(O)NR ^j R ^k , -NR ^j R ^k , -OR ^j , -S(O) ₂ NR ^j R ^k , -OC _1-6 alkyl-OR ^j , -OC _{1 -6} alkyl-NR ^j R ^k , -OC _1-6 alkyl-CO ₂ R ^j , -OC _1-6 alkyl-CONR ^j R ^k , -C _1-6 alkyl-OR ^j , -C _1-6 alkyl- NR ^j R ^k , -C _1-6 alkyl-CO ₂ R ^j , -C _1-6 alkyl-CONR ^j R ^k , and SF ₅ ,

where the C _1-6 alkyl portion of R ^y is optionally further substituted with OH, SO ₂ NH ₂ , CONH ₂ , CONOH, PO ₃ H ₂ , COO-C _1-8 alkyl, or CO ₂ H, where each R ^j and R ^k are independently selected from hydrogen, C _1-8 alkyl optionally substituted with 1-2 substituents selected from OH, SO ₂ NH ₂ , CONH ₂ , CONOH, PO ₃ H ₂ , COO-C _1-8 alkyl or CO ₂ H, and C _{1 -8} haloalkyl optionally substituted with 1-2- substituents selected from OH, SO ₂ NH ₂ , CONH ₂ , CONOH, PO ₃ H ₂ , COO-C _1-8 alkyl or CO ₂ H or when attached to one and on the same nitrogen atom, R ^j and R ^k can be combined with a nitrogen atom to form a five- or six-membered ring having 0 to 2 additional heteroatoms as ring members selected from N, O or S and optionally substituted with an oxo group; each R ⁱ is independently selected from the group consisting of -OH, C _1-8 alkyl, C _2-8 alkenyl, and C _1-8 haloalkyl, each of which may be optionally substituted with OH, SO ₂ NH ₂ , CONH ₂ , CONOH, PO ₃ H ₂ , COO-C _1-8 alkyl or CO ₂ H;

R ^g is selected from the group consisting of H, C _1-8 haloalkyl and C _1-8 alkyl;

R ^h is selected from -C _1-8 alkyl, C _1-8 haloalkyl, C _1-8 alkyl-COOH, C _1-8 alkyl-OH, C _1-8 alkyl-CONH ₂ , C _1-8 alkyl-SO ₂ NH ₂ , C _1-8 alkyl-PO ₃ H ₂ , C _1-8 alkyl-CONOH, C _1-8 alkyl-NR ^h1 R ^h2 , -C(O)-C _1-8 alkyl, -C(O) -C _1-8 alkyl-OH, -C(O)-C _1-8 alkyl-COOH, C _3-10 cycloalkyl, -C _3-10 cycloalkyl-COOH, -C _3-10 cycloalkyl-OH, C _{4- 8} heterocyclyl, -C _4-8 heterocyclyl-COOH, -C _4-8 heterocyclyl-OH, -C _1-8 alkyl-C _4-8 heterocyclyl, -C _1-8 alkyl-C _3-10 cycloalkyl, C _{5- 10} heteroaryl, -C _1-8 alkyl-C _5-10 heteroaryl, C ₁₀ carbocyclyl, -C _1-8 alkyl-C _6-10 aryl, -C _1-8 alkyl-(C=O)-C _6-10 aryl, -C _1-8 alkyl-NH(C=O)-C _1-8 alkenyl, -C _1-8 alkyl-NH(C=O)-C _1-8 alkyl, -C _1-8 alkyl-NH (C=O)-C _1-8 alkynyl, -C _1-8 alkyl-(C=O)-NH-C _1-8 alkyl-COOH, and -C _1-8 alkyl-(C=O)-NH -C _1-8 alkyl-OH optionally substituted with CO ₂ H; or

R ^h in combination with the N to which it is attached is a mono-, di- or tripeptide containing 1-3 natural amino acids and 0-2 non-natural amino acids, where

unnatural amino acids have an alpha carbon substituent selected from the group consisting of C _2-4 hydroxyalkyl, C _1-3 alkyl guanidinyl and C _1-4 alkyl heteroaryl,

the alpha carbon of each natural or non-natural amino acid is optionally further substituted with a methyl group, and

the terminal part of the mono-, di - or tripeptide is selected from the group consisting of C(O)OH, C(O)OC _1-6 alkyl and PO ₃ H ₂ where

R ^h1 and R ^h2 are each independently selected from the group consisting of H, C _1-6 alkyl and C _1-4 hydroxyalkyl;

C _1-8 alkyl portions of R ^h are optionally further substituted with 1-3 substituents independently selected from OH, COOH, SO ₂ NH ₂ , CONH ₂ , CONOH, COO-C _1-8 alkyl, PO ₃ H ₂ and C _5-6 heteroaryl optionally substituted with 1-2 C _1-3 alkyl substituents,

