TW201815417A - Combination use of anti-PD-1 antibody and IDO inhibitor in the preparation of a medicament for the treatment of tumor - Google Patents

Abstract

The present invention relates to combination use of anti-PD-1 antibody or its antigen-binding fragments and IDO inhibitor in the preparation of a medicament for the treatment of tumor. The composition of anti-PD-1 antibody or its antigen-binding fragments and IDO inhibitor can effectively resolve tumor heterogeneity, play a significant role in inhibiting tumor cells, and effectively inhibit the proliferation, migration or invasion of tumor cells.

Description

   Application of combination of PD-1 antibody and IDO inhibitor in preparing anti-tumor medicine   

The present invention relates to a PD-1 antibody or an antigen-binding fragment thereof and an IDO inhibitor compound (S) -2- (4- (4- (4- (6-fluoro-5H-imidazo [5,1-a] isoindole) Use of indole-5-yl) piperidin-1-yl) phenyl) -1H-pyrazol-1-yl) ethanol in the preparation of antitumor drugs.

Indoleamine-pyrrole-2,3-dioxygenase (IDO) is a heme-containing monomeric protein consisting of 403 amino acid residues, including two The folded α-helical domain, the large domain contains a catalytic pocket, and the substrate can interact with IDO in the catalytic pocket. IDO is closely related to a variety of cytokines such as interferon (IFN), interleukin (IL), and tumor necrosis factor, and they can start IDO under certain conditions. There is a regulatory point in the cell cycle of T-cells that is very sensitive to tryptophan levels. On the one hand, IDO depletes local tryptophan, causing T-cells to stagnate in the middle of the G1 phase, thereby inhibiting T cell proliferation; On the other hand, IDO catalyzes the production of canine urea, the main product of tryptophan metabolism, which induces changes in intracellular oxidants and antioxidants induced by oxygen free radicals and induces T-cell apoptosis, which is an inherent immune suppressive mechanism existing in the body. A large number of studies have shown that IDO is highly expressed in leukemia cells, inhibits local T cell proliferation, inhibits T-cell-mediated immune responses, blocks T-cell activation signal transduction, and thereby mediates tumor cells to escape the immune system. attack. Most human tumors have been found to constitutively express IDO. Therefore, IDO is a promising target for cancer immunotherapy.

IDO inhibitors have good application prospects as pharmaceuticals in the pharmaceutical industry. Patent application PCT / CN2016 / 079054 (application date 2016.04.12, publication number WO2016169421A1) provides a new type of highly efficient and low toxicity selective IDO inhibitor compounds. Has excellent effects and effects, especially excellent drug metabolism absorption activity, its chemical name is (S) -2- (4- (4- (4- (6-fluoro-5H-imidazo [5,1-a ] Isoindol-5-yl) piperidin-1-yl) phenyl) -1H-pyrazol-1-yl) ethanol, the structure is shown by the following formula (I)

Programmed death-1 (PD-1) is a protein receptor found on the surface of T cells and found in 1992. It is involved in the process of cell apoptosis. PD-1 belongs to the CD28 family. It has 23% amino acid homology to cytotoxic T Iymphocyte antigen 4, CTLA-4, but its expression is different from CTLA. It is mainly expressed in activated T cells, B cells, and myeloid cells. PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is mainly expressed on T cells, B cells, macrophages and dendritic cells (DCs). The expression of PD-L1 on activated cells can be up-regulated. The expression of PD-L2 is relatively limited, mainly on antigen-presenting cells, such as activated macrophages and dendritic cells. New research finds high expression of PD-L1 protein in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, and melanoma, and the expression level of PD-L1 is closely related to the clinical and prognosis of patients . Due to the role of PD-L1 as a second signaling pathway in inhibiting T cell proliferation, PD-1 / PD-L1 immunotherapy is a new type of anti-cancer immunotherapy currently attracting much attention. It aims to use the body's own immune system to fight cancer. Blocking the PD-1 / PD-L1 signaling pathway to kill cancer cells has the potential to treat many types of tumors, so blocking the binding between PD-L1 / PD-1 has become a very promising new field in tumor immunotherapy. Target. Patent application WO2015085847 (publication date 2015.06.18) discloses a PD-1 antibody with high affinity, high selectivity and high biological activity.

Traditional anticancer treatments (surgery, radiotherapy, and chemotherapy) have achieved certain clinical treatment effects compared with before, but due to the limitations of treatment methods, the cure of patients' tumors and the extension of survival time have not been achieved. In the treatment of local cancer, radiotherapy is effective, but often treats the symptoms and not the root cause when treating diffuse cancer. In these cases, chemotherapy is still the treatment of choice, but the severe toxic effects of chemotherapy on normal tissues often limit its clinical application.

