Biomarkers for cancer and uses thereof

Abstract

The present disclosure relates to the use of biomarkers to predict response to cancer (e.g., melanoma) treatment, to select a treatment for a cancer patient (e.g., using targeted therapy, e.g., using agents that target BRAF and / or MEK in combination with cancer immunotherapy (e.g., anti-PD-1 therapy)), to stratify cancer patients into different treatment groups, to treat cancer patients, and to predict clinical outcomes of cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 162,964, filed March 18, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on Mar. 4, 2022, is named C2160-7032WO_SL.txt, and is 51,904 bytes in size. [Background technology]

[0003] Cancer is a leading cause of death worldwide, accounting for over 8 million deaths annually. Melanoma is a malignant tumor resulting from the uncontrolled proliferation of pigment-producing cells. It is one of the most common cancers, its prevalence is increasing, and melanoma is responsible for the majority of skin cancer deaths.

[0004] Apart from traditional cancer therapies such as chemotherapy and radiation, new strategies are being developed to treat cancer. Among them are targeted therapy and cancer immunotherapy. It is important to find an appropriate treatment for cancer patients because cancer can grow and metastasize rapidly and surgical resection alone may not be sufficient for cure due to the presence of undetectable disseminated tumor cells. Despite advances in cancer therapy, there is a need to identify responders to cancer immunotherapy and targeted therapy in early and advanced cancers such as melanoma, as both treatment strategies are valuable treatment options. Summary of the Invention

[0005] The present disclosure provides biomarkers for predicting response to cancer treatment, e.g., melanoma treatment, for selecting a treatment for a cancer patient, e.g., melanoma patient, for stratifying a cancer patient, e.g., melanoma patient, into different treatment groups, for treating a cancer patient, e.g., melanoma patient, and for predicting clinical outcomes for patients with cancer, e.g., melanoma.

[0006] Thus, in one aspect, the disclosure features a method for identifying a patient with cancer who is likely to benefit from a treatment, the method includes obtaining a value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to CD8+ immune effector cell (e.g., CD8+ T cell) level or activity in a subject (e.g., in a sample from the subject), where a value greater than or equal to a reference value identifies the subject as likely to benefit from the treatment, and the treatment includes a targeted therapy in combination with a cancer immunotherapy.

[0007] In some embodiments, a subject is more likely to have an increased benefit from the treatment compared to a treatment comprising the targeted therapy without the cancer immunotherapy.

[0008] In another aspect, the disclosure features a method for selecting a treatment for a subject having cancer, the method includes obtaining a value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), and if the value is greater than or equal to the reference value, selecting a treatment for the subject that includes a targeted therapy in combination with a cancer immunotherapy.

[0009] In some embodiments of any of the above aspects, the method further includes administering to the subject an effective amount of the treatment (e.g., initiating administration or continuing administration). In some embodiments of any of the above aspects, the method further includes administering to the subject an altered dosing regimen of the treatment (e.g., a dosing regimen comprising a higher dose and / or more frequent administration than the reference dosing regimen).

[0010] In some embodiments of any of the above aspects, the method further includes discontinuing administration of the different treatment to the subject.

[0011] In some embodiments of any of the above aspects, the method further includes administering an additional treatment to the subject.

[0012] In some embodiments, the method further includes administering a pretreatment to the subject, where the pretreatment increases the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject).

[0013] In another aspect, the present disclosure features a method for treating a subject with cancer.In some embodiments, the method includes the following: when the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in the sample from the subject) is greater than or equal to a reference value, administer (e.g., start or continue administering) an effective amount of a treatment comprising a targeted treatment in combination with cancer immunotherapy to the subject, thereby treating the subject with cancer.

[0014] In another aspect, the present disclosure features a method for treating a subject with cancer.In some embodiments, the method includes: obtaining the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject); and if the value is greater than or equal to the reference value, administering (e.g., starting or continuing administration) an effective amount of a treatment comprising a targeted treatment in combination with cancer immunotherapy to the subject, thereby treating the subject with cancer.

[0015] In another aspect, the present disclosure features a method for treating a subject with cancer.In some embodiments, the method includes administering (e.g., starting or continuing) an effective amount of a targeting therapy to the subject; and receiving that the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in the sample from the subject) is greater than or equal to a reference value, administering (e.g., starting or continuing) an effective amount of a cancer immunotherapy to the subject, thereby treating the subject with cancer.

[0016] In another aspect, the present disclosure features a method for treating a subject with cancer.In some embodiments, the method includes administering (e.g., starting administration or continuing administration) an effective amount of a treatment comprising a targeted therapy in combination with cancer immunotherapy to the subject, and before administration, it is determined that the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is greater than or equal to a reference value, thereby treating the subject with cancer.

[0017] In another aspect, the disclosure features a method for treating a subject with cancer. In some embodiments, the method includes administering (e.g., commencing or continuing administration) an effective amount of a treatment comprising a targeted therapy in combination with a cancer immunotherapy to the subject, thereby treating the subject with cancer, wherein the subject is characterized by a value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to a CD8+ immune effector cell (e.g., CD8+ T cell) level or activity in the subject (e.g., in a sample from the subject) that is greater than or equal to a reference value.

[0018] In some embodiments, the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) comprises the ratio of the amount of CD4+ immune effector cells (e.g., CD4+ T cells) to the amount of CD8+ immune effector cells (e.g., CD8+ T cells), e.g., as measured by an assay disclosed herein, e.g., flow cytometry immunophenotyping. In certain embodiments, the value is greater than or equal to 2 (e.g., 2.01). In certain embodiments, the value is greater than or equal to 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10. In certain embodiments, the value is greater than or equal to 3.3 (e.g., 3.34). In certain embodiments, the value is greater than or equal to 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

[0019] In some embodiments, obtaining the value comprises determining the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject, e.g., in a sample from the subject. In certain embodiments, the sample from the subject comprises a blood sample (e.g., a peripheral blood sample containing peripheral blood mononuclear cells (PBMCs)) or a tumor sample. In certain embodiments, the value is obtained before administration of the treatment begins (e.g., is a baseline value). In certain embodiments, the value is obtained after administration of the treatment begins. In certain embodiments, the value is obtained 1, 2, 3, 4, 8, 10, 12, 20, 30, 40 weeks or more or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 months or more after administration of the treatment begins.

[0020] In some embodiments, the method further comprises obtaining a level or activity value of CD8+ tumor infiltrating lymphocytes (TILs) in the subject (e.g., in a sample from the subject), e.g., a tumor having a CD8+ TIL inflammatory phenotype. In certain embodiments, an increase in the level or activity value of CD8+ TILs compared to the reference value further identifies that the subject is likely to benefit from the treatment.

[0021] In some embodiments, the method further comprises obtaining a value of tumor mutation burden (TMB) in the subject (e.g., in a sample from the subject). In certain embodiments, an increase in the TMB value compared to the reference value further identifies that the subject is likely to benefit from the treatment.

[0022] In some embodiments, the value of TMB is greater than or equal to 10 mut / Mb, for example, greater than or equal to, or greater than 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mut / Mb.

[0023] In some embodiments, the method further comprises obtaining a level and / or activity value of PD-L1 in the subject (e.g., in a sample from the subject). In certain embodiments, a decrease in the level and / or activity value of PD-L1 compared to the reference value, together with, for example, an increase in the TMB value compared to the reference value, further identifies that the subject is likely to benefit from the treatment. In certain embodiments, the cancer has low or undetectable expression of PD-L1.

[0024] In some embodiments, the method further comprises obtaining the circulating tumor DNA (ctDNA) value in the subject (e.g., in a sample from the subject).In certain embodiments, an increase in the ctDNA value compared to the reference value further identifies that the subject is likely to benefit from the treatment.

[0025] In some embodiments, subjects who are likely to benefit from treatment or have increased benefit from treatment have improved progression-free survival (PFS), duration of objective response (DOR), and / or overall survival (OS) compared with subjects who are unlikely to benefit from treatment or have increased benefit from treatment.In some embodiments, subjects who are likely to benefit from treatment or have increased benefit from treatment have improved PFS, DOR, and / or OS compared with subjects who have not received treatment or subjects who have only received targeted therapy but not cancer immunotherapy.In certain embodiments, PFS, DOR, and / or OS are improved for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60 months or longer. In certain embodiments, PFS, DOR, and / or OS is improved by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 fold or more.

[0026] In some embodiments, the subject has been or is being treated with a targeted therapy.

[0027] In some embodiments, the subject was not or has not been treated with a targeted therapy.

[0028] In some embodiments, the subject has undergone or is undergoing cancer immunotherapy.

[0029] In some embodiments, the subject has not received or has not undergone cancer immunotherapy.

[0030] In some embodiments, the subject has received or is receiving a treatment that includes a targeted therapy in combination with a cancer immunotherapy.

[0031] In some embodiments, the subject has not received or has not received a treatment involving a targeted therapy in combination with the cancer immunotherapy.

[0032] In some embodiments, the subject has received or has received a targeted therapy and the cancer has recurred.

[0033] In some embodiments, the subject is a non-responder to a targeted therapy or has been identified as a non-responder to a targeted therapy.

[0034] In some embodiments, the subject is a partial responder to a targeted therapy or has been identified as a partial responder to a targeted therapy.

[0035] In some embodiments, the targeted therapy comprises a BRAF targeting agent and / or a MEK targeting agent. In certain embodiments, the targeted therapy comprises a BRAF targeting agent or a MEK targeting agent. In certain embodiments, the BRAF targeting agent is a BRAF inhibitor. In certain embodiments, the BRAF targeting agent inhibits wild-type BRAF and / or BRAF with V600 mutation (e.g., V600E mutation or V600K mutation). In certain embodiments, the BRAF targeting agent is dabrafenib, vemurafenib, encorafenib, ABM-1310, ARQ 736, ASN003, BGB-283, BGB-3245, CEP-32496, GDC-0879, LUT014, PLX4720, PLX8394, RO5212054, or its pharmaceutically acceptable salt. In certain embodiments, the BRAF targeting agent is dabrafenib. In certain embodiments, the BRAF targeting agent is vemurafenib. In certain embodiments, the BRAF targeting agent (e.g., dabrafenib) is administered (e.g., orally) at a dose between 25 mg and 300 mg (e.g., at a dose between 50 mg and 250 mg or between 100 mg and 200 mg), for example, twice a day. In certain embodiments, the MEK targeting agent is a MEK inhibitor. In certain embodiments, the agent that targets MEK is trametinib, cobimetinib, binimetinib, mirdametinib, pimasertib, refametinib, selumetinib, AS703988, AZD 8330, BI 847325, BIX 02188, BIX 02189, CI-1040, CS3006, E6201, FCN-159, G-38963, GDC-0623, HL-085, PD 98059, RO4987655, RO5126766, SHR 7390, TAK-733, U0126, WX-554, or a pharmaceutically acceptable salt thereof.In certain embodiments, the agent that targets MEK is trametinib. In certain embodiments, the agent that targets MEK is cobimetinib.In certain embodiments, the MEK targeting agent (e.g., trametinib) is administered (e.g., orally) at a dose between 0.1 mg and 5 mg (e.g., at a dose between 0.5 mg and 4 mg or between 1 mg and 3 mg, e.g., at a dose of 2 mg), e.g., once a day. In certain embodiments, the BRAF targeting agent is dabrafenib and the MEK targeting agent is trametinib. In certain embodiments, the BRAF targeting agent is vemurafenib and the MEK targeting agent is cobimetinib.

[0036] In some embodiments, the cancer immunotherapy comprises a PD-1 or PD-L1 binding antagonist.

[0037] In some embodiments, the cancer immunotherapy comprises a PD-1 inhibitor.

[0038] In some embodiments, the cancer immunotherapy comprises an anti-PD-1 antibody molecule. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising the VHCDR1 amino acid sequence of SEQ ID NO:501, the VHCDR2 amino acid sequence of SEQ ID NO:502, and the VHCDR3 amino acid sequence of SEQ ID NO:503; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:510, the VLCDR2 amino acid sequence of SEQ ID NO:511, and the VLCDR3 amino acid sequence of SEQ ID NO:512, each of which is disclosed in Table 1. In certain embodiments, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:506. In certain embodiments, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:520, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:520. In certain embodiments, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:516, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:516. In certain embodiments, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506 and a VL comprising the amino acid sequence of SEQ ID NO:520. In certain embodiments, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506 and a VL comprising the amino acid sequence of SEQ ID NO:516. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:508. In certain embodiments, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:522, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:522.In certain embodiments, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:518, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:518. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508 and a light chain comprising the amino acid sequence of SEQ ID NO:522. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508 and a light chain comprising the amino acid sequence of SEQ ID NO:518. In certain embodiments, the anti-PD-1 antibody molecule is administered to a subject at a dose of about 300 mg to 400 mg once every three weeks or once every four weeks (e.g., about 400 mg once every four weeks).

[0039] In some embodiments, the cancer immunotherapy includes a second cancer immunotherapeutic agent (e.g., a cancer immunotherapeutic agent described herein).

[0040] In some embodiments, the cancer is a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, or myeloma), or a metastasis thereof. In certain embodiments, the cancer is a melanoma or a metastasis thereof. In certain embodiments, the melanoma is stage I melanoma, stage II melanoma, stage III melanoma, or stage IV melanoma. In certain embodiments, the cancer is a cancer other than melanoma. In certain embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, or colorectal cancer, or a metastasis thereof. In certain embodiments, the cancer is refractory to a BRAF-targeting agent and / or a MEK-targeting agent. In certain embodiments, the cancer (e.g., melanoma) comprises a BRAF mutation. In certain embodiments, the BRAF mutation is a V600 mutation. In certain embodiments, the V600 mutation is a V600E or V600K mutation.

[0041] In some embodiments, the method further includes administering an additional treatment to the subject (e.g., a pretreatment to the subject (e.g., to increase the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject). In certain embodiments, the treatment is a first-, second-, third-, or fourth-line or above treatment. In certain embodiments, the treatment is an adjuvant treatment. In certain embodiments, the treatment is a neoadjuvant treatment.

[0042] In some embodiments, the methods include obtaining a value for immune activation level and / or activity, including, for example, TIL, PD-L1, CD8, IFNγ, or a T cell inflammatory gene expression signature as described herein.

[0043] In another aspect, the present disclosure features a method for identifying a subject with cancer who is likely to benefit from a treatment.The method includes obtaining a value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to CD8+ immune effector cell (e.g., CD8+ T cell) level or activity in a subject (e.g., in a sample from the subject), where a value smaller than the reference value identifies the subject as likely to benefit from the treatment, and the treatment includes targeted therapy (e.g., does not use cancer immunotherapy).In some embodiments, the subject is unlikely to have a substantial increase in benefit from a treatment that includes targeted therapy in combination with cancer immunotherapy.

[0044] In another aspect, the disclosure features a method for selecting a treatment for a subject with cancer.In some embodiments, the method includes: obtaining a value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject); and if the value is less than the reference value, selecting a treatment for the subject that includes a targeted treatment (e.g., not using cancer immunotherapy).

[0045] In some embodiments, the method further comprises administering (eg, commencing administration or continuing administration) an effective amount of a treatment to the subject.

[0046] In some embodiments, the method further includes administering to the subject an altered dosing regimen of the treatment (e.g., a dosing regimen comprising a higher dose and / or more frequent administration than the reference dosing regimen).

[0047] In some embodiments, the method further comprises discontinuing administration of the different treatment to the subject.

[0048] In some embodiments, the method further comprises administering an additional treatment to the subject.