The C ₁₀ carbocyclyl, C _5-10 heteroaryl, and C _6-10 aryl portions of R ^h are optionally substituted with 1-3 substituents independently selected from OH, B(OH) ₂ , COOH, SO ₂ NH ₂ , CONH ₂ , CONOH, PO ₃ H ₂ , COO-C _1-8 alkyl, C _1-4 alkyl, C _1-4 alkyl-OH, C _1-4 alkyl-SO ₂ NH ₂ , C _1-4 alkyl CONH ₂ , C _1-4 alkyl- CONOH, C _1-4 alkyl-PO ₃ H ₂ , C _1-4 alkyl-COOH and phenyl; And

The C _4-8 heterocyclyl and C _3-10 cycloalkyl portions of R ^h are optionally substituted with 1-4 R ^w substituents;

each substituent R ^w is independently selected from C _1-4 alkyl, C _1-4 alkyl-OH, C _1-4 alkyl-COOH, C _1-4 alkyl-SO ₂ NH ₂ , C _1-4 alkyl-CONH ₂ , C _1-4 alkyl-CONOH, C _1-4 alkyl-PO ₃ H, OH, COO-C _1-8 alkyl, COOH, SO ₂ NH ₂ , CONH ₂ , CONOH, PO ₃ H ₂ and oxo;

R ⁴ is selected from the group consisting of OC _1-8 alkyl, OC _1-8 haloalkyl, OC _1-8 alkyl-R ^z , C _6-10 aryl, C _5-10 heteroaryl, -OC _1-4 alkyl-C _{6 -10} aryl and -OC _1-4 alkyl-C _5-10 heteroaryl, where C _6-10 aryl and C _5-10 heteroaryl are optionally substituted with 1-5 R ^z ;

each R ^z is independently selected from the group consisting of halogen, -CN, -R ^m , -CO ₂ R ⁿ , -CONR ⁿ R ^p , -C(O)R ⁿ , -OC(O)NR ⁿ R ^p , - NR ⁿ C(O)R ^p , -NR ⁿ C(O) ₂ R ^m , -NR ⁿ -C(O)NR ⁿ R ^p , -NR ⁿ R ^p , -OR ⁿ , -OX ³ -OR ⁿ , -OX ³ -NR ⁿ R ^p , -OX ³ -CO ₂ R ⁿ , -OX ³ -CONR ⁿ R ^p , -X ³ -OR ⁿ , -X ³ -NR ⁿ R ^p , -X ³ -CO ₂ R ⁿ , -X ³ -CONR ⁿ R ^p , -SF ₅ , -S(O) ₂ R ⁿ R ^p , -S(O) ₂ NR ⁿ R ^p , and 3-7-membered carbocyclic or 4-7-membered a heterocyclic ring in which a 3-7 membered carbocyclic or 4-7 membered heterocyclic ring is optionally substituted with 1-5 R ^t , where each R ^t is independently selected from the group consisting of C _1-8 alkyl, C _1-8 haloalkyl, -CO ₂ R ⁿ , -CONR ⁿ R ^p , -C(O)R ⁿ , -OC(O)NR ⁿ R ^p , -NR ⁿ C(O)R ^p , -NR ⁿ C(O) ₂ R ^m , -NR ⁿ -C(O)NR ⁿ R ^p , -NR ⁿ R ^p , -OR ⁿ , -OX ³ -OR ⁿ , -OX ³ -NR ⁿ R ^p , -OX ³ -CO ₂ R ⁿ , - OX ³ -CONR ⁿ R ^p , -X ³ -OR ⁿ , -X ³ -NR ⁿ R ^p ,- X ³ -CO ₂ R ⁿ , -X ³ -CONR ⁿ R ^p , -SF ₅ , and -S( O) _2NR ⁿ R ^p ;

where each X3 is C _1-4 alkylene; each R ⁿ and R ^p is independently selected from hydrogen, C _1-8 alkyl and C _1-8 haloalkyl or, when attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a five- or six-membered ring, having 0 to 2 additional heteroatoms as ring members selected from N, O or S and optionally substituted with an oxo group; each R ^m is independently selected from the group consisting of C _1-8 alkyl, C _2-8 alkenyl and C _1-8 haloalkyl; and optionally, when two R ^z substituents are on adjacent atoms, they combine to form a fused five- or six-membered carbocyclic or heterocyclic ring, optionally substituted with an oxo group;

n is 0, 1, 2 or 3;

each R ⁵ is independently selected from the group consisting of halogen, -CN, -R ^q , -CO ₂ R ^r , -CONR ^r R ^s , -C(O)R ^r , -OC(O)NR ^r R ^s , - NR ^r C(O)R ^s , -NR ^r C(O) ₂ R ^q , -NR ^r -C(O)NR ^r R ^s , -NR ^r R ^s , -OR ^r , -OX ⁴ -OR ^r , -OX ⁴ -NR ^r R ^s , -OX ⁴ -CO ₂ R ^r , -OX ⁴ -CONR ^r R ^s , -X ⁴ -OR ^r , -X ⁴ -NR ^r R ^s , -X ⁴ -CO ₂ R ^r , -X ⁴ -CONR ^r R ^s , -SF ₅ , -S(O) ₂ NR ^r R ^s , where each X ⁴ is C _1-4 alkylene; each R ^r and R ^s is independently selected from hydrogen, C _1-8 alkyl and C _1-8 haloalkyl or, when attached to the same nitrogen atom, they may be combined with the nitrogen atom to form a five or six membered ring having 0 to 2 additional heteroatoms as ring members selected from N, O or S and optionally substituted with an oxo group; each R ^q is independently selected from the group consisting of C _1-8 alkyl and C _1-8 haloalkyl;