Combination medication is one of the important factors that affect the drug's effect. When two or more drugs are used in combination, both synergistic and antagonistic effects can be produced. IDO is highly expressed in antigen-presenting cells and tumor cells. The IDO pathway down-regulates the body's immune capacity by inhibiting T cell proliferation. Like other immune checkpoint inhibitors (CTLA-4, PD-1, and PD-L1), IDO is one of the important mechanisms for tumors to escape the immune system. Four IDO inhibitors are currently undergoing clinical trials. From the perspective of clinical effects, if the single-target effect of IDO is limited, the combination of drugs with immune checkpoint inhibitors may increase the efficacy through the possible combination mechanism described above. In June 2016, a phase III clinical trial of Incyte's IDO1 inhibitor and Merck's PD-1 antibody Keytruda as first-line therapy for advanced melanoma is underway, but there are problems with small sample sizes and non-randomized studies. Can it be successfully applied It is still worth in-depth research in clinical.

The technical problem to be solved by the present invention is to provide the use of a combination of a PD-1 antibody or an antigen-binding fragment thereof and an IDO inhibitor compound, specifically a PD-1 antibody or an antigen-binding fragment thereof and (S) -2- (4- (4 -(4- (6-fluoro-5H-imidazo [5,1-a] isoindole-5-yl) piperidin-1-yl) phenyl) -1H-pyrazol-1-yl) ethanol combination Use in the preparation of antitumor medicaments.

The invention provides a use of a PD-1 antibody or an antigen-binding fragment thereof in combination with an IDO inhibitor represented by compound (I) in the preparation of an antitumor drug, wherein the component PD-1 antibody or the antigen-binding fragment thereof comprises: An antibody light chain variable region comprising at least one LCDR selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; and the antibody heavy chain may Variable region, the antibody heavy chain variable region comprises at least one HCDR selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, the IDO inhibitor is of formula (I) Compound (S) -2- (4- (4- (4- (4- (6-fluoro-5H-imidazo [5,1-a] isoindol-5-yl) piperidin-1-yl) shown Phenyl) -1H-pyrazol-1-yl) ethanol or a pharmaceutically acceptable salt thereof, a solvent compound, or a stereoisomer thereof. In a specific embodiment of the present invention, the compound represented by formula (I) also uses a code 41 to indicate,

In a preferred embodiment of the present invention, the antibody light chain variable region of the PD-1 antibody or antigen-binding fragment thereof of the present invention comprises LCDR1 as shown in SEQ ID NO: 4.

In a preferred embodiment of the present invention, the antibody light chain variable region of the PD-1 antibody or antigen-binding fragment thereof of the present invention comprises LCDR2 as shown in SEQ ID NO: 5.

In a preferred embodiment of the present invention, the antibody light chain variable region of the PD-1 antibody or antigen-binding fragment thereof of the present invention comprises LCDR3 as shown in SEQ ID NO: 6.

In a preferred embodiment of the present invention, the antibody heavy chain variable region of the PD-1 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1 as shown in SEQ ID NO: 1.

In a preferred embodiment of the present invention, the antibody heavy chain variable region of the PD-1 antibody or antigen-binding fragment thereof of the present invention comprises HCDR2 as shown in SEQ ID NO: 2.

In a preferred embodiment of the present invention, the antibody heavy chain variable region of the PD-1 antibody or antigen-binding fragment thereof of the present invention comprises HCDR3 as shown in SEQ ID NO: 3.

The CDR sequences described above are shown in the following table:     

In a preferred embodiment of the present invention, the PD-1 antibody or antigen-binding fragment thereof of the present invention is a humanized antibody or a fragment thereof.

In a preferred embodiment of the present invention, the humanized antibody light chain sequence of the PD-1 antibody or antigen-binding fragment thereof of the present invention is the sequence shown in SEQ ID NO: 8 or a variant thereof; said The variant of the amino acid preferably has an amino acid change of 0-10 in the variable region of the light chain; more preferably, it is an amino acid change of A43S.

In a preferred embodiment of the present invention, the humanized antibody light chain sequence of the PD-1 antibody or antigen-binding fragment thereof of the present invention is the sequence shown in SEQ ID NO: 7 or a variant thereof; said The variant preferably has an amino acid change of 0-10 in the heavy chain variable region; more preferably the amino acid change of G44R.

The particularly preferred light chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: 8, and the heavy chain sequence is the sequence shown in SEQ ID NO: 7.

The sequences of the heavy and light chains of the aforementioned humanized antibodies are as follows:

Heavy chain SEQID NO: 7

Light chain SEQID NO: 8

In a preferred embodiment, the tumor is selected from a related disease or condition mediated by PD-1 and a disease of pathological characteristics of the tryptophan metabolic pathway mediated by IDO.

In a preferred embodiment, the tumor is selected from IDO overexpressing tumors. In a preferred embodiment, the tumor is resistant or insensitive to immunotherapy, and the immunotherapy is selected from PD-1 antibodies.

In a preferred embodiment, the tumor may be selected from the group consisting of lung cancer, gastric cancer, colon cancer, colon cancer, breast cancer, cervical cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, Kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, melanoma, glioma, glioblastoma, hepatocellular carcinoma, mastoid nephroma, head and neck tumor, leukemia, lymph Tumor, myeloma, or non-small cell lung cancer. A tumor that preferably expresses PD-L1 is more preferably breast cancer, lung cancer, gastric cancer, colon cancer, colon cancer, kidney cancer, melanoma or non-small cell lung cancer.