[0049] In some embodiments, the method further includes administering a pretreatment to the subject, where the pretreatment increases the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject).

[0050] In another aspect, the present disclosure features a method for treating a subject with cancer.In some embodiments, the method includes the following: when the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in the sample from the subject) is less than a reference value, administer (e.g., start or continue administering) an effective amount of a treatment comprising targeted therapy (e.g., not using cancer immunotherapy) to the subject, thereby treating the subject with cancer.

[0051] In another aspect, the disclosure features a method of treating a subject with cancer, the method includes obtaining a value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to CD8+ immune effector cell (e.g., CD8+ T cell) level or activity in the subject (e.g., in a sample from the subject), and if the value is less than the reference value, administering (e.g., starting or continuing administration) an effective amount of a therapy, including a targeted therapy (e.g., not using a cancer immunotherapy), to the subject, thereby treating the subject with cancer.

[0052] In another aspect, the disclosure features a method for treating a subject with cancer. The method includes administering (e.g., starting or continuing administration) an effective amount of a treatment comprising a targeted therapy in combination with a cancer immunotherapy to the subject; and upon the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) being less than a reference value, administering (e.g., starting or continuing administration) an effective amount of a targeted therapy (e.g., without a cancer immunotherapy) to the subject, thereby treating the subject with cancer.

[0053] In another aspect, the disclosure features a method of treating a subject with cancer, the method including administering (e.g., commencing administration or continuing administration) to a subject an effective amount of a therapy including a targeted therapy (e.g., not using cancer immunotherapy), where prior to administration, a value of a level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to a level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is determined to be less than a reference value, thereby treating the subject with cancer.

[0054] In another aspect, the disclosure features a method of treating a subject having cancer, the method including administering (e.g., commencing or continuing administration of) an effective amount of a therapy that includes a targeted therapy (e.g., not using a cancer immunotherapy) to the subject, thereby treating the subject having cancer, wherein the subject is characterized by a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) that is less than a reference value.

[0055] In some embodiments, the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) comprises the ratio of the amount of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the amount of CD8+ immune effector cells (e.g., CD8+ T cells), e.g., as measured by an assay disclosed herein, e.g., flow cytometry immunophenotyping. In certain embodiments, the value is less than about 3.3 (e.g., less than 3.34). In certain embodiments, the value is less than about 3, 2.5, 2, 1.5, 1, or 0.5. In certain embodiments, the value is less than about 2 (e.g., less than 2.01). In certain embodiments, the value is less than about 1.5, 1, or 0.5.

[0056] In some embodiments, obtaining the value comprises determining the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject, e.g., in a sample from the subject. In certain embodiments, the sample from the subject comprises a blood sample (e.g., a peripheral blood sample containing peripheral blood mononuclear cells (PBMCs)) or a tumor sample. In certain embodiments, the value is obtained before administration of the treatment begins (e.g., is a baseline value). In certain embodiments, the value is obtained after administration of the treatment begins. In certain embodiments, the value is obtained 1, 2, 3, 4, 8, 10, 12, 20, 30, 40 weeks or more or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 months or more after administration of the treatment begins.

[0057] In some embodiments, the method further comprises obtaining a value of TMB in the subject (e.g., in a sample from the subject). In certain embodiments, a decrease in the value of TMB compared to the reference value further identifies that the subject is likely to benefit from a treatment (e.g., without cancer immunotherapy). In certain embodiments, a decrease in the value of TMB compared to the reference value further identifies that the subject is unlikely to have a substantial increase in benefit from a treatment comprising a targeted therapy in combination with cancer immunotherapy. In certain embodiments, the value of TMB is less than 10 mut / Mb, for example, less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 mut / Mb, or less.

[0058] In some embodiments, the method further comprises obtaining a level and / or activity value of PD-L1 in the subject (e.g., in a sample from the subject). In certain embodiments, a decrease in the level and / or activity value of PD-L1, e.g., in conjunction with a decrease in the value of TMB, further identifies that the subject is likely to benefit from a treatment (e.g., without cancer immunotherapy). In certain embodiments, a decrease in the level and / or activity value of PD-L1, e.g., in conjunction with a decrease in the value of TMB, further identifies that the subject is unlikely to have a substantial increase in benefit from a treatment comprising a targeted therapy in combination with cancer immunotherapy. In certain embodiments, the cancer has low or undetectable expression of PD-L1.

[0059] In some embodiments, the subject has been or is being treated with a targeted therapy.

[0060] In some embodiments, the subject was not or has not been treated with a targeted therapy.

[0061] In some embodiments, the subject has undergone or is undergoing cancer immunotherapy.

[0062] In some embodiments, the subject has not received or has not undergone cancer immunotherapy.

[0063] In some embodiments, the subject has received or is receiving a treatment that includes a targeted therapy in combination with a cancer immunotherapy.

[0064] In some embodiments, the subject has not received or has not received a treatment involving a targeted therapy in combination with the cancer immunotherapy.

[0065] In some embodiments, the subject has received or has received a targeted therapy and the cancer has recurred.

[0066] In some embodiments, the subject is a non-responder to a targeted therapy or has been identified as a non-responder to a targeted therapy.

[0067] In some embodiments, the subject is a partial responder to a targeted therapy or has been identified as a partial responder to a targeted therapy.

[0068] In some embodiments, the targeted therapy comprises a BRAF targeting agent and / or a MEK targeting agent. In certain embodiments, the targeted therapy comprises a BRAF targeting agent or a MEK targeting agent. In certain embodiments, the BRAF targeting agent is a BRAF inhibitor. In certain embodiments, the BRAF targeting agent inhibits wild-type BRAF and / or BRAF with V600 mutation (e.g., V600E mutation or V600K mutation). In certain embodiments, the BRAF targeting agent is dabrafenib, vemurafenib, encorafenib, ABM-1310, ARQ 736, ASN003, BGB-283, BGB-3245, CEP-32496, GDC-0879, LUT014, PLX4720, PLX8394, RO5212054, or its pharmaceutically acceptable salt. In certain embodiments, the BRAF targeting agent is dabrafenib. In certain embodiments, the BRAF targeting agent is vemurafenib. In certain embodiments, the BRAF targeting agent (e.g., dabrafenib) is administered (e.g., orally) at a dose of between 25 mg and 300 mg (e.g., between 50 mg and 250 mg or between 100 mg and 200 mg, e.g., 150 mg), e.g., twice a day. In certain embodiments, the MEK targeting agent is a MEK inhibitor. In certain embodiments, the agent that targets MEK is trametinib, cobimetinib, binimetinib, mirdametinib, pimasertib, refametinib, selumetinib, AS703988, AZD 8330, BI 847325, BIX 02188, BIX 02189, CI-1040, CS3006, E6201, FCN-159, G-38963, GDC-0623, HL-085, PD 98059, RO4987655, RO5126766, SHR 7390, TAK-733, U0126, WX-554, or a pharmaceutically acceptable salt thereof.In certain embodiments, the agent that targets MEK is trametinib. In certain embodiments, the agent that targets MEK is cobimetinib.In certain embodiments, the MEK targeting agent (e.g., trametinib) is administered (e.g., orally) at a dose between 0.1 mg and 5 mg (e.g., at a dose between 0.5 mg and 4 mg or between 1 mg and 3 mg, e.g., at a dose of 2 mg), e.g., once a day. In certain embodiments, the BRAF targeting agent is dabrafenib and the MEK targeting agent is trametinib. In certain embodiments, the BRAF targeting agent is vemurafenib and the MEK targeting agent is cobimetinib.

[0069] In some embodiments, the cancer immunotherapy comprises a PD-1 or PD-L1 binding antagonist.

[0070] In some embodiments, the cancer immunotherapy comprises a PD-1 inhibitor.

[0071] In some embodiments, the cancer immunotherapy comprises an anti-PD-1 antibody molecule. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising the VHCDR1 amino acid sequence of SEQ ID NO:501, the VHCDR2 amino acid sequence of SEQ ID NO:502, and the VHCDR3 amino acid sequence of SEQ ID NO:503; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:510, the VLCDR2 amino acid sequence of SEQ ID NO:511, and the VLCDR3 amino acid sequence of SEQ ID NO:512, each of which is disclosed in Table 1. In certain embodiments, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:506. In certain embodiments, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:520, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:520. In certain embodiments, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:516, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:516. In certain embodiments, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506 and a VL comprising the amino acid sequence of SEQ ID NO:520. In certain embodiments, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506 and a VL comprising the amino acid sequence of SEQ ID NO:516. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:508. In certain embodiments, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:522, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:522.In certain embodiments, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:518, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:518. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508 and a light chain comprising the amino acid sequence of SEQ ID NO:522. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508 and a light chain comprising the amino acid sequence of SEQ ID NO:518. In certain embodiments, the anti-PD-1 antibody molecule is administered to a subject at a dose of about 300 mg to 400 mg once every three weeks or once every four weeks (e.g., about 400 mg once every four weeks).

[0072] In some embodiments, the cancer immunotherapy includes a second cancer immunotherapeutic agent (e.g., a cancer immunotherapeutic agent described herein).

[0073] In some embodiments, the cancer is a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, or myeloma), or a metastasis thereof. In certain embodiments, the cancer is a melanoma, or a metastasis thereof. In certain embodiments, the melanoma is stage I melanoma, stage II melanoma, stage III melanoma, or stage IV melanoma. In certain embodiments, the cancer is a cancer other than melanoma. In certain embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, or colorectal cancer, or a metastasis thereof.

[0074] In some embodiments, the cancer is refractory to an agent that targets BRAF and / or an agent that targets MEK.

[0075] In some embodiments, the cancer (e.g., melanoma) comprises a BRAF mutation. In certain embodiments, the BRAF mutation is a V600 mutation. In certain embodiments, the V600 mutation is a V600E or V600K mutation.

[0076] In some embodiments, the method further includes administering an additional treatment to the subject (e.g., a pretreatment to the subject (e.g., to increase the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject). In certain embodiments, the treatment is a first-, second-, third-, or fourth-line or above treatment. In certain embodiments, the treatment is an adjuvant treatment. In certain embodiments, the treatment is a neoadjuvant treatment.

[0077] In some embodiments, the methods include obtaining a value for immune activation level and / or activity, including, for example, TIL, PD-L1, CD8, IFNγ, or a T cell inflammatory gene expression signature as described herein.

[0078] In another aspect, the disclosure features a method of stratifying subjects with cancer into a first group and a second group, the method including obtaining a value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to CD8+ immune effector cell (e.g., CD8+ T cell) level or activity in a subject (e.g., in a sample from the subject), assigning subjects having a value less than the reference value to a first group likely to benefit from a treatment including a targeted therapy (e.g., without a cancer immunotherapy) and assigning subjects having a value greater than or equal to the reference value to a second group likely to benefit from a treatment including a targeted therapy in combination with a cancer immunotherapy.

[0079] In another aspect, the disclosure features a method of stratifying a subject having cancer into a first group and a second group to select a treatment, the method including obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), where a value less than the reference value identifies the subject as a member of the first group likely to benefit from a treatment including a targeted therapy (e.g., without cancer immunotherapy), and a value greater than or equal to the reference value identifies the subject as a member of the second group likely to benefit from a treatment including a targeted therapy in combination with an immunotherapy.

[0080] In some embodiments, the method further comprises administering (eg, commencing administration or continuing administration) an effective amount of a treatment to the subject.

[0081] In some embodiments, the method further includes administering to the subject an altered dosing regimen of the treatment (e.g., a dosing regimen comprising a higher dose and / or more frequent administration than the reference dosing regimen).

[0082] In some embodiments, the method further comprises discontinuing administration of the different treatment to the subject.

[0083] In some embodiments, the method further comprises administering an additional treatment to the subject.

[0084] In some embodiments, the method further includes administering a pretreatment to the subject, where the pretreatment increases the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject).

[0085] In another aspect, the present disclosure features a method for treating a subject with cancer.In some embodiments, the method includes the following: according to the value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to CD8+ immune effector cell (e.g., CD8+ T cell) level or activity in subject (e.g., in the sample from subject), administer (e.g., start or continue administering) an effective amount of treatment that includes targeted therapy (e.g., without cancer immunotherapy) to the subject that has a value that is smaller than the reference value; or administer (e.g., start or continue administering) an effective amount of treatment that includes targeted therapy in combination with cancer immunotherapy to the subject that has a value that is greater than or equal to the reference value, thereby treating the subject with cancer.

[0086] In another aspect, the present disclosure features a method for treating a subject with cancer. The method includes: obtaining the value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to CD8+ immune effector cell (e.g., CD8+ T cell) level or activity in the subject (e.g., in the sample from the subject); administering (e.g., starting or continuing administration) an effective amount of a treatment that includes a targeted treatment (e.g., without cancer immunotherapy) to the subject that has a value that is smaller than the reference value; or administering (e.g., starting or continuing administration) an effective amount of a treatment that includes a targeted treatment in combination with cancer immunotherapy to the subject that has a value that is greater than or equal to the reference value, thereby treating the subject with cancer.

[0087] In another aspect, the present disclosure features a method for treating a subject with cancer. The method includes administering (e.g., starting or continuing administration) an effective amount of a treatment comprising a targeted treatment (e.g., without cancer immunotherapy) to the subject; and receiving a value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), administering (e.g., starting or continuing administration) a treatment comprising a targeted treatment (e.g., without cancer immunotherapy) to the subject having a value less than the reference value, or administering (e.g., starting or continuing administration) a treatment comprising a targeted treatment in combination with cancer immunotherapy to the subject having a value greater than or equal to the reference value, thereby treating the subject with cancer.

[0088] In another aspect, the disclosure features a method for treating a subject with cancer. In some embodiments, the method includes administering to the subject an effective amount of a treatment comprising: (a) a targeted treatment (e.g., without cancer immunotherapy), and the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is determined to be less than a reference value before administration; or (b) a targeted treatment combined with cancer immunotherapy, and the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is determined to be greater than or equal to a reference value before administration, thereby treating the subject with cancer.

[0089] In another aspect, the disclosure features a method for treating a subject with cancer. In some aspects, the method includes administering to the subject an effective amount of a treatment comprising: (a) a targeted treatment (e.g., without cancer immunotherapy) to the subject, wherein the subject is characterized by a value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to the level or activity of CD8+ immune effector cell (e.g., CD8+ T cell) in the subject (e.g., in a sample from the subject) being smaller than a reference value; or (b) a targeted treatment in combination with cancer immunotherapy, wherein the subject is characterized by a value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to the level or activity of CD8+ immune effector cell (e.g., CD8+ T cell) in the subject (e.g., in a sample from the subject) being larger than or equal to a reference value, thereby treating the subject with cancer.

[0090] In some embodiments, the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) comprises the ratio of the amount of CD4+ immune effector cells (e.g., CD4+ T cells) to the amount of CD8+ immune effector cells (e.g., CD8+ T cells), e.g., as measured by an assay disclosed herein, e.g., flow cytometry immunophenotyping. In certain embodiments, the value is greater than or equal to 2 (e.g., 2.01). In certain embodiments, the value is greater than or equal to 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10. In certain embodiments, the value is greater than or equal to 3.3 (e.g., 3.34). In certain embodiments, the value is greater than or equal to 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10. In certain embodiments, the value is less than about 3.3 (e.g., less than about 3.34). In certain embodiments, the value is less than about 3, 2.5, 2, 1.5, 1, or 0.5. In certain embodiments, the value is less than about 2 (e.g., less than about 2.01). In certain embodiments, the value is less than about 1.5, 1, or 0.5.