R ^6a is selected from the group consisting of H, C _1-4 alkyl and C _1-4 haloalkyl;

each R ^6b is independently selected from the group consisting of F, C _1-4 alkyl, OR ^u , C _1-4 haloalkyl, NR ^u R ^v , where each R ^u and R ^v are independently selected from hydrogen, C _1-8 alkyl, and C _1-8 haloalkyl or, when attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a five- or six-membered ring having 0 to 2 additional heteroatoms as ring members selected from N, O or S, and optionally substituted with an oxo group; And

m is 0, 1, 2, 3, or 4.

In some embodiments, the small molecule PD-1/PD-L1 inhibitor is selected from the compounds or pharmaceutical compositions disclosed in WO 2018/005374 filed on June 26, 2017 by ChemoCentryx, the contents of which are incorporated herein for all purposes.

The PD-1 and/or PD-L1 inhibitors of the present invention may be formulated to slow degradation of a compound or antibody, or to minimize the immunogenicity of an antibody. Various techniques are known in the art to achieve these goals.

IV. Pharmaceutical compositions

Pharmaceutical compositions provided herein, such as those comprising compounds for modulating CCR2 activity and agents for blocking the PD-1/PD-L1 pathway, may contain a pharmaceutical carrier or diluent.

As used herein, the term "composition" is intended to cover a product comprising the specified ingredients in the specified amounts, as well as any product that is directly or indirectly formed from a combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not cause harm to the recipient.

Biological products, such as the antibodies of the present invention, may be formulated into a pharmaceutical composition containing a single antibody or antibodies, or a fragment thereof, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). The pharmaceutical composition of the invention may include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers and/or adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

Pharmaceutical compositions for use with the compounds and agents of the present invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy and drug delivery. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Typically, pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product to obtain the desired composition. In the pharmaceutical composition, the active compound of the invention is included in an amount sufficient to achieve the desired effect on the disease process or condition.

Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral administration, such as tablets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, and self-emulsifying agents as described in US Pat. No. 6,451,339, solid or soft capsules, syrups, elixirs, solutions, buccal patch, oral gel, chewing gum, chewable tablets, effervescent powder and effervescent tablets. Compositions intended for oral administration may be prepared in accordance with any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavors, colors, antioxidants, and preservatives for providing pharmaceutically elegant and palatable formulations. The tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents such as cellulose, silica, alumina, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example PVP, cellulose, PEG, starch, gelatin or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be enteric or otherwise coated, by known methods, to delay disintegration and absorption in the gastrointestinal tract and thereby provide sustained action over a longer period. For example, a release retardant material such as glyceryl monostearate or glyceryl distearate may be used. They can also be coated using the methods described in US Pat.

Compositions for oral administration may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil vehicle. such as peanut butter, liquid paraffin or olive oil. In addition, emulsions can be prepared with a water-immiscible ingredient such as oils and stabilized with surfactants such as mono-diglycerides, PEG esters and the like.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the preparation of aqueous suspensions. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersing or wetting agents can be a natural phosphatide, such as lecithin, or alkylene oxide condensates with fatty acids, such as polyoxyethylene stearate, or ethylene oxide condensates with long chain aliphatic alcohols, such as heptadecaethyleneoxyethanol, or ethylene oxide condensates with partial esters derived from fatty acids. acids and hexitol, such as polyoxyethylene sorbitan monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more colorants, one or more flavors, and one or more sweeteners, such as sucrose or saccharin.

Oil suspensions can be prepared by suspending the active ingredient in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweeteners such as those mentioned above and flavoring agents may be added to provide a palatable oral formulation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional adjuvants may also be present, such as sweeteners, flavors and colors.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin, or mixtures thereof. Suitable emulsifying agents may be natural gums such as gum arabic or gum tragacanth, natural phosphatides such as from soybeans, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweeteners and flavors.

Syrups and elixirs may be formulated with sweeteners such as glycerin, propylene glycol, sorbitol or sucrose. Such compositions may also contain softening, preservative and flavoring and coloring agents. Oral solutions can be prepared in combination with, for example, cyclodextrin, PEG and surfactants.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oily suspension. This suspension may be prepared in accordance with the prior art using those suitable dispersing or wetting agents and suspendida agents that have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fatty oils are commonly used as a solvent or suspending medium. Any aseptic fixed oil can be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds and agents of the present invention can also be used in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols. In addition, the compounds can be administered by delivery to the eye via solutions or ointments. In addition, transdermal delivery of the subject compounds can be carried out by means of iontophoretic patches and the like. For topical use, creams, ointments, gels, solutions or suspensions, etc. containing the compounds of the present invention are used. Used in this application, the term "topical application" also includes the use of means for washing and rinsing the mouth.