The amount of PD-1 antibody or antigen-binding fragment thereof in the present invention is not particularly limited, and may be, for example, 0.1-1000 mg, preferably from 50-600 mg, and preferably from 50 mg, 60 mg, 70 mg, 75 mg, 100 mg, 125 mg, 150 mg , 175mg, 200mg, 225mg, 250mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 600mg, more preferably from 100mg, 200mg, 400mg; it can also be 1-10mg / kg, preferably from 1mg / kg, 2mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, more preferably 3 mg / kg, 4 mg / kg, 5 mg / kg. The amount of IDO in the present invention is not particularly limited, and may be, for example, 0.1-1000mg, preferably 10mg, 20mg, 25mg, 50mg, 75mg, 100mg, 150mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 750mg, 800mg , 900mg, 1000mg, more preferably 50mg, 100mg, 200mg, 400mg, 800mg, 1000mg.

In combination administration, the weight ratio of the PD-1 antibody or its antigen-binding fragment to the IDO inhibitor represented by the general formula (I) is also not particularly limited, and may be, for example, 0.001 to 1,000, preferably 10 mg, 20 mg, or 25 mg. , 50mg, 75mg, 100mg, 150mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 750mg, 800mg, 900mg, 1000mg, more preferably 50mg, 100mg, 200mg, 400mg, 800mg, 1000mg.

The frequency of administration of the two can be determined according to the situation of the patient. In a preferred embodiment, the IDO inhibitor is BID (dosed twice a day) or once a day, and the PD-1 antibody is QOD (dosed every other day). Once) or Q3W (administered every three weeks) or Q2W (administered every 2 weeks).

The invention also relates to the use of a PD-1 antibody or an antigen-binding fragment thereof in combination with an IDO inhibitor represented by the general formula (I) in the preparation of an antitumor drug, wherein the PD-1 antibody or the antigen-binding fragment thereof and the general formula The IDO inhibitors shown in (I) are prepared into pharmaceutical compositions, for example, are formulated into tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injections, injections) Use sterile powders and concentrated solutions for injection), suppositories, inhalants or sprays.

In addition, the pharmaceutical preparations of the present invention can also be administered to patients or subjects in need of such treatment by any suitable mode of administration, such as oral, parenteral, rectal, pulmonary or topical administration. When used for oral administration, the pharmaceutical composition can be made into oral preparations, such as oral solid preparations, such as tablets, capsules, pills, granules, etc .; or oral liquid preparations, such as oral solutions, oral suspensions, etc. Agents, syrups, etc. When formulated into an oral preparation, the pharmaceutical preparation may further contain a suitable filler, a binder, a disintegrant, a lubricant, and the like. When used for parenteral administration, the pharmaceutical preparations can be made into injections, including injections, sterile powders for injections, and concentrated solutions for injections. When injectable, the pharmaceutical composition can be produced by conventional methods in the existing pharmaceutical field. When an injection is formulated, the pharmaceutical preparation may be added without an additional agent, or a suitable additional agent may be added according to the properties of the drug. When used for rectal administration, the pharmaceutical preparations can be made into suppositories and the like. When used for pulmonary administration, the pharmaceutical preparation can be made into an inhalant or a spray. A particularly preferred injectable form of the PD-1 antibody is an injection solution or a lyophilized powder needle, which contains the PD-1 antibody, a buffer, a stabilizer, and optionally a surfactant. The buffer may be selected from one or more of acetate, citrate, succinate, and phosphate. The stabilizer may be selected from sugars or amino acids, preferably disaccharides such as sucrose, lactose, trehalose, maltose. The surfactant is selected from the group consisting of polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, and polyoxyethylene sorbitan fatty acid ester. Preferably, the polyoxyethylene sorbitan fatty acid ester is polysorbate 20, 40, 60, or 80. , The best is polysorbate 20. Optimal injectable forms of PD-1 antibodies include PD-1 antibodies, acetate buffers, trehalose, and polysorbate 20.

In the present invention, the so-called "combination" is a method of administration, which includes various situations in which two drugs are administered sequentially or simultaneously. Here, the term "simultaneously" refers to the administration of PD-1 antibody and IDO inhibition in the same administration cycle. Agents, such as two drugs administered within 2 days, or within 1 day. The so-called "sequential administration" includes the case where the PD-1 antibody and the IDO inhibitor are administered separately in different administration cycles. These administration methods belong to the combined administration according to the present invention.

The invention also provides a method for treating tumors, which comprises administering the aforementioned PD-1 antibody and IDO inhibitor to a tumor patient in combination.

The present invention provides the PD-1 antibody in combination with the IDO inhibitor as a medicine for treating antitumor. The present invention also provides a medicine kit or a medicine packaging box, which contains the aforementioned IDO inhibitor and PD-1 antibody.

The present invention also provides a pharmaceutical composition comprising the aforementioned effective amount of the PD-1 antibody and the aforementioned IDO inhibitor, and one or more pharmaceutically acceptable excipients, diluents or carriers.