[0091] In some embodiments, obtaining the value comprises determining the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject, e.g., in a sample from the subject. In certain embodiments, the sample from the subject comprises a blood sample (e.g., a peripheral blood sample containing peripheral blood mononuclear cells (PBMCs)) or a tumor sample. In certain embodiments, the value is obtained before administration of the treatment begins (e.g., is a baseline value). In certain embodiments, the value is obtained after administration of the treatment begins. In certain embodiments, the value is obtained 1, 2, 3, 4, 8, 10, 12, 20, 30, 40 weeks or more or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 months or more after administration of the treatment begins.

[0092] In some embodiments, the method further comprises obtaining a level or activity value of CD8+ tumor infiltrating lymphocytes (TILs) in the subject (e.g., in a sample from the subject), e.g., a tumor having a CD8+ TIL inflammatory phenotype. In certain embodiments, an increase in the level or activity value of CD8+ TILs compared to the reference value further identifies that the subject is likely to benefit from the treatment.

[0093] In some embodiments, the method further comprises obtaining the value of tumor mutation burden (TMB) in the subject (e.g., in a sample from the subject).In certain embodiments, the increase in the value of TMB compared to the reference value further identifies that the subject is likely to benefit from treatment.In certain embodiments, the value of TMB is greater than or equal to 10mut / Mb, for example, greater than or equal to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25mut / Mb.

[0094] In some embodiments, the method further comprises obtaining a level and / or activity value of PD-L1 in the subject (e.g., in a sample from the subject). In certain embodiments, a decrease in the level and / or activity value of PD-L1 compared to the reference value, together with, for example, an increase in the value of TMB compared to the reference value, further identifies that the subject is likely to benefit from the treatment. In certain embodiments, the cancer has low or undetectable expression of PD-L1.

[0095] In some embodiments, the method further comprises obtaining a circulating tumor DNA (ctDNA) value in the subject (e.g., in a sample from the subject). In certain embodiments, an increase in the value of ctDNA compared to the reference value further identifies that the subject is likely to benefit from the treatment. In certain embodiments, a decrease in the value of TMB compared to the reference value further identifies that the subject is likely to benefit from the treatment (e.g., without cancer immunotherapy). In certain embodiments, a decrease in the value of TMB compared to the reference value further identifies that the subject is unlikely to have a substantial increase in benefit from the treatment comprising a targeted therapy in combination with cancer immunotherapy. In certain embodiments, the value of TMB is less than 10 mut / Mb, for example, less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 mut / Mb, or less.

[0096] In some embodiments, the method further comprises obtaining a level and / or activity value of PD-L1 in the subject (e.g., in a sample from the subject). In certain embodiments, a decrease in the level and / or activity value of PD-L1, e.g., in conjunction with a decrease in the value of TMB, further identifies that the subject is likely to benefit from a treatment (e.g., without cancer immunotherapy). In certain embodiments, a decrease in the level and / or activity value of PD-L1, e.g., in conjunction with a decrease in the value of TMB, further identifies that the subject is unlikely to have a substantial increase in benefit from a treatment comprising a targeted therapy in combination with cancer immunotherapy. In certain embodiments, the cancer has low or undetectable expression of PD-L1.

[0097] In some embodiments, subjects more likely to benefit from a treatment or more likely to have an increased benefit from a treatment have improved progression-free survival (PFS), duration of objective response (DOR), and / or overall survival (OS) compared to subjects less likely to benefit from a treatment or less likely to have an increased benefit from a treatment.

[0098] In some embodiments, subjects who are likely to benefit from treatment or who are likely to have increased benefit from treatment have improved PFS, DOR, and / or OS compared with subjects who have not received treatment or subjects who have only received targeted therapy but not cancer immunotherapy.In certain embodiments, PFS, DOR, and / or OS are improved by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60 months or longer.In certain embodiments, PFS, DOR, and / or OS are improved by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.

[0099] In some embodiments, the subject has been or is being treated with a targeted therapy.

[0100] In some embodiments, the subject was not or has not been treated with a targeted therapy.

[0101] In some embodiments, the subject has undergone or is undergoing cancer immunotherapy.

[0102] In some embodiments, the subject has not received or has not undergone cancer immunotherapy.

[0103] In some embodiments, the subject has received or is receiving a treatment that includes a targeted therapy in combination with a cancer immunotherapy.

[0104] In some embodiments, the subject has not received or has not received a treatment involving a targeted therapy in combination with the cancer immunotherapy.

[0105] In some embodiments, the subject has received or has received a targeted therapy and the cancer has recurred.

[0106] In some embodiments, the subject is a non-responder to a targeted therapy or has been identified as a non-responder to a targeted therapy.

[0107] In some embodiments, the subject is a partial responder to a targeted therapy or has been identified as a partial responder to a targeted therapy.

[0108] In some embodiments, the targeted therapy comprises a BRAF targeting agent and / or a MEK targeting agent. In certain embodiments, the targeted therapy comprises a BRAF targeting agent or a MEK targeting agent. In certain embodiments, the BRAF targeting agent is a BRAF inhibitor. In certain embodiments, the BRAF targeting agent inhibits wild-type BRAF and / or BRAF with V600 mutation (e.g., V600E mutation or V600K mutation). In certain embodiments, the BRAF targeting agent is dabrafenib, vemurafenib, encorafenib, ABM-1310, ARQ 736, ASN003, BGB-283, BGB-3245, CEP-32496, GDC-0879, LUT014, PLX4720, PLX8394, RO5212054, or its pharmaceutically acceptable salt. In certain embodiments, the BRAF targeting agent is dabrafenib. In certain embodiments, the BRAF targeting agent is vemurafenib. In certain embodiments, the BRAF targeting agent (e.g., dabrafenib) is administered (e.g., orally) at a dose of between 25 mg and 300 mg (e.g., between 50 mg and 250 mg or between 100 mg and 200 mg, e.g., 150 mg), e.g., twice a day. In certain embodiments, the MEK targeting agent is a MEK inhibitor. In certain embodiments, the agent that targets MEK is trametinib, cobimetinib, binimetinib, mirdametinib, pimasertib, refametinib, selumetinib, AS703988, AZD 8330, BI 847325, BIX 02188, BIX 02189, CI-1040, CS3006, E6201, FCN-159, G-38963, GDC-0623, HL-085, PD 98059, RO4987655, RO5126766, SHR 7390, TAK-733, U0126, WX-554, or a pharmaceutically acceptable salt thereof.In certain embodiments, the agent that targets MEK is trametinib. In certain embodiments, the agent that targets MEK is cobimetinib.In certain embodiments, the MEK targeting agent (e.g., trametinib) is administered (e.g., orally) at a dose between 0.1 mg and 5 mg (e.g., at a dose between 0.5 mg and 4 mg or between 1 mg and 3 mg, e.g., at a dose of 2 mg), e.g., once a day. In certain embodiments, the BRAF targeting agent is dabrafenib and the MEK targeting agent is trametinib. In certain embodiments, the BRAF targeting agent is vemurafenib and the MEK targeting agent is cobimetinib.

[0109] In some embodiments, the cancer immunotherapy comprises a PD-1 or PD-L1 binding antagonist.

[0110] In some embodiments, the cancer immunotherapy comprises a PD-1 inhibitor.

[0111] In some embodiments, the cancer immunotherapy comprises an anti-PD-1 antibody molecule. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising the VHCDR1 amino acid sequence of SEQ ID NO:501, the VHCDR2 amino acid sequence of SEQ ID NO:502, and the VHCDR3 amino acid sequence of SEQ ID NO:503; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:510, the VLCDR2 amino acid sequence of SEQ ID NO:511, and the VLCDR3 amino acid sequence of SEQ ID NO:512, each of which is disclosed in Table 1. In certain embodiments, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:506. In certain embodiments, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:520, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:520. In certain embodiments, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:516, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:516. In certain embodiments, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506 and a VL comprising the amino acid sequence of SEQ ID NO:520. In certain embodiments, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506 and a VL comprising the amino acid sequence of SEQ ID NO:516. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:508. In certain embodiments, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:522, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:522.In certain embodiments, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:518, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:518. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508 and a light chain comprising the amino acid sequence of SEQ ID NO:522. In certain embodiments, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508 and a light chain comprising the amino acid sequence of SEQ ID NO:518.

[0112] In some embodiments, the anti-PD-1 antibody molecule is administered to a subject at a dose of about 300 mg to 400 mg once every three weeks or once every four weeks (e.g., about 400 mg once every four weeks).

[0113] In some embodiments, the cancer immunotherapy includes a second cancer immunotherapeutic agent (e.g., a cancer immunotherapeutic agent described herein).

[0114] In some embodiments, the cancer is a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, or myeloma), or a metastasis thereof. In certain embodiments, the cancer is a melanoma, or a metastasis thereof. In certain embodiments, the melanoma is stage I melanoma, stage II melanoma, stage III melanoma, or stage IV melanoma. In certain embodiments, the cancer is a cancer other than melanoma. In certain embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, or colorectal cancer, or a metastasis thereof.

[0115] In some embodiments, the cancer is refractory to an agent that targets BRAF and / or an agent that targets MEK.

[0116] In some embodiments, the cancer (e.g., melanoma) comprises a BRAF mutation. In certain embodiments, the BRAF mutation is a V600 mutation. In certain embodiments, the V600 mutation is a V600E or V600K mutation.

[0117] In some embodiments, the method further includes administering an additional treatment to the subject (e.g., a pretreatment to the subject (e.g., to increase the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject). In certain embodiments, the treatment is a first-, second-, third-, or fourth-line or above treatment. In certain embodiments, the treatment is an adjuvant treatment. In certain embodiments, the treatment is a neoadjuvant treatment.

[0118] In some embodiments, the methods include obtaining a value for immune activation level and / or activity, including, for example, TIL, PD-L1, CD8, IFNγ, or a T cell inflammatory gene expression signature as described herein.

[0119] In another aspect, the present disclosure features a treatment comprising a targeted therapy in combination with cancer immunotherapy for use in a method for treating a subject with cancer.In some embodiments, the method comprises administering (e.g., starting administration or continuing administration) an effective amount of a treatment comprising a targeted therapy in combination with cancer immunotherapy to the subject, when the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in the sample from the subject) is greater than or equal to a reference value.

[0120] In another aspect, the present disclosure features a treatment comprising a targeted therapy in combination with cancer immunotherapy for use in a method for treating a subject with cancer.In some embodiments, the method includes obtaining the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject), and when the value is greater than or equal to the reference value, administering (e.g., starting administration or continuing administration) an effective amount of a treatment comprising a targeted therapy in combination with cancer immunotherapy to the subject.

[0121] In another aspect, the present disclosure features a treatment comprising a targeted therapy combined with cancer immunotherapy for use in a method for treating a subject with cancer.In some embodiments, the method includes administering (e.g., starting administration or continuing administration) an effective amount of a targeted therapy to the subject; and administering (e.g., starting administration or continuing administration) an effective amount of a cancer immunotherapy to the subject when the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in the sample from the subject) is greater than or equal to a reference value.

[0122] In another aspect, the disclosure features a treatment comprising a targeted therapy in combination with a cancer immunotherapy for use in a method of treating a subject with cancer. In some embodiments, the method includes administering (e.g., initiating administration or continuing administration) an effective amount of a treatment comprising a targeted therapy in combination with a cancer immunotherapy to a subject, and determining before administration that the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is greater than or equal to a reference value.

[0123] In another aspect, the disclosure features a treatment comprising a targeted therapy in combination with a cancer immunotherapy for use in a method of treating a subject with cancer. In some embodiments, the method includes administering (e.g., initiating or continuing administration) an effective amount of a treatment comprising a targeted therapy in combination with a cancer immunotherapy to a subject, wherein the subject is characterized by a value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to CD8+ immune effector cell (e.g., CD8+ T cell) level or activity in the subject (e.g., in a sample from the subject) that is greater than or equal to a reference value.

[0124] In another aspect, the present disclosure features a treatment comprising targeted therapy for use in a method for treating a subject with cancer.In some embodiments, the method comprises administering (e.g., starting administration or continuing administration) an effective amount of a treatment comprising targeted therapy (e.g., not using cancer immunotherapy) to the subject in response to the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in the sample from the subject) being smaller than a reference value.

[0125] In another aspect, the present disclosure features a treatment comprising targeted therapy for use in a method for treating a subject with cancer.In some embodiments, the method includes obtaining the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject), and if the value is smaller than the reference value, administering (e.g., starting administration or continuing administration) an effective amount of a treatment comprising targeted therapy (e.g., not using cancer immunotherapy) to the subject.

[0126] In another aspect, the present disclosure features a treatment comprising targeted therapy for use in a method for treating a subject with cancer.In some embodiments, the method includes administering (e.g., starting administration or continuing administration) an effective amount of a treatment comprising targeted therapy in combination with cancer immunotherapy to the subject; and administering (e.g., starting administration or continuing administration) an effective amount of targeted therapy (e.g., without cancer immunotherapy) to the subject when the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in the sample from the subject) is smaller than a reference value.

[0127] In another aspect, the disclosure features a treatment comprising a targeted therapy for use in a method of treating a subject with cancer. In some embodiments, the method includes administering (e.g., starting administration or continuing administration) an effective amount of a treatment comprising a targeted therapy (e.g., not using cancer immunotherapy) to a subject, and determining before administration that the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is less than a reference value.

[0128] In another aspect, the disclosure features a treatment comprising a targeted therapy for use in a method of treating a subject with cancer. In some embodiments, the method includes administering (e.g., initiating or continuing administration) an effective amount of a treatment comprising a targeted therapy (e.g., not using cancer immunotherapy) to a subject, wherein the subject is characterized by a value of CD4+ immune effector cell (e.g., CD4+ T cell) level or activity relative to CD8+ immune effector cell (e.g., CD8+ T cell) level or activity in the subject (e.g., in a sample from the subject) that is less than a reference value.

[0129] In another aspect, the present disclosure features a treatment comprising targeted therapy or a treatment comprising targeted therapy in combination with cancer immunotherapy for use in a method for treating a subject with cancer.In some embodiments, the method comprises the following: according to the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject), administer (e.g., start or continue administering) an effective amount of a treatment comprising targeted therapy (e.g., without cancer immunotherapy) to the subject having a value that is less than the reference value; or administer (e.g., start or continue administering) an effective amount of a treatment comprising targeted therapy in combination with cancer immunotherapy to the subject having a value that is greater than or equal to the reference value.

[0130] In another aspect, the present disclosure features a treatment comprising targeted therapy or comprising targeted therapy in combination with cancer immunotherapy for use in a method for treating a subject with cancer.In some embodiments, the method includes: obtaining the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject); administering (e.g., starting or continuing administration) an effective amount of treatment comprising targeted therapy (e.g., without cancer immunotherapy) to the subject having a value that is less than the reference value; or administering (e.g., starting or continuing administration) an effective amount of treatment comprising targeted therapy in combination with cancer immunotherapy to the subject having a value that is greater than or equal to the reference value.

[0131] In another aspect, the present disclosure features a treatment comprising targeted therapy or a treatment comprising targeted therapy combined with cancer immunotherapy for use in a method for treating a subject with cancer.In some embodiments, the method includes administering (e.g., starting administration or continuing administration) an effective amount of a treatment comprising targeted therapy (e.g., without immunotherapy) to a subject; and receiving the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), administering (e.g., starting administration or continuing administration) a treatment comprising targeted therapy (e.g., without cancer immunotherapy) to the subject having a value less than the reference value, or administering (e.g., starting administration or continuing administration) a treatment comprising targeted therapy combined with cancer immunotherapy to the subject having a value greater than or equal to the reference value.