The compounds of this invention may also be associated with a carrier which is suitable polymers as targeted drug carriers. Such polymers may include polyvinylpyrrolidone, a pyran copolymer, polyhydroxypropyl methacrylamide phenol, polyhydroxyethyl aspartamide phenol or polyethylene oxide polylysine substituted with palmitoyl residues. In addition, the compounds of the invention may be associated with a carrier which is a class of biodegradable polymers used to achieve controlled drug release, e.g. , polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels. Polymers and semi-permeable polymer matrices can be molded into molded articles such as valves, stents, tubes, prostheses, and the like. In one embodiment, a compound of the invention is associated with a polymeric or semi-permeable polymeric matrix that is formed into a stent or stent graft.

The compounds and agents of the invention may be formulated to be deposited in a medical device, which may include any of a variety of conventional grafts, stents, including stent grafts, catheters, balloons, baskets, or other device that may be deployed or permanently implanted in body cavities. As a specific example, it is necessary to have devices and methods that allow the compounds of the invention to be delivered to the area of the body that is being treated by the interventional technique. For example, the compound and agent can be delivered to the tumor or the tumor microenvironment.

In some embodiments, compounds and agents may be deposited in a medical device, such as a stent, and delivered to a treatment site to treat a body part. Stents are used as delivery vehicles for therapeutic agents. Intravascular stents are usually permanently implanted in coronary or peripheral vessels. Stent designs include those described in US Pat. Stents can also be used to deliver therapeutic agents to the site of contact with the vasculature, as disclosed, for example, in US patent No. 5102417 and international patent applications No. WO 91/12779 and WO 90/13332, US patent No. 5419760 and 5429634.

The term "deposited" means that the compound and agent are coated, adsorbed, placed or otherwise included in the device by methods known in the art. For example, the compound and agent may be embedded and released from within ("matrix type") or surrounded and released through ("reservoir type") polymeric materials that coat or surround the medical device. In the latter example, the compound and agent may be entrapped within polymeric materials or associated with polymeric materials using one or more of the techniques for obtaining such materials known in the art. In other formulations, the compound and agent may be bonded to the surface of the medical device without the need for coating through breakable bonds and release over time, may be removed by active mechanical or chemical processes, or be in a permanently immobilized form that presents an inhibitory agent at the site of implantation. .

In one embodiment, the compound and agent may be incorporated with polymeric compositions during the formation of biocompatible coatings for medical devices such as stents. Coatings made from these components are generally uniform and useful for application to a variety of implantable devices.

The polymer can be either a biostable polymer or a bioabsorbable polymer depending on the desired release rate or the desired degree of polymer stability, but a bioabsorbable polymer is preferred for this embodiment because, unlike a biostable polymer, it will not be present long after implantation, which could cause any adverse chronic local reactions. Bioabsorbable polymers that can be used include poly(L-lactic acid), polycaprolactone, polyglycolide (PGA), poly(lactide-co-glycolide) (PLLA/PGA), poly(hydroxybutyrate), poly(hydroxybutyrate-co-valerate) , polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid), poly(D-lactic acid), poly(L-lactic acid), poly(D,L-lactic acid), poly(D,L-lactide) (PLA) , poly(L-lactide) (PLLA), poly(glycolic acid-co-trimethylene carbonate) (PGA/PTMC), polyethylene oxide (PEO), polydioxanone (PDS), polyphosphoether, polyphosphoether urethane, poly(amino acids), cyanoacrylates, poly( trimethylene carbonate), poly(iminocarbonate), copoly(ester-esters) (e.g. PEO/PLA), polyalkylene oxalates, polyphosphazenes and biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen and hyaluronic acid, polyepsiloncaprolactone, polyhydroxybutyric acid, polyorthoesters , polyacetals, polydihydropyrans, polycyanoacrylates, cross-linked or amphipathic hydrogel block copolymers, and other suitable bioabsorbable polymers known in the art, but not limited to. In addition, biostable polymers with relatively low chronic tissue response can be used, such as polyurethanes, silicones, and polyesters, and other polymers can also be used if they can be dissolved and cured or polymerized on a medical device, such as polyolefins, polyisobutylene, and ethylene. - alpha-olefin copolymers; acrylic polymers and copolymers, vinyl halide polymers and copolymers such as polyvinyl chloride; polyvinylpyrrolidone; polyvinyl ethers such as polyvinyl methyl ether; polyvinylidene halides such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketones; polyvinyl aromatic compounds such as polystyrene, polyvinyl ethers such as polyvinyl acetate; copolymers of vinyl monomers with each other and with olefins such as ethylene methyl methacrylate copolymers, acrylonitrile styrene copolymers, ABS resins, and ethylene vinyl acetate copolymers; pyran copolymer; polyhydroxy-propyl-methacrylamide-phenol; polyhydroxyethyl aspartamide phenol; polyethylene oxide-polylysine substituted with palmitoyl residues; polyamides such as nylon 66 and polycaprolactam; alkyd resins, polycarbonates; polyoxymethylenes; polyimides; polyesters; epoxy resins, polyurethanes; viscose; triacetate silk; cellulose, cellulose acetate, cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; and carboxymethylcellulose.