Detailed description of the invention

To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Except where it is clear that it is otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

The three-letter and one-letter codes for amino acids used in the present invention are described in J. biol. Chem, 243, p3558 (1968).

The antibody according to the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The composition and arrangement of amino acids in the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain γ, and α chain , Ε chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into kappa chains or lambda chains by different constant regions. Each of the five types of Ig may have a κ chain or a λ chain.

In the present invention, the antibody light chain variable region according to the present invention may further include a light chain constant region, the light chain constant region comprising a human or murine κ, λ chain or a variant thereof.

In the present invention, the antibody heavy chain variable region according to the present invention may further include a heavy chain constant region, the heavy chain constant region comprising human or murine IgG1,2,3,4 or a variant thereof.

The sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of 3 CDR regions and 4 FR regions. The sequence from amine end to carboxy end is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the LCVR region and HCVR region of the antibody or antigen-binding fragment according to the invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3) or the numbers of Kabat and chothia Rules (HCDR1).

The term "humanized antibody", also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline Antibodies produced in antibody framework sequences. It can overcome the strong antibody variable antibody response induced by the chimeric antibody because it carries a large amount of mouse protein components. Such framework sequences can be obtained from a public DNA library including germline antibody gene sequences or published references. Germline DNA sequences of human heavy and light chain variable region genes are available in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), and in Kabat , EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In a preferred embodiment of the present invention, the CDR sequence of the PD-1 humanized antibody mouse is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

The "antigen-binding fragment" in the present invention refers to a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, and an Fv fragment sFv fragment that binds to human PD-1; The antibody is selected from one or more CDR regions of SEQ ID NO: 1 to SEQ ID NO: 6. The Fv fragment contains the variable region of the antibody heavy and light chains, but has no constant region, and has the smallest antibody fragment with all the antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The variable regions of two antibodies can also be linked into a single polypeptide chain with different linkers, called a single chain antibody (single chain antibody) or a single chain Fv (sFv). The term "binding to PD-1" in the present invention refers to the ability to interact with human PD-1. The term "antigen-binding site" of the present invention refers to a three-dimensional spatial site on the antigen that is discontinuous and is recognized by the antibody or antigen-binding fragment of the present invention.

"Administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals, humans, recipients Contact with a subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental methods. Treatment of cells includes contact of the reagent with the cells, and contact of the reagent with the fluid, wherein the fluid is in contact with the cells. "Administering" and "treating" also mean in vitro and ex vivo treatment of cells, such as cells, by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, preventive or prophylactic measures, research and diagnostic applications.

"Treatment" means the administration to a patient of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present invention, that the patient has one or more symptoms of the disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, a therapeutic agent is administered in a treated patient or population in an amount effective to alleviate the symptoms of one or more diseases, whether by inducing the deterioration of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") that is effective in alleviating the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired effect in the patient. It can be assessed whether the symptoms of the disease have been alleviated by any clinical testing method commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Embodiments of the present invention (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating symptoms of the target disease that each patient has, but according to any statistical test known in the art such as Student's t-test, chi-square test, basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra check sum Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include progeny. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of metastases. It should also be understood that due to intentional or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Included are mutant offspring that have the same functional or biological activity as those originally screened in the transformed cells. Where different names are meant, they are clearly visible from the context.

"As needed" or "as needed" means that the event or environment described later can, but need not, occur, and the description includes the place where the event or environment occurred or did not occur. For example, "including 1-3 antibody heavy chain variable regions as needed" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

The PD-1 antibody or its antigen-binding fragment of the present invention and the IDO inhibitor composition represented by compound (I) can effectively solve tumor heterogeneity, exert a significant effect on suppressing tumor cells, and effectively inhibit tumor cell proliferation, migration, or invasion.

Fig. 1 is a graph showing the inhibitory action curve of the combination of PD-1 antibody of the present invention and an IDO inhibitor (also referred to as compound 41 in the examples) on the growth of MC38 tumors.

The present invention is further described below with reference to examples, but these examples do not limit the scope of the present invention. The experimental methods without specific conditions in the examples of the present invention generally follow conventional conditions, such as the manual of antibody technology experiments and molecular breeding manuals of Cold Spring Harbor; or according to the conditions recommended by the raw material or commodity manufacturers. The reagents without specific sources are conventional reagents purchased on the market.