[0132] In another aspect, the disclosure features a treatment that includes a targeted therapy or a treatment that includes a targeted therapy in combination with a cancer immunotherapy for use in a method of treating a subject having cancer comprising administering to a subject an effective amount of the treatment that includes: (a) a targeted therapy (e.g., without a cancer immunotherapy), where prior to administration, it has been determined that a value for a level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to a level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is less than a reference value; or (b) a targeted therapy in combination with a cancer immunotherapy, where prior to administration, it has been determined that a value for a level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to a level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is greater than or equal to a reference value.

[0133] In another aspect, the disclosure features a treatment comprising a targeted therapy or in combination with a targeted therapy for use in a method of treating a subject having cancer comprising administering to a subject an effective amount of the treatment comprising: (a) a targeted therapy to the subject (e.g., without cancer immunotherapy), where the subject is characterized or characterized by a value for a level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to a level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) that is less than a reference value; or (b) a targeted therapy in combination with a cancer immunotherapy, where the subject is characterized or characterized by a value for a level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to a level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) that is greater than or equal to a reference value. [Brief description of the drawings]

[0134] [Figure 1] Figure 1 is a consortium diagram of the clinical trial. BID, twice a day; Q4W, once every 4 weeks; QD, once a day. [Figure 2-1]Figures 2A-2C are a series of plots showing flow cytometry and cytokine analysis of plasma and peripheral blood mononuclear cells at baseline and after 4 weeks of treatment. Immunophenotyping of peripheral blood mononuclear cells using markers for T cell activation and proliferation (N=323), and cytokine profiling of plasma samples taken at baseline and after 4 weeks of treatment (N=468). Proliferating CD8+ / PD-1+ T cells (2A), activated cytotoxic CD8+ T cells (2B), and plasma IFN-γ (2C) are shown. CD=classification of cell surface antigens; HLA-DR=human leukocyte antigen, DR subtype; IFN=interferon; PD-1=programmed death receptor 1; placebo-DabTram=placebo plus dabrafenib and trametinib; sparta-DabTram=spartalizumab plus dabrafenib and trametinib. [Figure 2-2] Same as above. [Figure 3-1] Figures 3A-3C are a series of plots showing further results from immunophenotyping of peripheral blood mononuclear cells at baseline and after 4 weeks of treatment. Immunophenotyping of peripheral blood mononuclear cells using markers for T cell activation and proliferation in plasma samples taken at baseline and after 4 weeks of treatment (N=323). Levels of proliferating CD8+ T cells (Figure 3A), activated CD4+ T cells (Figure 3B), and proliferating PD-1+ / CD4+ T cells (Figure 3C) are shown. CD=classification of cell surface antigens; HLA-DR=human leukocyte antigen, DR subtype; PD-1=programmed death 1; placebo-DabTram=placebo plus dabrafenib and trametinib; sparta-DabTram=spartalizumab plus dabrafenib and trametinib. [Figure 3-2] Same as above. [Figure 4-1]Figures 4A-4E are a series of plots showing further results from cytokine profiling at baseline and after 4 weeks of treatment. Shown are levels of CXCL10 (N=474) (Figure 4A), IL-15 (N=473) (Figure 4B), IL-18 (N=467) (Figure 4C), IL-17A (N=473) (Figure 4D), and IL-12p40 (N=473) (Figure 4E) in plasma samples taken at baseline and after 4 weeks of treatment. CXCL=chemokine ligand of the CXC motif; IL=interleukin; placebo-DabTram=placebo with dabrafenib and trametinib; sparta-DabTram=spartalizumab with dabrafenib and trametinib. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1] Figures 5A and 5B are a pair of plots showing progression-free survival based on baseline peripheral CD4+ / CD8+ ratio and treatment arm. Kaplan-Meier estimates of progression-free survival based on baseline peripheral blood CD4+ / CD8+ ratio in peripheral blood mononuclear cell samples from patients randomized to the placebo-DabTram arm (N=208) (Figure 5A) or sparta-DabTram arm (N=200) (Figure 5B) are shown. HR = hazard ratio; placebo-DabTram = placebo plus dabrafenib and trametinib; sparta-DabTram = spartalizumab plus dabrafenib and trametinib. [Figure 5-2] Same as above. [Figure 6-1]Figures 6A and 6B are a pair of plots showing progression-free survival based on baseline peripheral CD4+ / CD8+ T-cell ratio. Kaplan-Meier estimates of progression-free survival in patients randomized to either the sparta-DabTram arm or the placebo-DabTram arm reflecting peripheral blood mononuclear cell samples with baseline CD4+ / CD8+ T-cell ratios equal to or greater than the median baseline ratio of 2.9 (N=204) (Figure 6A) or less than the median baseline ratio of 2.9 (N=204) (Figure 6B) are shown. Figure 6C shows the relationship between the cut points selected for patient selection and the hazard ratios of the treatment arms. Hazard ratios are shown in black and 95% confidence intervals in red. The two blue dots indicate the range of the optimized cutoff between 2.02 and 3.34. CD=cell surface classification; HR=hazard ratio; placebo-DabTram=placebo plus dabrafenib and trametinib; sparta-DabTram=spartalizumab plus dabrafenib and trametinib. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 7-1] Figures 7A-7B are a series of plots showing progression-free survival and overall survival based on combined PD-L1 status and TMB. Kaplan-Meier estimates of progression-free survival (Figure 7A) and overall survival (Figure 7B) based on PD-L1 status (<1% [negative] or ≥1% [positive]) and TMB (<10 mut / Mb [low] or ≥10 mut / Mb [high]) are shown. PD-L1 negative / TMB high, N=62; PD-L1 negative / TMB low, N=109; PD-L1 positive / TMB high, N=110; PD-L1 positive / TMB low, N=119. HR=hazard ratio; mut=mutation; NE=not estimable; PD-L1=programmed death-ligand 1; placebo-DabTram=placebo plus dabrafenib and trametinib; sparta-DabTram=spartalizumab plus dabrafenib and trametinib; TMB=tumor mutation burden. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 7-6] Same as above. [Figure 7-7] Same as above. [Figure 7-8] Same as above. [Figure 8-1] Figures 8A-8D are a series of plots showing progression-free survival after next-line treatment based on PD-L1 / TMB subgroups. Kaplan-Meier estimates of progression-free survival after next-line treatment (PFS2) in patients whose tumors were PD-L1 negative / TMB high (N=62) (Figure 8A), PD-L1 negative / TMB low (N=109) (Figure 8B), PD-L1 positive / TMB high (N=110) (Figure 8C), or PD-L1 positive / TMB low (N=119) (Figure 8D) tumors are shown. HR = hazard ratio; NE = not estimable; PD-L1 = programmed death-ligand 1; placebo-DabTram = placebo plus dabrafenib and trametinib; sparta-DabTram = spartalizumab plus dabrafenib and trametinib; TMB = tumor mutation burden. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 9-1] Figures 9A-9E are a series of plots and images showing analysis of the tumor microenvironment across combined PD-L1 / TMB-defined subgroups. Comparison of T cell inflammatory signature levels (N=362) (Figure 9A) and antigen-presenting cell numbers based on HLA-DR (N=320) (Figure 9B) and CD11b (N=320) (Figure 9C) expression within tumor samples from PD-L1 / TMB-defined subgroups. Antigen-presenting cells were analyzed using multiplex fluorescent immunohistochemistry, and examples of high (Figure 9D) and low expressers (Figure 9E) are shown. CD = clustering; CPM = counts per million; DAPI = 4',6-diamidino-2-phenylindole; GES = gene expression signature; HLA-DR = human leukocyte antigen, DR subtype; PD-L1 = programmed death ligand 1; TMB = tumor mutation burden. [Figure 9-2]Same as above. [Figure 9-3] Same as above. [Figure 10] Figures 10A and 10B are a pair of plots showing an analysis of tumor mutational burden (N=384) (Figure 10A) and age (N=455) (Figure 10B) based on BRAF V600K status vs. V600E mutation status. mut / Mb = mutations per megabase; TMB = tumor mutational burden. [Figure 11-1] Figures 11A and 11B are a pair of plots showing progression-free survival based on BRAF V600E / K mutation status. Kaplan-Meier estimates of progression-free survival in patients with confirmed BRAF V600E mutant disease (N=402) (Figure 11A) or V600K mutant disease (N=53) (Figure 11B) based on central review are shown. HR = hazard ratio; NE = not estimable; placebo-DabTram = placebo plus dabrafenib and trametinib; sparta-DabTram = spartalizumab plus dabrafenib and trametinib. [Figure 11-2] Same as above. [Figure 12-1] Figures 12A and 12B are a pair of plots showing overall survival based on BRAF V600E / K mutation status. Kaplan-Meier estimates of overall survival in patients with confirmed BRAF V600E (N=402) (Figure 12A) or V600K (N=53) (Figure 12B) mutant disease based on central review are shown. HR = hazard ratio; NE = not estimable; placebo-DabTram = placebo plus dabrafenib and trametinib; sparta-DabTram = spartalizumab plus dabrafenib and trametinib. [Figure 12-2] Same as above. [Figure 13]Figure 13 is a plot showing downregulation of the SPRY signaling pathway in patients with BRAF V600K mutation disease. Evaluation of the SPRY signaling pathway gene expression signature in tumor samples (N=369) included analysis of MAPK3, SPRY2, HRAS, MAP2K1, RAF1, SPRY4, SPRY3, EGF, MAPK1, SHC1, PTPRB, SPRY1, GRB2, EGFR, CBL, RASA1, and SOS1 genes. GES=gene expression signature. [Figure 14-1] Figures 14A and 14B include a Venn diagram and a pair of plots showing progression-free survival benefit based on gene expression signature levels. Among the top 100 gene expression signatures in each arm, there was an overlap of 49 signatures (listed in Table E) that were highly expressed in tumor samples from both sparta-DabTram-treated and placebo-DabTram-treated patients. Examples of the prognostic and predictive value of these signatures are shown in Figure 14B, as exemplified by Kaplan-Meier estimates of progression-free survival based on the expression of the T cell inflammation signature (low expression, N=110; high expression, N=323). GES=gene expression signature; HR=hazard ratio; NE=not estimable; placebo-DabTram=placebo with dabrafenib and trametinib; sparta-DabTram=spartalizumab with dabrafenib and trametinib. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 15-1]Figures 15A and 15B include a series of images and plots showing the characterization of T cell phenotypes (15A) and their impact on prognosis (15B). Representative samples of inflammatory, exclusion, and desert T cell phenotypes obtained from digital pathology immunohistochemistry of tumor- and stromal-infiltrating lymphocytes are shown in Figure 15A. Kaplan-Meier estimates of progression-free survival based on these phenotypes in the placebo-DabTram (top; inflammatory, n=73; exclusion, n=72; desert, n=62) and sparta-DabTram (bottom; inflammatory, n=66; exclusion, n=91; desert, n=52) treatment arms are shown in Figure 15B. HR = hazard ratio; placebo-DabTram = placebo plus dabrafenib and trametinib; sparta-DabTram = spartalizumab plus dabrafenib and trametinib. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above. [Figure 15-5] Same as above. [Figure 15-6] Same as above. [Figure 16-1] Figures 16A-16C are a series of plots showing the benefit of treatment based on T cell phenotype. Kaplan-Meier estimates of progression-free survival and overall survival based on inflammatory (N=139) (Figure 16A), elimination (N=163) (Figure 16B), and immune desert (N=115) (16C) CD8+ tumor-infiltrating lymphocyte phenotype in each treatment arm are shown. CD = surface antigen classification; HR = hazard ratio; placebo-DabTram = placebo plus dabrafenib and trametinib; sparta-DabTram = spartalizumab plus dabrafenib and trametinib. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above. [Figure 16-5] Same as above. [Figure 16-6] Same as above. [Figure 17]Figures 17A-17C are a series of plots showing that baseline ctDNA shedding correlates with disease burden. Baseline ctDNA shedding based on lactate dehydrogenase levels (N=391) (Figure 17A), sum of lesion diameters (N=474) (Figure 17B), and number of target lesions (N=474) (Figure 17C). ctDNA = circulating tumor DNA; LDH, lactate dehydrogenase. [Figure 18-1] Figures 18A and 18B are a pair of plots showing baseline ctDNA shedding based on best overall response and treatment arm. Baseline ctDNA shedding based on best overall response for patients treated with placebo-DabTram (N=246) (Figure 18A) or sparta-DabTram (N=234) (Figure 18B). BOR=best overall response; cfDNA=cell-free DNA; CR=complete response; ctDNA=circulating tumor DNA; PD=progressive disease; placebo-DabTram=placebo with dabrafenib and trametinib; PR=partial response; SD=stable disease; sparta-DabTram=spartalizumab with dabrafenib and trametinib; UNK=unknown. [Figure 18-2] Same as above. [Figure 19-1]Figures 19A and 19B are a series of plots showing the predictive and prognostic value of ctDNA shedding at baseline and on treatment. Kaplan-Meier estimates of progression-free survival and overall survival based on ctDNA shedding at baseline and on treatment in either the placebo-DabTram or sparta-DabTram arm are shown in Figure 19A. "No shedding" indicates that ctDNA shedding was not observed at baseline or at week 8 (placebo-DabTram, n=53; sparta-DabTram, n=56), "Loss at W8" indicates that shedding was observed at baseline but not at week 8 (placebo-DabTram, n=122; sparta-DabTram, n=136), and "Loss at W8" indicates that shedding was observed at both baseline and week 8 (placebo-DabTram, n=27; sparta-DabTram, n=16). Kaplan-Meier estimates of progression-free survival and overall survival based on treatment with placebo-DabTram or sparta-DabTram for patients with (N=342) or without (N=138) baseline ctDNA shedding are shown in Figure 19B. ctDNA = circulating tumor DNA; HR = hazard ratio; placebo-DabTram = placebo plus dabrafenib and trametinib; sparta-DabTram = spartalizumab plus dabrafenib and trametinib. [Figure 19-2] Same as above. [Figure 19-3] Same as above. [Figure 19-4] Same as above. [Figure 19-5] Same as above. [Figure 19-6] Same as above. [Figure 19-7] Same as above. [Figure 19-8] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0135] Detailed Description The present disclosure provides methods and compositions for the treatment and diagnosis of cancer, particularly melanoma. The present disclosure is based at least in part on the discovery that adding cancer immunotherapy to targeted therapy can extend progression-free survival in patients with high baseline CD4+ / CD8+ ratios. Without wishing to be bound by theory, in some embodiments, blood CD4+ / CD8+ ratios appear to be associated with response to treatments that include targeted therapy and cancer immunotherapy, and are shown to be a useful non-invasive predictive and prognostic indicator for cancer patients. In these patients, treatment with targeted therapy in combination with cancer immunotherapy may improve progression-free survival compared to treatment with targeted therapy alone.

[0136] Thus, determining the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) can be used as a biomarker (e.g., predictive biomarker) in the treatment of subjects with cancer (e.g., melanoma), to determine whether a subject with cancer (e.g., melanoma) is likely to respond to treatment with a cancer therapy (e.g., melanoma therapy) that includes a targeted therapy (e.g., a targeted therapy including an agent that targets BRAF and / or an agent that targets MEK) in combination with a cancer immunotherapy, to optimize the therapeutic efficacy of a cancer therapy (e.g., melanoma therapy) that includes a targeted therapy (e.g., a targeted therapy including an agent that targets BRAF and / or an agent that targets MEK) in combination with a cancer immunotherapy, and for patient selection for a cancer therapy (e.g., melanoma therapy) that includes a targeted therapy (e.g., a targeted therapy including an agent that targets BRAF and / or an agent that targets MEK) in combination with a cancer immunotherapy.