In some embodiments, the compound and agent are formulated to be released from the polymer coating into the environment in which the medical device is located. Preferably, the compound and agent are released in a controlled manner over an extended period of time (eg, weeks or months) using at least one of several well known techniques involving polymeric carriers or layers to control elution. Some of these methods have been previously described in US Patent Application No. 20040243225.

V. Ways of using combination therapy.

In another aspect, the present invention provides a combination therapy for the treatment of cancer. The combination therapy comprises a therapeutically effective amount of a CCR2 antagonist and a therapeutically effective amount of a PD-1 and/or PD-L1 inhibitor. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of cancer.

Depending on the condition of the disease and the condition of the subject, the compounds, antibodies, and formulations of the present invention may be administered orally, parenterally (e.g., by intramuscular, intraperitoneal, intravenous, intracerebral, intrasternal injection or infusion, subcutaneous injection, or implantation), inhalation, intranasal, vaginally, rectally, sublingually or topically. In addition, the compounds and antibodies may be formulated, singly or together, in suitable unit dosage formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and solvents suitable for each route of administration. The present invention also contemplates the possibility of administering the compounds and antibodies of the present invention as depot formulations.

It is understood that the specific dose level and frequency of administration for any particular patient may vary and will depend on a variety of factors, including the activity of the particular compound used, the metabolic stability and duration of action of that compound, age, body weight, hereditary characteristics, general health, sex, diet, method and time of administration, rate of elimination, combination of drugs, severity of the particular condition, and individual receiving therapy.

In the treatment of cancers, such as solid tumors, that require chemokine receptor modulation, a suitable dosage level of a CCR2 antagonist will generally be about 0.001 to 100 mg per kg of patient body weight per day, which can be administered as a single or multiple dose. Preferably, the dosage level will be from about 0.01 to about 25 mg/kg per day; more preferably from about 0.05 to 10 mg/kg per day. A suitable dosage level may be about 0.01 to 25 mg/kg per day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day. In this range, the dosage may be from 0.005 to 0.05; 0.05 to 0.5 or 0.5 to 5.0 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing from 1.0 to 1000 mg of the active ingredient, in particular 1.0; 5.0; 10.0; 15.0; 20.0; 25.0; 50.0; 75.0; 100.0; 150.0; 200.0; 250.0; 300.0; 400.0; 500.0; 600.0; 750.0; 800.0; 900.0 and 1000.0 mg of the active ingredient for symptomatic dosage adjustments for the patient to be treated. The compounds may be administered on a schedule of 1 to 4 times per day, preferably once or twice per day.

A suitable dosage level for a PD-1 inhibitor and/or a PD-L1 inhibitor is typically about 0.0001 to 100 mg/kg, typically about 0.001 to 20 mg/kg, and more preferably about 0.01 to 10 mg/kg body weight. subject's body. Preferably the dose is in the range of 0.1-10 mg/kg body weight. For example, dosages may be 0.1; 0.3; 1; 3; 5 or 10 mg/kg body weight, and more preferably 0.3; 1; 3 or 10 mg/kg body weight. Dosing regimens can generally be designed to achieve effects that result in sustained receptor occupancy (RO) based on typical antibody pharmacokinetic properties. An exemplary antibody treatment regimen includes administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months. For example, the dosing regimen may include administration of the antibody: (i) every two weeks in 6-week cycles; (ii) every four weeks for six doses, then every three months; (iii) every three weeks; (iv) 3-10 mg/kg body weight once, then 1 mg/kg body weight every 2-3 weeks. Given that an IgG4 antibody typically has a half-life of 2-3 weeks, the preferred dosage regimen for an anti-PD-1 or anti-PD-L1 antibody is 0.3-10 mg/kg body weight, preferably 3-10 mg/kg body weight, more preferably 3 mg/kg body weight by intravenous administration, with the antibody administered every 14 days for 6-week or 12-week cycles until a complete response or confirmation of progressive disease. An exemplary small molecule treatment regimen is administering daily, twice a week, three times a week, or once a week. Dosage and schedule may change during the course of treatment.

In some embodiments, two or more antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the indicated ranges. The antibody can be administered multiple times. The intervals between single doses can be, for example, once a week, every 2 weeks, every 3 weeks, monthly, every three months or once a year. The intervals may also be irregular as measured by the patient's blood levels of antibodies to the target antigen. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 mg/ml, and in some methods, about 25-300 mg/ml.

The therapeutic compound and agent described herein in combination therapy may be administered either alone or in a pharmaceutical composition that contains the therapeutic compound and agent and one or more pharmaceutically acceptable carriers, excipients and diluents.

In some embodiments, each therapeutic compound and agent is provided in an amount that is sub-therapeutic when used alone or without the other compound. Those of skill in the art will appreciate that "combinations" may include combinations in treatment (i.e., two or more drugs may be administered as a mixture, or at least simultaneously, or at least administered to a subject at different times, but such that both were present in the subject at the same time).