The HC sequence of the PD-1 antibody of the present invention is (SEQ ID NO: 7) and the LC sequence is (SEQ ID NO: 8), as shown below: SEQID NO: 7

SEQID NO: 8

The IDO inhibitor compound (S) -2- (4- (4- (4- (4- (6-fluoro-5H-imidazo [5,1-a] isoindole)- 5-yl) piperidin-1-yl) phenyl) -1H-pyrazol-1-yl) ethanol Refer to Example 40 in Patent Application PCT / CN2016 / 079054 (application date 2016.04.12, publication number WO2016169421A1), 41), the specific method is as follows:

( S ) -2- (4- (4- (4- (4- (6-fluoro-5H-imidazo [5,1- a ] isoindole-5-yl) piperidin-1-yl) phenyl)- Preparation of 1 H -pyrazol-1-yl) ethanol

(1) 6-fluoro-5- (piperidin-4-yl) -5 H -imidazo [5,1- a ] isoindole ditrifluoroacetate (compound 1g) (see patent application PCT / CN2016 / 079054 (application date 2016.04.12, publication number WO2016169421A1), the preparation method in Example 1)

    First step     4-((2-Bromo-6-fluorobenzene) (hydroxy) methyl) piperidine-1-carboxylic acid third butyl ester 1c

Diisopropylamino lithium (32.5 mL, 65.0 mmol) was added to tetrahydrofuran (50 mL), and the preformed 1-bromo-3-fluorobenzene 1a (8.75 g, 50.0 mmol, 25 mL) was added dropwise at -78 ° C. The solution was stirred at -78 ° C for 1 hour. Then, a tetrahydrofuran solution of the preformed tetramethylpiperidine-1-carboxylic acid third butyl ester 1b (8.75 g, 50.0 mmol, 25 mL) was added dropwise at -78 ° C, and the mixture was stirred at -78 ° C for 1 hour. After the reaction was completed, methanol (25 mL) was added dropwise to quench the reaction at -78 ° C. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using an eluent system (n-hexane and ethyl acetate) to obtain Compound 1c (16.3 g, yellow syrup solid, yield 84.0%).

MS m / z (LC-MS): 332.0 [M-56]

    Second step     4-((2-Bromo-6-fluorophenyl) (p-toluenesulfonyloxy) methyl) piperidine-1-carboxylic acid third butyl ester 1d

Compound 1c (15 g, 38.63 mmol) was dissolved in tetrahydrofuran (350 mL), sodium hydride (3.09 g, 77.26 mmol) was added in portions, and stirred until no gas was evolved. A pre-prepared solution of p-toluenesulfonyl chloride (8.10 g, 42.49 mmol, 250 mL) in tetrahydrofuran was added dropwise, stirred at room temperature for 30 minutes, refluxed for 4 hours, and stirred at 70 ° C for 48 hours. After the reaction was completed, the reaction solution was cooled to 0 ° C, and the reaction was quenched by adding water (50 mL) dropwise. A saturated sodium chloride solution (50 mL) was added to separate the layers. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by chromatography using an eluent system (n-hexane and ethyl acetate) to obtain compound 1d (6.6 g, pale yellow viscous solid, yield: 31.80%).

MS m / z (LC-MS): 314.0 / 316.0 [M-56-TsO]

    third step     4-((2-Bromo-6-fluorobenzene) ( 1H -imidazol-1-yl) methyl) piperidine-1-carboxylic acid third butyl ester 1e

Imidazole (12.5 g, 184.3 mmol) was dissolved in N , N -dimethylformamide (50 mL), sodium hydride (7.40 g, 184.3 mmol) was added in portions, and the mixture was stirred at room temperature for 1 hour. A solution of compound 1d (10.0 g, 18.43 mmol, 20 mL) in N , N -dimethylformamide was stirred at 100 ° C. for 12 hours. After the reaction was completed, ethyl acetate (300 mL) was added, and the organic phase was washed with saturated sodium chloride solution (150 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by the system (dichloromethane and methanol) to obtain compound 1e (1.90 g, brown sticky solid, yield: 23.5%).

MS m / z (LC-MS): 438.1 / 440.1 [M + 1]

    the fourth step     4- (6-fluoro-5 H -imidazo [5,1-a] isoindol-5-yl) piperidine-1-carboxylic acid third butyl ester 1f

Compound 1e (1.90 g, 4.33 mmol), N, N -dicyclohexylmethylamine (1.35 g, 6.93 mmol), and triphenylphosphine (908 mg, 3.46 mmol) were added to N , N -dimethylformamide In a solution (10 mL), palladium acetate (390 mg, 1.74 mmol) was added under an argon atmosphere, and the mixture was stirred at 100 ° C for 4.5 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using an eluent system (n-hexane and ethyl acetate) to obtain compound 1f (1.30 g, yellow sticky solid, yield: 83.8 %).

MS m / z (LC-MS): 358.1 [M + 1]

    the fifth step     6-fluoro-5- (piperidin-4-yl) -5 H -imidazo [5,1- a ] isoindole ditrifluoroacetate 1g

Compound 1f (1.30 g, 3.64 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added dropwise, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain 1 g of a crude compound (1.77 g, brown sticky solid). The product was directly subjected to the next reaction without purification.