[0137] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0138] It should be understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0139] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0140] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0141] "About" and "approximately" are generally intended to mean an acceptable degree of error for a measured quantity given the nature or precision of the measurement. Exemplary degrees of error are within 20% (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0142] By "combination" or "in combination with," it is not intended to imply that the treatments or therapeutic agents must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope described herein. The therapeutic agents of the combination can be administered simultaneously with, before, or after one or more other additional treatments or therapeutic agents. The therapeutic agents or treatment protocols can be administered in any order. Generally, each agent is administered at a dose and / or time schedule determined for that agent. It will further be understood that the additional therapeutic agents utilized in the combination may be administered together in a single composition or separately in different compositions. In general, it is expected that the additional therapeutic agents utilized in the combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in the combination will be lower than the levels utilized individually.

[0143] In some embodiments, the additional therapeutic agent is administered at a therapeutic dose or a sub-therapeutic dose. In certain embodiments, the concentration of the second or third therapeutic agent required to achieve inhibition, e.g., growth inhibition, is lower than when the second or third therapeutic agent is administered in combination with the first therapeutic agent, and is lower than when the second or third therapeutic agent is administered individually. In certain embodiments, the concentration of the first therapeutic agent required to achieve inhibition, e.g., growth inhibition, is lower than when the first therapeutic agent is administered in combination with the second or third therapeutic agent, and is lower than when the first therapeutic agent is administered individually. In certain embodiments, the concentration of the second or third therapeutic agent required to achieve inhibition, e.g., growth inhibition, in the combination therapy is lower than the therapeutic dose of the second or third therapeutic agent as a monotherapy, e.g., 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% lower. In certain embodiments, the concentration of the first therapeutic agent required to achieve inhibition, e.g., growth inhibition, in a combination therapy is lower than the therapeutic dose of the first therapeutic agent as a monotherapy, e.g., 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% lower.

[0144] The terms "inhibition", "inhibitor", "binding antagonist" or "antagonist" include a reduction in a particular parameter, such as the activity of a given molecule, such as an immune checkpoint inhibitor. For example, inhibition of at least 5%, 10%, 20%, 30%, 40% or more of the activity, such as the activity of a given molecule, such as an inhibitory molecule, is included in the term. Thus, inhibition need not be 100%.

[0145] The terms "activation," "activator," and "agonist" include an increase in a particular parameter, e.g., the activity of a given molecule, e.g., a costimulatory molecule. For example, an increase in activity, e.g., costimulatory activity, of at least 5%, 10%, 25%, 50%, 75% or more is included in the terms.

[0146] The term "anti-cancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in the survival time of cancer cells, or an amelioration of various physiological symptoms associated with the cancerous condition. An "anti-cancer effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to forestall the onset of cancer.

[0147] The term "anti-tumor effect" refers to a biological effect that can be manifested by a variety of means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0148] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal 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, but are not limited to, melanoma, lung cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse tumors or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant as well as malignant cancers and tumors. The term "cancer," as used herein, includes primary malignant cells or tumors (e.g., those in which cells have not migrated to sites within a subject's body other than the site of the original malignant tumor or tumor) and secondary malignant cells or tumors (e.g., those resulting from metastasis, the migration of malignant or tumor cells to a secondary site different from the site of the original tumor).

[0149] As used herein, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and / or duration of a disorder, e.g., a proliferative disorder, or the amelioration of one or more symptoms (preferably one or more discernible symptoms) of a disorder resulting from the administration of one or more therapies. In specific embodiments, the terms "treat", "treatment" and "treating" refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, not necessarily discernible by the patient. In other embodiments, the terms "treat", "treatment" and "treating" refer to the inhibition of the progression of a proliferative disorder, either physical, e.g., by stabilization of a discernible symptom, physiological, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat", "treatment" and "treating" refer to the reduction or stabilization of tumor size or cancerous cell number.

[0150] The compositions and methods of the present disclosure encompass polypeptides and nucleic acids having a designated sequence or a sequence that is substantially identical or similar thereto, for example, a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or higher to the designated sequence. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimal number of amino acid residues that are i) identical to or ii) conservatively substituted for the aligned amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences may have a common structural domain and / or a common functional activity. For example, an amino acid sequence that contains a common structural domain that has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, for example, a sequence provided herein.

[0151] In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient number or a minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences code for a polypeptide having a common functional activity or code for a common structural polypeptide domain or common functional polypeptide activity. For example, a nucleotide sequence that has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, such as a sequence provided herein.

[0152] The term "amino acid" is intended to include all molecules, whether natural or synthetic, that contain both amino and acid functionality and can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes both D- or L-optical isomers and peptidomimetics.

[0153] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein (when single chain) to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic procedures.

[0154] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence", and "polynucleotide" are used interchangeably. They refer to polymeric forms of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may be single-stranded or double-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. A nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin that does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.

[0155] Various aspects of the disclosure are described in further detail below. Additional definitions are set forth throughout the specification.

[0156] Therapeutic Uses and Biomarkers In one aspect, a method is provided herein that includes: (i) identifying a subject (e.g., a subject having a cancer, e.g., a cancer described herein) that is likely to benefit from a treatment (e.g., a treatment comprising a targeted therapy in combination with a cancer immunotherapy or without a cancer immunotherapy); (ii) identifying a subject that is likely to have an increased benefit from a treatment (e.g., a treatment comprising a targeted therapy in combination with a cancer immunotherapy compared to a treatment comprising a targeted therapy without a cancer immunotherapy agent); (iii) selecting a treatment for the subject; (iv) treating the subject; and (v) stratifying the subject. The method includes evaluating one or more biomarkers, wherein the one or more biomarkers indicate a likelihood of benefit or efficacy of the treatment.

[0157] In some embodiments, the method includes obtaining, determining, and / or evaluating one or more (e.g., two, three, four, or all) of the following: (i) the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells); (ii) a value of a level or activity of immune activation, including TIL, PD-L1, a CD8 phenotype defined by analysis of digital pathology images (e.g., inflammatory, exclusion, and desert based on analysis of digital pathology images), IFNγ, or a T cell inflammatory gene expression signature, e.g., as described herein, e.g., a tumor having a CD8+ TIL inflammatory phenotype; (iii) the value of the loading period (TMB); (iv) PD-L1 level and / or activity; and / or (v) Circulating tumor DNA (ctDNA) levels.

[0158] In some embodiments, the method comprises obtaining, determining and / or evaluating a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells). In some embodiments, the method further comprises obtaining, determining and / or evaluating any one of (ii), (iii), (iv), or (v). In some embodiments, the method further comprises obtaining, determining and / or evaluating any two of (ii), (iii), (iv), or (v). In some embodiments, the method further comprises obtaining, determining and / or evaluating any three of (ii), (iii), (iv), or (v). In some embodiments, the method further comprises obtaining, determining and / or evaluating all of (ii), (iii), (iv), and (v).

[0159] Thus, in some embodiments, a method of identifying a subject having cancer who is likely to benefit from a treatment includes obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject) using digital pathology image analysis using tumor markup areas to define absolute numbers of CD8+ TILs within tumor nests and / or stromal compartments.

[0160] In some embodiments, a value greater than or equal to the reference value identifies the subject as likely to benefit from a treatment that includes a targeted therapy (e.g., includes an agent that targets BRAF and / or an agent that targets MEK) in combination with a cancer immunotherapy (e.g., an anti-PD-1 therapy). In some embodiments, the subject is likely to have an increased benefit from the treatment compared to a treatment that includes a targeted therapy without the cancer immunotherapy.

[0161] In some embodiments, a value greater than or equal to the reference value identifies the subject as likely to benefit from the treatment, and the treatment is a cancer immunotherapy (e.g., a PD-1 binding antagonist) as a monotherapy. In some embodiments, the subject is likely to have an increased benefit from the treatment compared to a treatment other than the cancer immunotherapy.

[0162] In some embodiments, a value less than the reference value identifies the subject as unlikely to have a substantial increase in benefit from a treatment comprising a targeted therapy (e.g., comprising an agent that targets BRAF and / or an agent that targets MEK) in combination with the cancer immunotherapy.

[0163] In some embodiments, a method of selecting a treatment for a subject with cancer includes obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), and if the value is greater than or equal to the reference value, selecting a treatment for the subject that includes a targeted therapy in combination with a cancer immunotherapy.

[0164] In some embodiments, a method of selecting a treatment for a subject with cancer includes obtaining a value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), and if the value is greater than or equal to the reference value, selecting the treatment for the subject, where the treatment is a cancer immunotherapy (e.g., a PD-1 binding antagonist) as a monotherapy.

[0165] In some embodiments, a method of selecting a treatment for a subject with cancer includes obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), and selecting a treatment for the subject that includes a targeted therapy (e.g., no cancer immunotherapy) if the value is less than the reference value.

[0166] In some embodiments, upon a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject) being greater than or equal to a reference value, the method of treating includes administering (e.g., commencing or continuing administration of) an effective amount of a therapy comprising a targeted therapy in combination with a cancer immunotherapy to the subject, thereby treating the subject with cancer.

[0167] In some embodiments, upon a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject) being greater than or equal to a reference value, a method of treating includes administering (e.g., commencing or continuing administration of) an effective amount of a therapy to the subject, thereby treating the subject with cancer, where the therapy is a cancer immunotherapy (e.g., a PD-1 binding antagonist) as a monotherapy.

[0168] In some embodiments, a method of treating a subject with cancer includes obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), and if the value is greater than or equal to the reference value, administering (e.g., commencing or continuing administration of) an effective amount of a therapy comprising a targeted therapy in combination with a cancer immunotherapy to the subject, thereby treating the subject with cancer.

[0169] In some embodiments, a method of treating a subject having cancer includes obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject); and if the value is greater than or equal to the reference value, administering (e.g., commencing or continuing administration) an effective amount of a therapy to the subject, thereby treating the subject having cancer, where the therapy is a cancer immunotherapy (e.g., a PD-1 binding antagonist) as a monotherapy.

[0170] In some embodiments, a method of treating a subject with cancer includes administering (e.g., initiating administration or continuing administration) an effective amount of a targeted therapy to the subject, and, in response to a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) being greater than or equal to a reference value, administering (e.g., initiating administration or continuing administration) an effective amount of a cancer immunotherapy to the subject, thereby treating the subject with cancer.

[0171] In some embodiments, a method of treating a subject having cancer includes administering (e.g., commencing administration or continuing administration) to a subject an effective amount of a therapy comprising a targeted therapy in combination with a cancer immunotherapy, where prior to administration, it is determined that a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is greater than or equal to a reference value.

[0172] In some embodiments, a method of treating a subject having cancer includes administering (e.g., commencing administration or continuing administration) an effective amount of a therapy to a subject, where the therapy is a cancer immunotherapy (e.g., a PD-1 binding antagonist) as a monotherapy, and where prior to administration, it is determined that a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is greater than or equal to a reference value.

[0173] In some embodiments, a method of treating a subject having cancer includes administering to a subject (e.g., commencing or continuing administration of) an effective amount of a therapy comprising a targeted therapy in combination with a cancer immunotherapy, thereby treating the subject having cancer, wherein the subject is characterized by a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) that is greater than or equal to a reference value.

[0174] In some embodiments, a method of treating a subject having cancer includes administering (e.g., commencing or continuing administration) an effective amount of a therapy to the subject, thereby treating the subject having cancer, where the therapy is a cancer immunotherapy (e.g., a PD-1 binding antagonist) as a monotherapy, and the subject is characterized by a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) that is greater than or equal to a reference value.

[0175] In some embodiments, a method of treating a subject with cancer in response to a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) being less than a reference value, comprises administering (e.g., commencing or continuing administration of) an effective amount of a therapy comprising a targeted therapy (e.g., not using a cancer immunotherapy) to the subject, thereby treating the subject with cancer.

[0176] In some embodiments, a method of treating a subject with cancer includes obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), and if the value is less than the reference value, administering (e.g., commencing or continuing administration of) an effective amount of a therapy comprising a targeted therapy (e.g., not a cancer immunotherapy) to the subject, thereby treating the subject with cancer.

[0177] In some embodiments, a method of treating a subject having cancer includes administering (e.g., commencing administration or continuing administration) to the subject an effective amount of a therapy that includes a targeted therapy in combination with a cancer immunotherapy; and, in response to a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) being less than a reference value, administering (e.g., commencing administration or continuing administration) to the subject an effective amount of the targeted therapy (e.g., without the cancer immunotherapy), thereby treating the subject having cancer.

[0178] In some embodiments, a method of treating a subject having cancer includes administering (e.g., commencing administration or continuing administration) to a subject an effective amount of a therapy that includes a targeted therapy (e.g., not using a cancer immunotherapy), where prior to administration, it has been determined that a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is less than a reference value, thereby treating the subject having cancer.

[0179] In some embodiments, a method of treating a subject having cancer includes administering (e.g., commencing or continuing administration) to a subject an effective amount of a therapy that includes a targeted therapy (e.g., not using a cancer immunotherapy), thereby treating the subject having cancer, wherein the subject is characterized by a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) that is less than a reference value, thereby treating the subject having cancer.

[0180] In some embodiments, upon receiving a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject), a method of treating includes administering (e.g., initiating or continuing administration) an effective amount of a treatment that includes a targeted therapy (e.g., without a cancer immunotherapy) to a subject having a value less than the reference value; or administering (e.g., initiating or continuing administration) an effective amount of a treatment that includes a targeted therapy in combination with a cancer immunotherapy to a subject having a value greater than or equal to the reference value, thereby treating the subject with cancer.

[0181] In some embodiments, a method of treating a subject with cancer includes obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject); administering (e.g., initiating or continuing administration) an effective amount of a therapy that includes a targeted therapy (e.g., without a cancer immunotherapy) to subjects with a value less than the reference value; or administering (e.g., initiating or continuing administration) an effective amount of a therapy that includes a targeted therapy in combination with a cancer immunotherapy to subjects with a value greater than or equal to the reference value, thereby treating the subject with cancer.

[0182] In some embodiments, a method of treating a subject having cancer includes administering (e.g., initiating or continuing administration) to the subject an effective amount of a treatment that includes a targeted therapy (e.g., without a cancer immunotherapy); and receiving a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject), administering (e.g., initiating or continuing administration) a treatment that includes the targeted therapy (e.g., without a cancer immunotherapy) to subjects having a value less than the reference value, or administering (e.g., initiating or continuing administration) a treatment that includes the targeted therapy in combination with a cancer immunotherapy to subjects having a value greater than or equal to the reference value, thereby treating the subject having cancer.

[0183] In some embodiments, the method of treating a subject having cancer includes: (a) a targeted therapy (e.g., not using a cancer immunotherapy), where prior to administration, it has been determined that the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) is less than a reference value; (b) a targeted therapy in combination with a cancer immunotherapy, where prior to administration, a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject) is determined to be greater than or equal to a reference value, thereby treating the subject with cancer; or (c) a cancer immunotherapy (e.g., as a monotherapy), where prior to administration, a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject) is determined to be greater than or equal to a reference value, thereby treating the subject with cancer. administering to the subject an effective amount of a treatment comprising Includes.