Similarly, the compounds, agents and compositions of the present invention can be used in combination with other drugs that are used to treat, prevent, suppress or attenuate cancer. Such other drugs may be administered in the manner and in the amount normally used therefor, simultaneously or sequentially with a compound, agent or composition of the present invention. When a compound, agent or composition of the present invention is used simultaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound, agent or composition of the present invention is preferred. Accordingly, pharmaceutical compositions may include those that also contain one or more other active ingredients or therapeutic agents, in addition to the compound, agent or composition of the present invention.

Combination therapy includes the combined use of a CCR2 antagonist and a PD-1 and/or PD-L1 inhibitor, the sequential use of a CCR2 antagonist and a PD-1 and/or PD-L1 inhibitor, the use of a composition containing a CCR2 antagonist and a PD-1 and/or PD inhibitor -L1, or the simultaneous use of separate compositions, where one composition contains a CCR2 antagonist, and the other composition contains a PD-1 and/or PD-L1 inhibitor.

Co-administration includes the use of the CCR2 antagonist of the present invention for 0.5; 1, 2, 4, 6, 8, 10, 12, 16, 20 or 24 hours after application of the PD-1 and/or PD-L1 inhibitor of the present invention. Co-administration also includes application simultaneously, approximately simultaneously (eg, within about 1, 5, 10, 15, 20, or 30 minutes of each other), or consecutively, in any order. In addition, each of the CCR2 antagonist and PD-1 and/or PD-L1 inhibitor can be administered once a day, two, three or more times a day to provide a preferred dosage level per day.

VI. Sets

In some aspects, provided herein are kits containing a CCR2 chemokine receptor antagonist and a PD-1 and/or PD-L1 inhibitor disclosed herein that are useful in the treatment of cancer. The kit may contain a pharmaceutical composition comprising a CCR2 chemokine receptor antagonist compound, such as a small molecule CCR2 inhibitor, and a pharmaceutical composition comprising PD-1 and/or PD-L1, such as an inhibitory antibody. In some cases, the kit includes written materials, such as instructions for use of the compound, antibody, or pharmaceutical compositions thereof. The kit may include, but is not limited to, buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any of the methods disclosed herein.

Suitable CCR2 chemokine receptor antagonist and PD-1 and/or PD-L1 inhibitors include the compounds described herein.

VII. Examples

Example 1 The use of a CCR2 inhibitor enhances anti-PD-1 therapy.

CT26 tumors are highly infiltrated with cytotoxic T cells and other leukocytes but nonetheless grow rapidly in Balb/c mice after subcutaneous implantation. These tumors partially respond to treatment with anti-PD-1 and anti-CTLA monoclonal antibodies.

Six days after subcutaneous implantation of CT26 in the flanks of 9-week-old female Balb/c mice (2.5×10 ⁵ /mouse), recipients were randomized by tumor size and treatment was initiated. Mice received anti-PD-1 (RPM1-14 from BioXcell, Inc., catalog number BE0146) by intraperitoneal injection on days 7, 10, 17 and 21 (200 μg/mouse) and received the CCR2 antagonist, Compound 1 (30 or 60 mg/kg) or control solution by gavage every 24 hours starting on day 7.

We have found that the curative effects of anti-PD-1 therapy are markedly enhanced by specific blockade of the chemokine receptor 2 (CCR2) with a small molecule antagonist. This combined anti-PD-1/CCR2i approach significantly reduces tumor size and increases long-term survival rates, with over 50% of mice (up to 73%) showing complete regression of the previously established tumor. The effects of this combination therapy depend on the presence of CD8 ⁺ T cells, as tumors do not respond to therapy in CD8-depleted mice. The antitumor response against CT26 is specific: long-term survivors are resistant to re-inoculation with a CT26 tumor (even without further use of any of the drugs), but not resistant to a 4T1 breast tumor. CCR2 antagonism alters the tumor microenvironment, reducing the amount of M-SCMP per gram of tumor (CCR2 ^hi population, phenotypically defined as CD11b ⁺ /Ly6G ^- /Ly6C ^hi ). Tumor size reduction is inversely proportional to the ratio of CD8 T cells to M-SCMP. Model CT26 data are presented in the following paragraphs.

In FIG. 1 shows immunohistochemical analysis of normal and tumor human tissue using monoclonal antibodies with human CCR2. Panels A and B show normal colon tissue at 40X and 200X, respectively. Panels C, D, E and F show typical human colon cancer tissue at the magnifications indicated. Specific staining of CCR2 was observed on 78 malignant tumors (diagnosed grade I-III) in patients with colon cancer, but not on normal control tissues of the colon.

CT26 is a Balb/c derived colon adenocarcinoma that generates tumors when injected subcutaneously in Balb/c mice. Individual cells were isolated from established subcutaneous CT26 tumors (28 days post-injection) by mincing, briefly digesting the tissue with collagenase-D, and passing the suspension through a 100 μm sieve. Cells were stained for flow cytometry and gated for live CD45 ⁺ tumor-infiltrating leukocytes (FIG. 2). Comparison between mAb staining against mouse CCR2 mAb (FIG. 2A) and comparison with isotype control (FIG. 2B) revealed CCR2-specific staining in a CD11b ⁺ leukocyte subpopulation.