MS m / z (LC-MS): 258.3 [M + 1]

(2) 4- (4-bromophenyl) -1- (2-((tetrahydro-2 H -pyran-2-yl) oxy) ethyl) -1 H -pyrazole (compound 40a ) ( (See the preparation method disclosed in CN104755477A (publication date 2015.07.01) page 44 )

(3) ( S ) -2- (4- (4- (4- (4- (6-fluoro- 5H -imidazo [5,1- a ] isoindole-5-yl) piperidin-1-yl) phenyl) -1 H - pyrazol-1-yl) ethanol (compound 41)

    First step    

4- (4-Bromophenyl) -1- (2-((tetrahydro-2 H -pyran-2-yl) oxy) ethyl) -1 H -pyrazole 40a (14.8 g, 42 mmol) , 6-fluoro-5- (piperidin-4-yl) -5 H -imidazo [5,1- a ] isoindole 17a (13.9 g, 42 mmol) was added N , N -dimethylformamide ( 300 mL), tritributylphosphine tetrafluoroborate (1.863 g, 64.5 mmol) and potassium phosphate (35 g, 168 mmol) were added and replaced with argon for three times. Tris (dibenzylideneacetone) dipalladium (2.92g, 3.19mmol) was added, and the argon gas was substituted once. The reaction solution was heated to 110 ° C and stirred for 2 hours. After the reaction, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to remove N , N -dimethylformamide, and the resulting residue was purified by silica gel column chromatography using an eluent system (dichloromethane and methanol) to obtain a compound. 40b (6.38 g, grey oil, yield: 29%).

    Second step    

Compound 40b (9 g, 17.1 mmol) was dissolved in methanol (100 mL), and concentrated hydrochloric acid (12 M , 5.7 mL) was added. The reaction solution was raised to 45 ° C and stirred for 1 hour. After the reaction was completed, the reaction solution was cooled to room temperature, and saturated sodium carbonate was added to adjust the pH of the reaction solution to 8, and the filtrate was concentrated under reduced pressure. The obtained product was purified by silica gel column chromatography with an eluent system (dichloromethane and methanol). The residue gave compound 40c (5.2 g, yellow solid, yield: 65%).

    third step    

Compound 40c (1.4g, 3.16mmol) was prepared chiral (Separation conditions: Chiral preparation column Superchiral S-AS (Chiralway), 2cm ID × 25cm Length, 5 μm ; mobile phase: CO ₂ / MeOH / DEA = 60/40 / 0.05 (v / v / v), flow rate: 50 mL / min), the corresponding components were collected, and concentrated under reduced pressure to obtain compound 40 (630 mg, yellow solid) and compound 41 (652 mg, yellow solid).

Experimental Example 1. The effect of the composition of the present invention on subcutaneous xenografts of mouse colon cancer (MC38) hPD-1 transgenic mice

Test article: PD-1 antibody lyophilized powder (prepared according to the method in patent application PCT / CN2016 / 098982, application date 2016.09.14, publication number WO2017054646A1), IDO inhibitor compound 41, and its preparation method see the preparation example.

Control drug: IDO inhibitor compound INCB024360 (structure is ) Prepared with reference to the method disclosed in Example 3 on page 51 of patent WO2010005958A1 (publication date 2010.01.14); NLG0919 (structure is ) Prepared with reference to the method disclosed in Example 25 on page 121 of the specification WO2012142237A1 (publication date 2010.10.18).

Experimental animals: human-derived PD-1 gene transgenic mice, 7-16 weeks, 80, male and female. The transgenic mice were purchased from Cephrim. All of these human PD-1 mice were bred in a special pathogen free (SPF) laboratory animal barrier environment provided by INNOVIVE. The breeding and operation of experimental animals are carried out in accordance with laboratory standards approved by the Laboratory Animal Care and Use Committee (IACUC).

Sources of reagents and other materials: β-cyclodextrin (HPB, oral) was purchased from Roquette; sodium carboxymethyl cellulose (CMC-Na); N, N-dimethylacetamide (DMA); IgG was derived from human The serum was purchased from Sigma-Aldrich; all cell culture media and other auxiliary components were purchased from Invitrogen.

Establishment of tumor model: Resuscitate a tube of rat-derived rectal cancer cell line MC38, which has been frozen in advance, into a T-25 long-neck culture flask, and add 10% bovine serum, 1% penicillin, 2mM glutamine, 1 DMEM (high sugar) cell culture solution containing% sodium pyruvate, 1% non-essential amino acid, and 1% vitamins, and cultured in a 37 ° C incubator containing 5% carbon dioxide. The culture medium is changed twice a week. The cells are subcultured until there are enough cell numbers to plant the mice. The requirements for planting cells are: 1) during the rapid growth period (usually 70% to 90% of the bottom area of the bottle); 2) low number of passages (usually 2 to 4 generations); 3) the day before inoculation Replace with new culture medium; 4) Survival rate is above 95%.

When MC38 cells were transplanted into mice (day 0), MC38 cells were first digested and collected according to standard cell culture procedures, washed twice with PBS, then counted, and finally formulated with PBS to a concentration of 5 × 10 ⁶ MC38 single cell suspension. Prior to seeding, the cell suspension was placed in an ice bath. Each hPD-1 transgenic mouse was inoculated with 0.1 mL of 500,000 (5 × 10 ⁵ ) MC38 cells subcutaneously on the right side.

Test solution preparation: 0.5% CMC-Na, 10% cyclodextrin and 5% glucose aqueous solution were prepared in advance and stored in a refrigerator at 4 ° C.