[0184] In some embodiments, the method of treating a subject having cancer includes: (a) a targeted therapy (e.g., not using cancer immunotherapy) to a subject, wherein the subject is characterized or has been characterized as having a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) that is less than a reference value; (b) a targeted therapy in combination with a cancer immunotherapy, wherein the subject is characterized or characterized as having a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) that is greater than or equal to a reference value, thereby treating the subject with cancer; or (c) Cancer immunotherapy (e.g., as a monotherapy), in which a subject is characterized or characterized by a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject) that is greater than or equal to a reference value, thereby treating the subject with cancer. administering to the subject an effective amount of a treatment comprising Includes.

[0185] In some embodiments, a method of stratifying a subject having cancer into a first group and a second group comprises: (i) obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject); (ii) assigning subjects having a value less than the reference value to a first group that is more likely to benefit from a treatment that includes a targeted therapy (e.g., no cancer immunotherapy); (iii) allocating subjects having a value greater than or equal to the reference value to a second group that is more likely to benefit from a treatment comprising the targeted therapy in combination with the cancer immunotherapy; Includes.

[0186] In some embodiments, a method of stratifying a subject having cancer into a first group and a second group comprises: (i) obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject); (ii) assigning subjects having a value less than the reference value to a first group that is more likely to benefit from a treatment that includes a targeted therapy (e.g., no cancer immunotherapy); (iii) allocating subjects having a value greater than or equal to the reference value to a second group that is more likely to benefit from a treatment that includes cancer immunotherapy; Includes.

[0187] In some embodiments, a method for stratifying a subject having cancer into a first group and a second group and selecting a treatment comprises: (i) obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject); a value less than the reference value identifies the subject as a member of a first group likely to benefit from a treatment that includes a targeted therapy (e.g., not a cancer immunotherapy); A value greater than or equal to the reference value identifies the subject as a member of a second group likely to benefit from a treatment comprising the targeted therapy in combination with the immunotherapy. Includes.

[0188] In some embodiments, a method for stratifying a subject having cancer into a first group and a second group and selecting a treatment comprises: (i) obtaining a value for the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a subject (e.g., in a sample from the subject); a value less than the reference value identifies the subject as a member of a first group likely to benefit from a treatment that includes a targeted therapy (e.g., without a cancer immunotherapy); A value greater than or equal to the reference value identifies the subject as a member of a second group likely to benefit from treatment, including immunotherapy. Includes.

[0189] In some embodiments, the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) includes the ratio of the amount of CD4+ immune effector cells (e.g., CD4+ T cells) to the amount of CD8+ immune effector cells (e.g., CD8+ T cells), and can be measured by any assay disclosed herein, e.g., flow cytometric immunophenotyping.

[0190] In some embodiments, a value greater than or equal to 2.01 (e.g., a value greater than or equal to 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10) identifies that the subject is likely to benefit from a treatment comprising a targeted therapy (e.g., a treatment comprising an agent that targets BRAF and / or an agent that targets MEK) in combination with a cancer immunotherapy (e.g., an anti-PD-1 binding antagonist). In some embodiments, a value greater than or equal to 3.34 (e.g., a value greater than or equal to 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10) identifies that the subject is likely to benefit from a treatment comprising a targeted therapy (e.g., a treatment comprising an agent that targets BRAF and / or an agent that targets MEK) in combination with a cancer immunotherapy (e.g., an anti-PD-1 binding antagonist).

[0191] In some embodiments, a value greater than or equal to 2.01 (e.g., greater than or equal to 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10) identifies that the subject is likely to benefit from a treatment that includes a cancer immunotherapy (e.g., an anti-PD-1 binding antagonist). In some embodiments, a value greater than or equal to 3.34 (e.g., greater than or equal to 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10) identifies that the subject is likely to benefit from a treatment that includes a cancer immunotherapy (e.g., an anti-PD-1 binding antagonist).

[0192] In some embodiments, obtaining the value comprises determining the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) in the subject relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in a sample from the subject.

[0193] In some embodiments, the sample from the subject comprises a blood sample (such as a peripheral blood sample containing peripheral blood mononuclear cells (PBMCs)) or a tumor sample.

[0194] In some embodiments, the value is obtained before administration of the treatment begins (e.g., is a baseline value). In some embodiments, the value is obtained after administration of the treatment begins. For example, the value may be obtained 1, 2, 3, 4, 8, 10, 12, 20, 30, 40 weeks or more or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 months or more after administration of the treatment begins.

[0195] In certain embodiments of the methods described herein, the methods further comprise obtaining, determining, and / or evaluating one or more additional biomarkers.

[0196] In some embodiments, the method further comprises obtaining, determining, and / or evaluating a level or activity value of CD8+ tumor infiltrating lymphocytes (TILs) in the subject (e.g., in a sample from the subject), e.g., a tumor having a CD8+ TIL inflammatory phenotype. In some embodiments, an increase in the level or activity value of CD8+ TILs compared to a reference value further identifies that the subject is likely to benefit from the treatment.

[0197] In some embodiments, the method further comprises obtaining, determining, and / or evaluating a level and / or activity value of PD-L1 in the subject (e.g., in a sample from the subject). In some embodiments, a decrease in the level and / or activity value of PD-L1 compared to the reference value further identifies that the subject is likely to benefit from the treatment, e.g., in conjunction with an increase in the TMB value compared to the reference value. In some embodiments, the cancer has low or undetectable expression of PD-L1.

[0198] In some embodiments, the method further comprises obtaining, determining, and / or evaluating a value for a level and / or activity of immune activation including, for example, TIL, PD-L1, CD8, IFNγ, or a T cell inflammatory gene expression signature as described herein.

[0199] Tumors with CD8+ TILs (e.g., tumors with an inflammatory phenotype) can be characterized and quantified by multiple markers; for example, increases in tumor-infiltrating lymphocytes (determined by H / E staining or CD8 gene / protein expression levels), interferon gamma and related markers, PD-L1 (protein or gene expression), combinations of markers in other known checkpoints (e.g., LAG-3, TIM-3) or signatures (e.g., IFN gamma, T cell, inflammatory gene expression signatures). Responses to immunotherapy occur primarily in patients with such pre-existing intratumoral T cell adaptive immune responses.

[0200] IFN-γ is a cytokine that is not only important for the host response to viral infection but also plays a key role in cancer-associated immunity. IFN-γ is secreted by immune cells in the tumor microenvironment and coordinates the processes of innate and adaptive antitumor responses (e.g., enhancing MHC class I expression and contributing to the recruitment of effector cells). At the same time, the same IFN-γ signaling process can induce feedback inhibition. As part of this feedback loop, IFN-γ signaling allows the activation of the PD-1 signaling axis by direct upregulation of the ligands PD-L1 and PD-L2 in tumor, immune infiltrate, and stromal cells, ultimately impairing antitumor immunity.

[0201] There are various detection methods for immune genes such as CD8 and PD-L1: for example, IHC, flow cytometry, mRNA expression in samples such as tissues, blood, exosomes. For example, PD-L1 protein can be tested by immunohistochemistry (IHC) using different antibody clones, such as PD-L1 IHC 22C3 PharmDx kit (Dako North America, Carpinteria, CA, USA), PD-L1 28-8 PharmDx kit (Dako North America), PD-L1 SP263 Ventana test (Ventana Medical Systems Inc., Tucson, AZ, USA), and PD-L1 SP142 Ventana test (Ventana Medical Systems Inc., Tucson, AZ, USA). PD-L1 protein levels can be investigated, for example, in tumors and immune cells.

[0202] Multiple signatures (e.g., T cell inflammation, IFNγ, T cell / CD8 gene expression signatures) have been described in the literature (e.g., T cell inflammation in Cristescu et al., “Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy”, Science 2018). These gene signatures are novel methods to capture the complexity of the dynamic immune response to tumors by distinguishing tumors with a pre-existing inflammatory component from non-inflammatory tumors. Inspection of the gene lists of these signatures suggests that there is considerable overlap in selected genes, particularly in biological features (e.g., including IFN-γ signaling, cytolytic activity, antigen presentation, and T cell trafficking, as well as inhibitory mechanisms evident in T cell homeostasis), and that these signatures are all highly correlated and identify tumors with ongoing adaptive Th1 and cytotoxic CD8+ T cell responses. Those skilled in the art will appreciate that these gene expression signatures may be replaced by other T cell inflammatory and IFN-y signatures (e.g., Cristescu et al., "Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy", Science 2018; Ayers et al. IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade, The Journal of Clinical Investigation 2017).

[0203] Several other IFNγ signatures have been reported; one example is the six-gene signature published by Ayers et al. (IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade, The Journal of Clinical Investigation 2017): IDO1, CXCL10, CXCL9, HLA-DRA, STAT1, IFNG.

[0204] The well-established T cell inflammatory signature is composed of 18 inflammatory genes associated with antigen presentation, chemokine expression, cytolytic activity, and adaptive immune resistance, including CCL5, CD27, CD274 (PD-L1), CD276 (B7-H3), CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, LAG3, NKG7, PDCD1LG2 (PDL2), PSMB10, STAT1, and TIGIT. The T cell inflammation signature can be used to stratify patients into those with low T cell inflammation signature levels and those with high T cell inflammation signature levels (cutoff value greater than or equal to -0.318 = high, cutoff value less than -0.318 = low) (e.g., Cristescu et al., “Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy”, Science 2018; Ayers et al. IFN-γ-related mRNA profile -predicts clinical response to PD-1 blockade, The Journal of Clinical Investigation 2017).

[0205] Those skilled in the art will understand that similar cut-off values ​​can be established for other IFN-y and T cell signatures described in this patent specification and literature. For example, PD-L1 cut-off values ​​of ≧1%, ≧5%, ≧10% (e.g., 1%) can be used to define low / high immune activation.

[0206] In some embodiments, the method further comprises obtaining, determining, and / or evaluating the value of tumor mutation burden (TMB) in the subject (e.g., in a sample from the subject).In some embodiments, an increase in the TMB value compared to the reference value further identifies that the subject is likely to benefit from treatment.In some embodiments, the TMB value is greater than or equal to 10mut / Mb, for example, greater than or equal to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25mut / Mb.

[0207] In some embodiments, the method further comprises obtaining or assessing a value of tumor mutation burden (TMB) in the subject (e.g., in a sample from the subject). In some embodiments, a decrease in the value of TMB compared to the reference value further identifies that the subject is more likely to benefit from a treatment (e.g., without cancer immunotherapy). In some embodiments, a decrease in the value of TMB compared to the reference value further identifies that the subject is less likely to have a substantial increase in benefit from a treatment comprising a targeted therapy in combination with cancer immunotherapy. In some embodiments, the value of TMB is less than 10 mut / Mb, for example, less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 mut / Mb, or less.

[0208] As used herein, the terms "tumor mutation burden," "tumor mutational burden," "mutation load," "mutational load," or "TMB" may be understood interchangeably and refer to the level (e.g., number) of alterations (e.g., one or more alterations, e.g., one or more somatic alterations) per preselected unit (e.g., per megabase (Mb)) in a given set of genes (e.g., in the coding regions of a given set of genes) detected from a tumor (e.g., a tumor tissue sample, such as a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh frozen tumor sample, or a blood sample containing tumor cells, tumor RNA, DNA, or protein). TMB and its measurement are disclosed in WO 2018 / 068028, the entirety of which is incorporated herein by reference.

[0209] The TMB score can be measured, for example, based on the whole genome or exome, or based on a subset of the genome or exome. In certain embodiments, the TMB score measured based on a subset of the genome or exome can be extrapolated to determine the mutation load of the whole genome or exome. In some embodiments, the TMB score refers to the level of cumulative somatic mutations in an individual (e.g., an animal, e.g., a human). The TMB score may refer to the cumulative somatic mutations in a patient with cancer (e.g., melanoma). In some embodiments, the TMB score refers to the cumulative mutations in the whole genome of an individual. In some embodiments, the TMB score refers to the cumulative mutations in a particular sample (e.g., a tumor sample, e.g., a melanoma sample) taken from a patient.

[0210] The term "somatic mutation" or "somatic alteration" refers to a genetic alteration present in somatic tissue (e.g., cells outside the germline). Examples of genetic alterations include, but are not limited to, point mutations (e.g., exchange of a single nucleotide for another nucleotide (e.g., silent mutations, missense mutations, and nonsense mutations)), insertions and deletions (e.g., addition and / or removal of one or more nucleotides (e.g., indels)), amplifications, gene duplications, copy number alterations (CNAs), rearrangements, and splice site mutations. The presence of certain mutations may be associated with disease conditions (e.g., cancer, e.g., melanoma).

[0211] In certain embodiments, the somatic alteration is a silent mutation (e.g., a synonymous alteration). In other embodiments, the somatic alteration is a nonsynonymous single nucleotide variation (SNV). In other embodiments, the somatic alteration is a passenger mutation (e.g., an alteration that has no detectable effect on the fitness of the clone). In certain embodiments, the somatic alteration is a variation of unknown significance (VUS), e.g., an alteration whose pathogenicity cannot be confirmed or denied. In certain embodiments, the somatic alteration has not been identified to be associated with a cancer phenotype.

[0212] In certain embodiments, the somatic alteration is not associated with, or is not known to be associated with, an effect on cell division, growth, or survival, while in other embodiments, the somatic alteration is associated with an effect on cell division, growth, or survival.

[0213] In certain embodiments, the number of somatic alterations excludes functional alterations in the subgenomic interval. In some embodiments, a functional alteration is an alteration that has an effect on cell division, growth, or survival (e.g., promotes cell division, growth, or survival) compared to a reference sequence (e.g., a wild-type or unmutated sequence). In certain embodiments, a functional alteration is identified as such by being included in a database of functional alterations, such as the COSMIC database (see Forbes et al. Nucl. Acids Res.43 (D1): D805-D811, 2015, which is incorporated by reference in its entirety). In other embodiments, a functional alteration is an alteration with a known functional context (e.g., occurs as a known somatic alteration in the COSMIC database). In certain embodiments, a functional alteration is an alteration with an appropriate functional context (e.g., a truncation in a tumor suppressor gene). In certain embodiments, a functional alteration is a driver mutation (e.g., an alteration that confers a selective advantage to a clone in its microenvironment by increasing cell survival or reproduction). In other embodiments, the functional alteration is an alteration that can cause clonal expansion. In certain embodiments, the functional alteration is an alteration that can cause one, two, three, four, five, or all six of the following: (a) self-sufficiency in growth signals; (b) reduced, e.g., insensitivity to anti-growth signals; (c) reduced apoptosis; (d) increased replicative capacity; (e) sustained angiogenesis; or (f) tissue invasion or metastasis.

[0214] In certain embodiments, all functional alterations in all genes (e.g., oncogenes) in a given set of genes are excluded. In certain embodiments, the number of somatic alterations excludes alterations that exist in the sample at a frequency threshold (e.g., less than 5%, less than 3%, less than 1%). In certain embodiments, the number of somatic alterations excludes germline mutations in the subgenomic interval. In certain embodiments, the germline alterations are SNPs, base substitutions, insertions, deletions, indels, or silent mutations (e.g., synonymous mutations).