Gating of the CCR ²⁺ population (FIG. 3B) shows that these cells express high levels of Ly6C and lack Ly6G; which are the hallmarks of M-SCMP. Such cells are involved in the anergy induction of tumor-specific cytotoxic T-cell populations.

In FIG. 4 shows direct gating for M-CMPS cells isolated from CT26 tumors showing stable expression of CCR2. Panel A: Gating of live CD45 ⁺ CT26 infiltrating cells in the CD11b ⁺ population. Panel B: Gating of the LyC ^hi /Ly6G ^- population. Panel C: Histogram of CCR2 staining (right) overlaid with mAb stained isotype control (left) in a Ly6C ^hi / ^Ly6G population.

In FIG. 5 shows the overall study design for anti-PD-1 + Compound 1 in the CT26 model.

In FIG. 6 shows that Compound 1 administered by gavage at a dose of 30 mg/kg per day provides minimal plasma levels equal to or greater than those required for complete receptor coverage. Panel A shows the levels of Compound 1 in plasma on day 3 of application. Panel B shows Compound 1 23 days after application.

In FIG. 7 shows that the combination of Compound 1 and α-PD-1 results in a reduction in tumor volumes. Panel A shows the results for mice treated with 1% HPMC + isotype. Panel B shows the results for mice treated with 1% HPMC + α-PD-1. Panel C shows the results for mice treated with 30 mg/kg of Compound 1 + isotype. Panel D shows the results for mice treated with 30 mg/kg Compound 1 + α-PD-1. The dotted line indicates the largest tumor volume observed in the Compound 1 + α-PD-1 group. "1% HPMC" is the control solution for Compound 1, "Isotype" is the isotype control at a dose corresponding to α-PD-1. As seen in FIG. 7, α-PD-1 alone reduces CT26 tumor volume, but the combination with Compound 1 enhances this effect.

Tetramer staining of peripheral blood lymphocytes with MHC class I molecules and their associated peptides for the immunodominant CT26 antigen demonstrates a CT26-specific CD8 T cell response in tumor bearing mice (FIG. 8). AH1, an immunodominant peptide for the cytotoxic T-cell response against CT26, is derived from the gp70 protein of the MuLV retrovirus endogenous to CT26. AH1-specific T cell receptors are mostly absent from the peripheral blood CD8 T cells of intact mice, but are abundant in the same population of CT26 tumor-bearing mice.

In FIG. 9 shows that the reduction in tumor size induced by Compound 1 + α-PD-1 therapy requires CD8 T cells. Panel A: Shows tumor volume in mice treated with Control Solution + α-PD-1 + α-CD8. Panel B: shows tumor volume in mice treated with 30 mg/kg Compound 1 + α-PD-1 + isotype control. Panel C: shows tumor volume in mice treated with 30 mg/kg of Compound 1 + α-PD-1 + α-CD8.

In FIG. 10 shows that despite the involvement of cytotoxic T cells in tumor size reduction, the number of tumor CD8 T cells does not change significantly with treatment. Tumor-infiltrating cytotoxic T cells (Thy1 ⁺ /CD8 ⁺ ) were determined by weighing tumors before dissociation, which allowed the calculation of the number of cells per gram of tumor.

In FIG. 11 shows that Compound 1 reduces the amount of M-SCMP in the CT26 microenvironment by day 24. The number of M-SCMP was determined by weighing the tumors before dissociation, which made it possible to calculate the number of cells per gram of tumor. As can be seen from the figure, Compound 1 alone reduces the population of M-SCMP, but the combination with α-PD-1 enhances the effect.

In FIG. 12 shows that the ratio of CD8 T cells to M-SCMP is significantly increased with combination treatment. The ratio of CD8 T cells to M-CMMP was determined from the number of cells shown in FIG. 10 and FIG. 11. The ratio in control mice (veh + iso) was 1:1, which means one M-SCMP for each CD8 T cell. The combination treatment reduced M-SCMP in favor of CD8 T cells, yielding 100 CD8 T cells for each M-SCMP. Treatment with Compound 1 alone produced 10 CD8 T cells for each M-SCMP cell. Treatment with α-PD-1 produced CD8 T cells for every M-SCM cell.

In FIG. 13 shows that the number of survivors for a long time with CT26 in response to α-PD-1 is increased with combined treatment with CCR2. On day 83, 6 survivors remained in the α-PD-1 + Compound 1 group, while only 2 survivors remained in the α-PD-1 + Veh group. Subgroups of mice taken on day 27 for cell analysis were excluded from this survival analysis. One mouse from the Iso + Veh group and one from the α-PD-1 + 598 group did not develop tumor at all, and these two mice were excluded from this analysis. The Gehan-Breslow-Wilcoxon test was used to determine the p value between the red (middle) and blue (upper) curves.

Surviving CT26 mice pre-treated with α-PD-1 with or without Compound 1, tumor-free for 2-4 weeks, were re-inoculated with CT26 in the right flank and inoculated with 4T1 (for the first time) in the left flank. An increase in CT26 was observed only in mice that were not previously exposed to CT26 (Fig. 14B). Growth in 4T1 was noted in all mice regardless of previous exposure to CT26 (FIG. 14A).