PD-1 antibody: PD-1 antibody lyophilized powder (200 mg) was first added to water for injection (5 mL) to obtain a 40 mg / mL PD-1 antibody concentrated solution. The PD-1 antibody solution to be administered should be prepared fresh each time. Take the PD-1 antibody concentrate (100 μL), add 13.2 mL of a 5% glucose solution (1: 133.3 times dilution), and mix to obtain a 0.3 mg / mL solution. Each mouse was injected intraperitoneally (ip) with a volume of 10 mL / Kg, with a final dose of 3 mg / kg / time.

Human plasma IgG: IgG lyophilized powder (21 mg) was dissolved in physiological saline (7 mL) to obtain a 3 mg / mL IgG concentrated solution. This concentrated solution was divided into 7 1mL test tubes and stored at -20 ° C. For each administration, take 1 tube of concentrated solution and melt at room temperature. Dilute with saline 1:10 (0.5 mL + 4.5 mL physiological saline). After mixing, a 0.3 mg / mL solution was obtained. Each mouse was injected intraperitoneally (ip) with a volume of 10 mL / Kg, with a final dose of 3 mg / kg / time.

All other samples are prepared every other day.

INCB024360 DMA solution: Firstly call INCB024360 compound (300mg), then add DMA (0.9mL), mix thoroughly and completely dissolve to obtain 333mg / mL INCB024360 concentrated solution, and place at room temperature. Every other day, take INCB024360 concentrated solution (240mL), and slowly add 10% cyclodextrin aqueous solution (3.76mL) while rotating at low speed. If it does not dissolve, you can dissolve it in a short-term ultrasonic water bath. A 20 mg / mL dosing solution was obtained. Each mouse received an oral volume of 5 mL / Kg with a final dose of 100 mg / Kg / time.

NLG0919 suspension: Weigh the NLG0919 compound (120 mg) and add 0.5% CMC-Na (6 mL). Then it was sonicated in a water bath to obtain a 20 mg / mL fine particle NLG0919 suspension. Place at room temperature. Each mouse received an oral volume of 5 mL / Kg with a final dose of 100 mg / Kg / time.

Compound 41 suspension: Compound 41 compound (120 mg) was weighed and 0.5% CMC-Na (15 mL) was added. Then the water bath was sonicated to obtain 8 mg / mL fine particles of NLG0919 suspension. Dilute the above suspension with 0.5% CMC-Na 1: 2 times (3mL plus 3mL) and 1: 4 times (1.5mL plus 4.5mL) to obtain 4mg / mL and 2mg / mL fine particles of compound 41 accordingly. Suspension. Place at room temperature. Each mouse received an oral volume of 5 mL / Kg with final doses of 40, 20, and 10 mg / Kg / time.

PD-1 antibody or hIgG is given every other day in the morning. The other three compounds, INCB024360, NLG0919, and compound 41, were administered twice in the morning and evening, with an interval of 8 hours between morning and evening.

After the tumor volume reached about 100 mm ³ , group administration was started. After the MC38 cells were transplanted, the tumor growth was observed every day. When the tumor appears, measure the size of the tumor until it grows to about 100mm ³ (calculate the tumor volume formula; tumor volume = 1 × w × h × 0.5236, where 1 = length, w = width, h = height, unit mm), Sixty-four MC80-transplanted mice were selected and randomly divided into eight groups (4 for each sex, # 1 to # 4 are female; # 5 to # 8 are male). This day is set to be the eighth day. The specific experimental protocol design of the test product according to the preset single or combined use, dosage and route is shown in the following table:

ip is intraperitoneal injection; po is oral.

Collection of tissue samples: On the second day (the 22nd day of the experiment) after the administration of this experiment, all the experimental mice continued to do the PK / PD study. In brief, six groups of mice (groups 3 to 8) were given only one dose (INCB024360, NLG0919, and compound 41) at the oral dose of this group. Of the 8 mice in each group, the first and second mice were given no medicine and only served as the zero control. The third to fifth mice were orally administered 2 hours in advance, and the sixth to eighth mice were orally administered 8 hours in advance. In this way, the mice died of carbon dioxide at about the same time. The whole blood sample of the mouse was immediately taken out through a cardiac puncture, put into an anticoagulation test tube, and then placed in an ice bath until centrifuged. The blood samples were centrifuged in a centrifuge at 15,000 rpm for 5 minutes, and the supernatant (serum) in the test tube was taken out and placed in a new set of test tubes. Finally, all serum samples were stored in an ultra-low temperature freezer at -80 ° C until they were sent to Shanghai Hengrui Pharmaceutical Co., Ltd. At the same time, tumors were stripped from all mice, weighed on a balance and recorded. After all tumors have been removed, individual tumors are photographed and recorded. The tumors are then packaged separately and stored in -80 ° C ultra-low temperature freezers.

Experimental endpoint: Tumor volume and body weight of mice were measured twice a week.