[0215] In certain embodiments, germline alterations are excluded by using a method that does not use a comparison to a matched normal sequence. In other embodiments, germline alterations are excluded by a method that includes the use of an algorithm. In certain embodiments, germline alterations are identified as such by inclusion in a database of germline alterations, such as the dbSNP database (see Sherry et al. Nucleic Acids Res.29(1): 308-311, 2001, which is incorporated by reference in its entirety). In other embodiments, germline alterations are identified as such by inclusion in the ExAC database (see Exome Aggregation Consortium et al. bioRxiv preprint, October 30, 2015, which is incorporated by reference in its entirety). In some embodiments, germline alterations are identified as such by inclusion in the ESP database (Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA). In some embodiments, germline alterations are identified by modeling the tumor content of a sample (see Riester et al. Source Code Biol Med. 2016 Dec 15;11:13). In some embodiments, germline alterations are identified from sequencing of samples from individuals without cancer.

[0216] As used herein, a "low TMB score" refers to a TMB score that is at or below a reference TMB score, whereas a "high TMB score" refers to a TMB score that exceeds the reference TMB score.

[0217] As used herein, a "low immune activation score" refers to an immune activation score that is at or below a reference immune activation score, whereas a "high immune activation score" refers to an immune activation score that exceeds the reference immune activation score.

[0218] As used herein, the term "reference TMB score" refers to a TMB score that is compared with another TMB score, for example, to make a diagnosis, prediction, prognosis, and / or treatment decision.For example, a reference TMB score may be the TMB score of a reference sample, a reference population, and / or a predetermined value.In some cases, the individual's responsiveness to treatment with a targeted therapy is significantly improved at or below a cutoff value relative to the individual's responsiveness to treatment with a non-targeted therapy.In some cases, the individual's responsiveness to treatment with a non-targeted therapy is significantly improved above a cutoff value relative to the individual's responsiveness to treatment with a targeted therapy.

[0219] It will be appreciated by those of skill in the art that the numerical value of the reference TMB score may vary depending on the type of cancer, the methodology used to measure the TMB score, and / or the statistical methods used to generate the TMB score.

[0220] The term "equivalent TMB value" refers to a numerical value corresponding to the TMB score, which can be calculated by dividing the count of somatic variants (e.g., somatic mutations) by the number of sequenced bases (e.g., about 1.5 Mb assessed by a targeted panel). In general, it should be understood that the TMB score is linearly related to the size of the sequenced genomic region. Such equivalent TMB value indicates an equivalent degree of tumor mutation burden compared to the TMB score, and can be used interchangeably in the methods described herein, for example, to predict the response of cancer patients to targeted therapy (e.g., targeted therapy including agents that target BRAF and / or MEK, e.g., dabrafenib and trametinib, or, e.g., vemurafenib and cobimetinib). As an example, in some embodiments, the equivalent TMB value is a normalized TMB value, which can be calculated by dividing the count of somatic variants (e.g., somatic mutations) by the number of sequenced bases. For example, the equivalent TMB value can be expressed as the number of somatic mutations counted over a defined number of sequencing bases (e.g., about 1.5 Mb assessed by a targeted panel). It should be understood that the TMB score described herein (e.g., the TMB score expressed as the number of somatic mutations counted over a defined number of sequencing bases (e.g., 1.5 Mb in the targeted panel described herein)) encompasses the equivalent TMB value obtained using different methodologies (e.g., whole exome sequencing or whole genome sequencing). As an example, in a whole exome panel, the target region may be approximately 50 Mb, and a sample with about 500 somatic mutations detected is equivalent to a TMB score of about 10 mutations / Mb.

[0221] In some embodiments, the method further comprises obtaining, determining, and / or evaluating a level and / or activity of PD-L1 in the subject (e.g., in a sample from the subject). In some embodiments, a decrease in the level and / or activity of PD-L1, e.g., in conjunction with a decrease in the value of TMB, further identifies that the subject is unlikely to have a substantial increase in benefit from a treatment comprising a targeted therapy in combination with a cancer immunotherapy. In some embodiments, the cancer has low or undetectable expression of PD-L1.

[0222] In some embodiments, the method further involves obtaining, determining, and / or evaluating the level of circulating tumor DNA (ctDNA) in the subject (e.g., in a sample from the subject).In some embodiments, an increase in the level of ctDNA compared to the reference value further identifies that the subject is likely to benefit from a treatment that includes a targeted therapy in combination with cancer immunotherapy.

[0223] The term "detection" includes any means of detection, including direct and indirect detection.

[0224] The term "biomarker" as used herein refers to an indicator, such as a predictive, diagnostic, and / or prognostic indicator, that can be detected in a sample, such as a specific gene (alteration and expression level) or a protein encoded by said gene, or one or more somatic mutations of said specific gene. A biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and / or clinical features (e.g., responsiveness to treatment, including targeted therapy, including agents targeting BRAF and / or agents targeting MEK in combination with cancer immunotherapy (e.g., PD-1 binding antagonists)).

[0225] The term "sample" or "biological sample" as used herein refers to a composition obtained or derived from a subject and / or individual of interest that contains cellular and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid (CSF), saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof. In one embodiment, "sample" refers to a "tissue sample" or a "cell sample". In another embodiment, "sample" refers to a "blood sample".

[0226] "Tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue, such as from fresh, frozen and / or preserved organs, tissue samples, biopsies, and / or aspirates; blood or any blood component, such as plasma; bodily fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time during the subject's pregnancy or development. The tissue sample may be primary or cultured cells, or a cell line. Optionally, the tissue or cell sample is obtained from a diseased tissue / organ. For example, a "tumor sample" is a tissue sample obtained from a tumor or other cancerous tissue. The tissue sample may contain a mixed population of cell types (e.g., tumor and non-tumor cells, cancerous and non-cancerous cells). The tissue sample may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. In some cases, the tissue sample or tumor tissue sample is not a sample or blood component, such as a blood sample or plasma. In a preferred embodiment, the tissue or cell sample is a tumor sample.

[0227] "Tumor cell" as used herein refers to any tumor cell present in a tumor or a sample thereof. Tumor cells may be distinguished from other cells that may be present in a tumor sample, such as stromal cells and tumor-infiltrating immune cells, using methods known in the art and / or described herein.

[0228] The terms "amount" and "level" generally refer to the amount of a biomarker in a biological sample. This can be measured by methods known to those skilled in the art and are also disclosed herein. For example, gene or protein expression levels can be analyzed by methods such as flow cytometry, IHC, qRT-PCR, Nanostring, and other methods known to those skilled in the art.

[0229] "Expression" generally refers to the process by which information (e.g., genetically encoded information and / or epigenetic information) is converted into structures present and operational within a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or modification of a polynucleotide and / or polypeptide (e.g., post-translational modification of a polypeptide). Fragments of transcribed polynucleotides, translated polypeptides, or modifications of polynucleotides and / or polypeptides (e.g., post-translational modification of a polypeptide) will also be considered to be expressed, regardless of whether they originate from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a polypeptide, e.g., proteolysis. "Expressed genes" also include those that are transcribed into polynucleotides as mRNA and then translated into a polypeptide, and those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer RNA and ribosomal RNA).

[0230] "Increased expression," "increased expression levels," "increased levels," "elevated expression," "elevated expression levels," or "elevated levels" refer to increased expression or increased levels of a biomarker in an individual compared to a control, e.g., an individual(s) not afflicted with a disease or disorder (e.g., cancer), or an internal control (e.g., a housekeeping biomarker).

[0231] "Decreased expression," "reduced expression levels," "reduced levels," "reduced expression," "reduced expression levels," or "reduced levels" refer to a decrease in expression or a decrease in the level of a biomarker in an individual compared to a control, e.g., an individual(s) not afflicted with a disease or disorder (e.g., cancer), or an internal control (e.g., a housekeeping biomarker).

[0232] As used herein, the term "reference level" refers to a value to which a sample value is compared, for example, to make a diagnostic, predictive, prognostic, and / or therapeutic decision. For example, a reference level may be a value in a reference sample, a reference population, and / or a predetermined value. A "reference sample", "reference tissue", "reference cell", "control sample", "control tissue", or "control cell" as used herein refers to a sample, tissue, cell, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference tissue, reference cell, control sample, control tissue, or control cell is obtained from a healthy and / or non-disease part (e.g., tissue or cell) of the body of the same subject or individual. For example, the reference sample, reference tissue, reference cell, control sample, control tissue, or control cell may be a healthy and / or non-disease tissue or cell adjacent to a diseased tissue or cell (e.g., tissue or cell adjacent to a tumor). In another embodiment, the reference sample is obtained from an untreated tissue and / or cell of the body of the same subject or individual. In yet another embodiment, the reference sample, reference tissue, reference cell, control sample, control tissue, or control cell is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of an individual other than the same subject or individual. In yet another embodiment, the reference sample, reference tissue, reference cell, control sample, control tissue, or control cell is obtained from untreated tissue and / or cell of the body of an individual other than the same subject or individual.

[0233] In some embodiments, the methods described herein involve administering (e.g., initiating or continuing administration of) an effective amount of a therapy to the subject (e.g., a targeted therapy comprising an agent that targets BRAF and / or an agent that targets MEK, with or without a cancer immunotherapy (e.g., a PD-1 binding antagonist). In other embodiments, the methods involve administering to the subject an altered dosing regimen of the therapy (e.g., a dosing regimen comprising higher doses and / or more frequent administration than a reference dosing regimen). In some embodiments, the methods involve discontinuing administration of a different therapy to the subject.

[0234] In some embodiments, the methods described herein include administering to the subject an additional treatment. In some embodiments, the methods involve administering to the subject a pretreatment, where the pretreatment increases the value of the level or activity of CD4+ immune effector cells (e.g., CD4+ T cells) relative to the level or activity of CD8+ immune effector cells (e.g., CD8+ T cells) in the subject (e.g., in a sample from the subject).

[0235] In some embodiments, the treatment is a first, second, third, or fourth line or above treatment, hi some embodiments, the treatment is an adjuvant or neoadjuvant treatment.

[0236] Selecting a target In some embodiments, the subject described herein is a subject with cancer. The subject can be a mammal, such as a primate, such as a higher primate, such as a human (e.g., a patient with or at risk of having a disorder described herein, such as cancer). In one embodiment, the subject does not need to boost an immune response. In one embodiment, the subject has or at risk of having a disorder described herein, such as a cancer described herein. In certain embodiments, the subject is or at risk of being immunocompromised. For example, the subject is or has undergone chemotherapy treatment and / or radiation therapy. Alternatively, or in combination, the subject is or at risk of being immunocompromised as a result of infection.

[0237] In certain embodiments, the cancer treated according to the methods described herein includes, but is not limited to, a solid tumor (e.g., melanoma, lung cancer (e.g., NSCLC), pancreatic cancer, or colorectal cancer), a hematological cancer (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), or metastases thereof. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignant tumors of various organ systems, such as those affecting the lung, breast, ovary, lymphatic system, gastrointestinal tract (e.g., colon), anus, genital and urogenital tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neuronal or glial cells), head and neck, skin (e.g., melanoma), and pancreas, including sarcomas and carcinomas, such as adenocarcinomas, and adenocarcinomas, including malignant tumors such as colon cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (clear cell renal cell carcinoma or non-clear cell renal cell carcinoma)), liver cancer, lung cancer (e.g., non-small cell lung cancer (squamous or non-squamous non-small cell lung cancer)), small intestine cancer, and esophageal cancer. The cancer may be early stage, intermediate stage, late stage, or metastatic.

[0238] In one embodiment, the cancer is melanoma, for example, aggressive melanoma. In one embodiment, the cancer is aggressive or unresectable melanoma that does not respond to other treatments (i.e., refractory). In another embodiment, the cancer is melanoma with a BRAF mutation (e.g., a BRAF V600 mutation). In yet another embodiment, the combination disclosed herein (e.g., a combination comprising an anti-PD-1 antibody molecule) comprises an agent that targets BRAF (e.g., vemurafenib or dabrafenib) and / or an agent that targets MEK (e.g., trametinib).

[0239] In certain embodiments, the cancer is a cancer (e.g., melanoma) with a BRAF mutation, for example, a BRAF mutation described herein. In certain embodiments, the BRAF mutation is a V600 mutation. In certain embodiments, the BRAF mutation is located in the activation segment of the kinase domain. In other embodiments, the BRAF mutation results in increased kinase activity, and is optionally transformed in vitro. In some embodiments, the BRAF mutation is selected from a V600E mutation, a V600K mutation, or a V600D mutation. In certain embodiments, the BRAF mutation is a V600E mutation. In other embodiments, the combination is used to treat a cancer (e.g., melanoma) other than a wild-type BRAF cancer (e.g., wild-type BRAF melanoma). In certain embodiments, the BRAF mutation is a mutation that is sensitive or responsive to an inhibitor of BRAF, an inhibitor of MEK, or both.

[0240] Exemplary BRAF mutations include, but are not limited to, BRAF c.1779_1780delTGinsGA(D594N), BRAF c.1780G>C(D594H), BRAF c.1780G>A(D594N), BRAF c.1781A>G(D594G), BRAF c.1781A>T(D594V), BRAF c.1782T>A(D594E), BRAF c.1782T>G(D594E), BRAF c.1789C>G(L597V), BRAF c.1789_1790delCTinsTC(L597S), BRAF c.1790T>A(L597Q), BRAF c.1790T>G(L597R), BRAF These include BRAF c.1798G>A(V600M), BRAF c.1798_1799delGTinsAA(V600K), BRAF c.1798_1799delGTinsAG(V600R), BRAF c.1799T>A(V600E), BRAF c.1799T>G(V600G), BRAF c.1799_1800delTGinsAT(V600D), BRAF c.1799_1800delTGinsAA(V600E), or BRAF c.1801A>G(K601E). Other exemplary BRAF mutations include BRAF fusions, as described, for example, in Botton et al. Pigment Cell Melanoma Res. 2013; 26(6):845-51; Hutchinson et al. Clin Cancer Res. 2013; 19(24):6696-702).

[0241] In one embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and / or non-squamous histology, or NSCLC adenocarcinoma)), skin cancer (e.g., Merkel cell carcinoma or melanoma (e.g., advanced melanoma)), kidney cancer (e.g., renal cancer (e.g., renal cell carcinoma)), liver cancer, myeloma (e.g., multiple myeloma), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of the estrogen receptor, progesterone receptor, or Her2 / neu, e.g., triple-negative breast cancer), colorectal cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC), anal cancer, gastroesophageal cancer, thyroid cancer (e.g., anaplastic thyroid carcinoma), cervical cancer, neuroendocrine tumors (NETs) (e.g., atypical lung carcinoid tumors), lymphoproliferative disorders (e.g., post-transplant lymphoproliferative disorders) or hematological cancer, T-cell lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or leukemia (e.g., myeloid leukemia or lymphocytic leukemia).

[0242] In another embodiment, the cancer is selected to form a carcinoma (eg, an advanced or metastatic carcinoma), a melanoma, or a lung cancer, eg, a non-small cell lung cancer.

[0243] In one embodiment, the cancer is lung cancer, for example non-small cell lung cancer or small cell lung cancer.In some embodiments, the non-small cell lung cancer is stage I (e.g., stage Ia or Ib), stage II (e.g., stage IIa or IIb), stage III (e.g., stage IIIa or IIIb) or stage IV non-small cell lung cancer.

[0244] In another embodiment, the cancer is liver cancer, e.g., advanced liver cancer, with or without associated viral infection, e.g., chronic viral hepatitis.

[0245] In another embodiment, the cancer is prostate cancer, for example aggressive prostate cancer.

[0246] In yet another embodiment, the cancer is a myeloma, for example multiple myeloma.

[0247] In yet another embodiment, the cancer is a renal cancer, e.g., a renal cell carcinoma (RCC) (e.g., metastatic RCC, non-clear cell renal cell carcinoma (nccRCC), or clear cell renal cell carcinoma (CCRCC)).