These data are consistent with the hypothesis that CCR2 antagonism enhances anti-PD-1 therapy by preventing the accumulation of M-SCMP in the tumor, thus reducing their inhibitory effect on cytotoxic T cells.

In addition to colon cancer, CCR2 blockade has shown efficacy in mouse models of glioblastoma and pancreatic cancer, as well as in human pancreatic cancer.

While the foregoing invention has been described in some detail for purposes of illustration and example for clarity of understanding, one skilled in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference. If there is a conflict between the present application and the reference herein, the present application takes precedence.

Claims (46)

1. A method for enhancing the therapeutic effect of a PD-1 and/or PD-L1 inhibitor in the treatment of colorectal cancer in a mammal, comprising administering a therapeutically effective amount of a CCR2 chemokine receptor antagonist and a therapeutically effective amount of a PD-1 and/or PD-L1 inhibitor to the mammal, wherein said CCR2 chemokine receptor antagonist has the formula selected from the group consisting of:

or their pharmaceutically acceptable salts. 2. The method of claim 1, wherein said CCR2 chemokine receptor antagonist has the formula

or a pharmaceutically acceptable salt thereof. 3. The method of claim 1, wherein said CCR2 chemokine receptor antagonist has the formula

or a pharmaceutically acceptable salt thereof. 4. The method of claim 1, wherein said CCR2 chemokine receptor antagonist has the formula

or a pharmaceutically acceptable salt thereof. 5. The method according to any one of paragraphs. 1-4, wherein said PD-1 and/or PD-L1 inhibitor is a PD-1 inhibitor. 6. The method of claim 5 wherein the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, IBI-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001 , GB-226 and STI-1110. 7. The method of claim 5 wherein the PD-1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. 8. The method according to any one of paragraphs. 1-4, wherein said PD-1 and/or PD-L1 inhibitor is a PD-L1 inhibitor. 9. The method of claim 8 wherein the PD-L1 inhibitor is selected from the group consisting of durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, CA-327, STI -1014 and KY-1003. 10. The method according to any one of paragraphs. 1-9, wherein the CCR2 chemokine receptor antagonist and PD-1 and/or PD-L1 inhibitor are co-administered. 11. The method of claim 10 wherein the CCR2 chemokine receptor antagonist and the PD-1 and/or PD-L1 inhibitor are administered in a combination formulation. 12. The method according to any one of paragraphs. 1-9, wherein the CCR2 chemokine receptor antagonist and the PD-1 and/or PD-L1 inhibitor are administered sequentially. 13. The method of claim 12 wherein the CCR2 chemokine receptor antagonist is administered prior to administration of the PD-1 and/or PD-L1 inhibitor. 14. The method of claim 12 wherein the CCR2 chemokine receptor antagonist is administered following administration of the PD-1 and/or PD-L1 inhibitor. 15. The method according to any one of paragraphs. 1-14, wherein the CCR2 chemokine receptor antagonist is administered orally and the PD-1 and/or PD-L1 inhibitor is administered intravenously. 16. The method according to any one of paragraphs. 1-15, where the mammal is a human. 17. The method according to any one of paragraphs. 1-16, where the therapeutic effect is to reduce the size of the tumor. 18. The use of a therapeutically effective amount of a CCR2 chemokine receptor antagonist to enhance the therapeutic effect of a PD-1 and/or PD-L1 inhibitor in the treatment of colorectal cancer in a subject, wherein said CCR2 chemokine receptor antagonist has the formula selected from the group consisting of:

or their pharmaceutically acceptable salts. 19. Use of claim 18 wherein the CCR2 chemokine receptor antagonist is used in a composition comprising a CCR2 chemokine receptor antagonist, a PD-1 and/or PD-L1 inhibitor and a pharmaceutically acceptable carrier or excipient. 20. Use according to claim 18 or 19, where the PD-1 and/or PD-L1 inhibitor is selected from any inhibitor according to claims 5-9. 21. Application according to any one of paragraphs. 18-20, where the therapeutic effect is to reduce the size of the tumor. 22. A kit for enhancing the therapeutic effect of a PD-1 and/or PD-L1 inhibitor in the treatment of a subject suffering from colorectal cancer, comprising a therapeutically effective amount of a CCR2 chemokine receptor antagonist and a therapeutically effective amount of a PD-1 and/or PD-L1 inhibitor, with instructions for effective application wherein said CCR2 chemokine receptor antagonist has the formula selected from the group consisting of:

or their pharmaceutically acceptable salts. 23. The set according to p. 22, where the PD-1 and/or PD-L1 inhibitor is selected from any inhibitor according to paragraphs. 5-9. 24. The kit of claim 22 or 23, wherein the CCR2 chemokine receptor antagonist and PD-1 and/or PD-L1 inhibitor are in a co-administration formulation. 25. The kit according to claim 22 or 23, wherein the CCR2 chemokine receptor antagonist and the PD-1 and/or PD-L1 inhibitor are in a sequential formulation.

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