If any mouse has one of the following conditions, then the mouse will be suspended from the experiment and the data collection will be removed: 1) if the tumor volume of the mouse exceeds 10% of its weight or exceeds 1500mm ³ ; 2) if the mouse Show toxic reactions, and can not perform basic daily actions; 3) If the weight of the mouse has dropped by more than 10% of the body weight before the start of the experiment.

Data analysis: The data of this experiment were analyzed by two-way ANOVA method between the blank group, the treatment group, and the treatment group, and then compared with Bonferroni's multiple groups. All statistics The analysis was run on GraphPad Prism statistical software (Prism 6 for Windows, Version 6.0, GraphPad Software Inc., San Diego, CA).

Experimental results:

Experimental conclusion: Grouping and treatment began on the 8th day (D8) after vaccination. According to the experimental data in Table 1 above, the maximum tumor inhibition rates of PD-1 antibody or compound 41 alone were 41.9% and 43.3%, respectively. The maximum tumor inhibition rate of the combination of PD-1 antibody and IDO inhibitor compound 41 was on the 22nd day. The inhibitory effects of the control IDO inhibitor compound INCB024360 (100 mg / kg) and NLG0919 (100 mg / kg) in combination with PD-1 antibody The tumor rates were 43.3% and 57.4%, respectively. Compound 41 combined with PD-1 antibody inhibited tumor growth in a dose-dependent manner (10, 20, 40 mg / kg). The maximum tumor inhibition rates were 63.7%, 69.0%, and 84.3%. . The above combination group was statistically significant compared with the control group or the PD-1 antibody monotherapy group on the 22nd day. The efficacy of 10 mg / kg IDO inhibitor compound 41 was equivalent to that of 100 mg / kg IDO / TDO inhibitor NLG0919. Better than 100mg / kg IDO inhibitor INCB024360.

In summary, the inhibitory effect of the PD-1 antibody of the present invention combined with IDO inhibitor compound 41 on colon cancer (MC38) cells is significantly better than that of single-component PD-1 antibody or compound 41 and better than that of control group PD. -1 antibody in combination with INCB024360 or NLG0919100, therefore, the combination of PD-1 antibody of the present invention and IDO inhibitors has a significant antitumor effect.

<110> Suzhou Shengdia Biopharmaceutical Co., Ltd., Jiangsu Hengrui Pharmaceutical Co., Ltd., Shanghai Hengrui Pharmaceutical Co., Ltd.

<120> Use of a combination of PD-1 antibody and IDO inhibitor in the preparation of antitumor medicine

<130> 2017

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<170> PatentIn version 3.3

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Claims (15)

A use of a PD-1 antibody or an antigen-binding fragment thereof in combination with an IDO inhibitor in the preparation of an antitumor drug, characterized in that the PD-1 antibody or the antigen-binding fragment thereof comprises: an antibody light chain variable region, and the antibody light The chain variable region comprises at least one LCDR selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; and an antibody heavy chain variable region, the antibody heavy chain variable region Comprising at least one HCDR selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, the IDO inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable compound thereof Salts, solvent compounds, or stereoisomers thereof,     The use according to item 1 of the patent application scope, wherein the antibody light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, as shown in SEQ ID NO: 6 LCDR3 shown.         The use according to item 1 of the patent application scope, wherein the antibody heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and as shown in SEQ ID NO: 3 HCDR3 shown.         The use according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or a fragment thereof.         The use according to item 4 of the scope of patent application, wherein the humanized antibody light chain sequence is the sequence shown in SEQ ID NO: 8 or a variant thereof.         The use according to item 5 of the scope of patent application, wherein the variant has a 0-10 amino acid change in the variable region of the light chain.         The use according to item 6 of the scope of patent application, wherein the variant is an amino acid change of A43S in the variable region of the light chain.         The use according to item 4 of the scope of patent application, wherein the humanized antibody heavy chain sequence is the sequence shown in SEQ ID NO: 7 or a variant thereof.         The use as described in claim 8 of the scope of patent application, wherein the variant has a 0-10 amino acid change in the heavy chain variable region.         The use according to item 9 of the scope of patent application, wherein the variant is an amino acid change of G44R in the variable region of the heavy chain.         The use according to item 4 of the scope of patent application, wherein the light chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: 8, and the heavy chain sequence is the sequence shown in SEQ ID NO: 7.         The use according to any one of claims 1 to 11, wherein the tumor is selected from PD-1 mediated and / or IDO mediated tumors.         The use according to any one of claims 1 to 11, wherein the tumor includes lung cancer, gastric cancer, colon cancer, colon cancer, breast cancer, cervical cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer , Oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, melanoma, glioma, glioblastoma, hepatocellular carcinoma, mastoid kidney Sex tumor, head and neck tumor, leukemia, lymphoma, myeloma, or non-small cell lung cancer.         The use according to item 13 of the scope of patent application, wherein the tumor includes breast cancer, lung cancer, gastric cancer, colon cancer, colon cancer, kidney cancer, melanoma or non-small cell lung cancer.         The use according to any one of claims 1 to 13, wherein the tumor is a tumor expressing PD-L1.