[0248] In some embodiments, the subject has been or is being treated with a targeted therapy (e.g., a therapy comprising an agent that targets BRAF and / or an agent that targets MEK). In some embodiments, the subject has not been or is not being treated with a targeted therapy.

[0249] In some embodiments, the subject has received or is receiving cancer immunotherapy (e.g., a PD-1 binding antagonist or a PD-L1 binding antagonist). In some embodiments, the subject has not received or is not receiving cancer immunotherapy.

[0250] In some embodiments, the subject has received or is receiving a treatment that includes a targeted therapy in combination with a cancer immunotherapy. In some embodiments, the subject has not received or is not receiving a treatment that includes a targeted therapy in combination with a cancer immunotherapy.

[0251] In some embodiments, the subject has undergone or has undergone targeted therapy and the cancer has recurred. In some embodiments, the subject is a non-responder to the targeted therapy or has been identified as a non-responder to the targeted therapy. In some embodiments, the subject is a partial responder to the targeted therapy or has been identified as a partial responder to the targeted therapy.

[0252] Response to treatment In some embodiments, treatment according to the methods described herein may result in one or more of the following: increased antigen presentation, increased effector cell function (e.g., one or more of T cell proliferation, IFN-γ secretion, or cytolytic function), inhibition of regulatory T cell function, affecting the activity of multiple cell types such as regulatory T cells, effector T cells, and NK cells, increased tumor-infiltrating lymphocytes, increased T cell receptor-mediated proliferation, and reduced immune evasion by cancerous cells. In one embodiment, the use of PD-1 inhibitors in combination inhibits, reduces, or neutralizes one or more activities of PD-1, resulting in blocking or reducing immune checkpoints. Thus, such methods can be used to treat or prevent disorders in which it is desired to enhance the immune response in a subject.

[0253] Thus, in another aspect, a method of modulating an immune response in a subject is provided. The method comprises administering to the subject a combination disclosed herein (e.g., a combination comprising a therapeutically effective amount of an anti-PD-1 antibody molecule), alone or in combination with one or more agents or procedures, such that the immune response in the subject is modulated. In one embodiment, the antibody molecule enhances, stimulates or increases the immune response in the subject.

[0254] In one aspect, a method of treating (e.g., one or more of reducing, inhibiting, or delaying progression of) a cancer or tumor in a subject is provided, the method comprising administering to the subject a combination disclosed herein (e.g., a combination comprising a therapeutically effective amount of an anti-PD-1 antibody molecule).

[0255] In some embodiments of the methods described herein, subjects more likely to benefit from the treatment or more likely to have an increased benefit from the treatment have improved progression-free survival (PFS), duration of objective response (DOR), and / or overall survival (OS) compared to subjects less likely to benefit from the treatment or less likely to have an increased benefit from the treatment.

[0256] In some embodiments, subjects who are more likely to benefit from a treatment or who are more likely to have an increased benefit from a treatment have improved PFS, DOR, and / or OS compared to subjects who did not receive the treatment or subjects who received only the targeted therapy but not the cancer immunotherapy.

[0257] In some embodiments, PFS, DOR, and / or OS is improved by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60 months or longer. In some embodiments, PFS, DOR, and / or OS is improved by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 fold or more.

[0258] In some embodiments, treatment according to the methods described herein results in an improvement in progression-free survival (PFS), duration of objective response (DOR), and / or overall survival (OS). In some embodiments, PFS, DOR, and / or OS are improved by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60 months or longer. In some embodiments, PFS, DOR, and / or OS are improved by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.

[0259] "Individual response" or "response" can be assessed using any endpoint that indicates benefit to an individual, including, but not limited to, (1) inhibition to some extent, including slowing or complete halt, of disease progression (e.g., cancer progression); (2) reduction in tumor size; (3) inhibition (i.e., reduction, slowing, or complete halt) of cancer cell invasion into adjacent peripheral organs and / or tissues; (4) inhibition (i.e., reduction, slowing, or complete halt) of metastasis; (5) alleviation to some extent of one or more symptoms associated with a disease or disorder (e.g., cancer); (6) increase or prolongation of survival time, including overall survival, progression-free survival, and recurrence-free survival; and / or (7) reduction in mortality at a given time point following treatment.

[0260] An "effective response" of a patient to treatment with a pharmaceutical agent or a patient's "responsiveness" and similar expressions refer to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of an increase in survival (including overall survival and / or progression-free survival and / or regression-free survival), an objective response (including a complete or partial response), or an improvement in the signs or symptoms of cancer.

[0261] "Objective response" refers to a measurable response, including complete response (CR) or partial response (PR). In some embodiments, "objective response rate (ORR)" refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0262] By "complete response" or "CR" is intended the disappearance of all signs of cancer (e.g., disappearance of all target lesions) in response to treatment. This does not necessarily mean that the cancer has been cured.

[0263] "Sustained response" refers to the sustained effect of reducing tumor growth after treatment is stopped.For example, the tumor size may be the same size or smaller than the size at the beginning of administration of the medicament.In some embodiments, the sustained response has a duration at least as long as the treatment period, at least 1.5 times, 2.0 times, 2.5 times, or 3.0 times the treatment period, or longer.

[0264] As used herein, "reducing or inhibiting cancer recurrence" means reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression. As disclosed herein, cancer recurrence and / or cancer progression includes, but is not limited to, cancer metastasis.

[0265] The term "survival" refers to the patient being alive, and includes overall survival, progression-free survival, and recurrence-free survival.

[0266] As used herein, "recurrence-free survival" or "RFS" refers to the length of time after complete surgical removal of a tumor that is free of any recurrence of disease. During and after treatment during which no signs or symptoms of the disease (e.g., cancer) being treated appear. Recurrence-free survival may include the amount of time that a patient experiences a complete or partial response, as well as the amount of time that a patient experiences stable disease.

[0267] As used herein, "overall survival" or "OS" refers to the percentage of individuals in a group who are likely to be alive after a particular period of time.

[0268] By "prolonging survival" is meant increasing the overall survival or progression-free survival and recurrence-free survival of a treated patient relative to untreated patients (i.e., relative to patients not treated with a pharmaceutical agent), or relative to patients who do not have a specified level of somatic mutations, and / or relative to patients treated with an anti-tumor agent.

[0269] The term "cancer therapy" refers to a therapy useful for treating cancer. In some embodiments, the cancer therapy is a combination therapy.

[0270] In some embodiments, a subject "likely to benefit" from a particular treatment is one who is more likely or more likely to respond to that treatment.

[0271] As used herein, the terms "individual," "patient," and "subject" are used interchangeably and refer to any single animal, more preferably a mammal, for which treatment is desired (including, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human animals such as non-human primates). In certain embodiments, the individual or patient herein is a human.

[0272] As used herein, "administering" and "administration" refer to a method of giving a dosage of a compound (e.g., an antagonist) or a pharmaceutical composition (e.g., a pharmaceutical composition comprising an antagonist) to a subject (e.g., a patient). Administering can be by any suitable means, including parenteral, pulmonary, and nasal administration, as well as intralesional administration if localized treatment is desired. Parenteral injections include, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, injections such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic. Various administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

[0273] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents, with at least a portion of the administration overlapping in time.Concurrent administration therefore includes a dosing regimen in which one or more agents are administered after one or more other agents are discontinued.

[0274] By "reducing or inhibiting" is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. Reducing or inhibiting can refer, for example, to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.

[0275] The term "based on," as used herein, means that information regarding one or more biomarkers is used to inform treatment decisions, information provided in package inserts, or marketing / promotional guidance, etc.

[0276] targeted therapy In certain embodiments, the treatment described herein comprises a targeted treatment.

[0277] As used herein, "targeted therapy" refers to a cancer treatment that blocks the growth and spread of cancer by interfering with specific molecules ("molecular targets") involved in the growth, progression, recurrence, and / or spread of cancer. Targeted therapy may also be referred to as "molecularly targeted drugs," "molecularly targeted therapy," "precision medicine," or similar names.

[0278] As used herein, the term "drug" is understood to mean a substance that produces a desired effect in a tissue, system, animal, mammal, human, or other subject. It should also be understood that an "agent" may be a single compound or a combination or composition of two or more compounds.

[0279] As used herein, "agents that target BRAF" refer to agents that directly or indirectly target, reduce or inhibit the activity and / or function of BRAF. Exemplary agents that target BRAF include, but are not limited to, compounds, proteins or antibodies that target BRAF. In some embodiments, the agent that targets BRAF is a "BRAF inhibitor."

[0280] As used herein, an "agent that targets MEK" refers to an agent that directly or indirectly targets, reduces or inhibits the activity and / or function of MEK. Exemplary agents that target MEK include, but are not limited to, compounds, proteins or antibodies that target MEK. In some embodiments, the agent that targets MEK is a "MEK inhibitor."

[0281] The BRAF inhibitor and / or MEK inhibitor described herein may contain one or more chiral atoms or may otherwise be capable of existing as enantiomers.Therefore, the compounds of the present disclosure include mixtures of enantiomers, as well as purified enantiomers or mixtures enriched as enantiomers.It is also understood that all tautomers and mixtures of tautomers are included within the scope of the BRAF inhibitor and / or MEK inhibitor described herein.

[0282] It is also understood that the BRAF inhibitor and / or MEK inhibitor described herein may exist as a solvate, either separately or both. As used herein, the term "solvate" refers to a complex of variable stoichiometry formed by a solute (in this disclosure, a compound of formula (I) or (II) or (III) or (IV) or a salt thereof) and a solvent. For the purposes of this disclosure, such a solvent may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, dimethylsulfolide, ethanol and acetic acid. In one embodiment, the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvents include, but are not limited to, water, ethanol and acetic acid. In another embodiment, the solvent used is water.

[0283] The BRAF inhibitor and / or MEK inhibitor described herein may have the ability to crystallize in more than one form, a characteristic known as polymorphism, and it is understood that such polymorphic forms ("polymorphs") are within the scope of the therapeutic agent described herein.Polymorphism can generally occur as a response to changes in temperature or pressure or both, and can also occur due to variations in the crystallization process.Polymorphs can be distinguished by various physical characteristics known in the art, such as X-ray diffraction patterns, solubility, and melting point.

[0284] Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of the present disclosure. Salts of the compounds of the present disclosure may include acid addition salts derived from the nitrogen of a substituent in the compounds of the present disclosure. Representative salts include the following salts: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, tartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate. Salts include, but are not limited to, phenylalanine, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, monopotassium maleate, mucate, napsylate, nitrate, N-methylglucanate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate / diphosphate, polygalacturonate, potassium salt, salicylate, sodium salt, stearate, acetate, succinate, tannate, tartrate, theocrine salt, tosylate, triethiodide salt, trimethylammonium salt and valerate salt. Other salts that are not pharmaceutically acceptable may be useful in the preparation of the compounds of the present disclosure, and these form a further aspect of the present disclosure. Salts can be readily prepared by those skilled in the art.

[0285] For use in treatment, it is possible that the BRAF inhibitor and / or MEK inhibitor described herein can be administered as a drug substance, but it is also possible that the active ingredient is present as a pharmaceutical composition.Thus, the present disclosure further provides a pharmaceutical composition comprising the BRAF inhibitor and / or MEK inhibitor described herein and one or more pharma-ceutically acceptable carriers, diluents, or excipients.The carrier(s), diluent(s) or excipient(s) must be acceptable in the sense that they are compatible with other components of the formulation, can be pharma-ceutically formulated, and are not harmful to the recipient thereof.According to another aspect of the present disclosure, there is also provided a process for the preparation of a pharmaceutical composition, comprising mixing the BRAF inhibitor and / or MEK inhibitor described herein with one or more pharma-ceutically acceptable carriers, diluents, or excipients.Such elements of the pharmaceutical composition utilized may be present in separate pharmaceutical combinations or may be formulated together in one pharmaceutical composition. Accordingly, the present disclosure further provides combinations of pharmaceutical compositions, one of which comprises a BRAF inhibitor (e.g., a BRAF inhibitor described herein) and one or more pharma- ceutically acceptable carriers, diluents, or excipients, as well as pharmaceutical compositions containing a MEK inhibitor (e.g., a MEK inhibitor described herein) and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0286] The BRAF inhibitor and / or MEK inhibitor described herein may be employed in combination according to the present disclosure by simultaneously administering in a unitary pharmaceutical composition that contains both compounds.Alternatively, the combination may be administered separately in a sequential manner, for example, in separate pharmaceutical compositions that each contain either a BRAF inhibitor or a MEK inhibitor, such that the BRAF inhibitor is administered first and the MEK inhibitor is administered second, or the MEK inhibitor is administered first and the BRAF inhibitor is administered second.Such sequential administration may be close in time (for example, simultaneously) or distant in time.

[0287] Furthermore, it does not matter if the combined compounds are administered in the same dosage form, for example, one compound may be administered intravenously and the other compound may be administered orally. Preferably, both compounds are administered orally.

[0288] Exemplary BRAF Inhibitors In some embodiments, the targeted therapy described herein comprises an agent that targets BRAF (e.g., an inhibitor of BRAF). In some embodiments, the targeted therapy is used to treat cancer, such as a cancer described herein, such as a skin cancer (e.g., melanoma). Without wishing to be bound by theory, in some embodiments, it is believed that the mitogen-activated protein kinase (MAPK) pathway is abnormally activated in some human cancers, for example, by mutations in BRAF kinase, which are found in nearly 50% of metastatic melanomas. In some embodiments, the targeted therapy is used to treat cancers that have a BRAF mutation, for example, a BRAF mutation described herein. In some embodiments, the targeted therapy is used to treat melanoma, for example, unresectable or metastatic melanoma. In some embodiments, the targeted therapy is used to treat melanoma in a subject, where the melanoma has a BRAF mutation (e.g., a BRAF V600 mutation).

[0289] In some embodiments, the BRAF inhibitor is dabrafenib. Dabrafenib is also known as GSK2118436, or TAFINLAR® (CAS number 1195765-45-7). The chemical formula of dabrafenib is the compound of formula (II):

[0290] [ka] or a pharma- ceutically acceptable salt thereof. For convenience, the group of potential compounds and salts will be referred to collectively as dabrafenib, meaning that reference to dabrafenib refers alternatively to either that compound or a pharma- ceutically acceptable salt thereof.

[0291] Depending on naming conventions, dabrafenib may be properly referred to as N-{3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide.

[0292] Dabrafenib, together with its pharma- ceutically acceptable salts, is disclosed and claimed in PCT patent application PCT / US09 / 42682 as being useful as an inhibitor of BRAF activity, particularly in the treatment of cancer. Dabrafenib is embodied in Examples 58a to 58e of the present application. This PCT application was published on November 12, 2009 as WO 2009 / 137391, which is incorporated herein by reference. Dabrafenib may be prepared according to the method described in WO 2011 / 047238 (see, for example, pages 15-21), which is incorporated herein by reference.

[0293] Dabrafenib is an orally bioavailable, potent and selective RAF kinase inhibitor of human wild-type BRAF and CRAF enzymes, as well as mutant forms of BRAF enzymes, such as BRAF V600E, BRAF V600K and BRAF V600D. The mode of action of dabrafenib is consistent with competitive inhibition of ATP binding. In certain embodiments, the combination is used to treat subjects who have been determined to have cancer (e.g., melanoma) with BRAF mutation, for example, BRAF mutation (e.g., BRAF V600 mutation) as described herein. In other embodiments, the combination is used to treat cancer (e.g., melanoma) other than wild-type BRAF cancer (e.g., wild-type BRAF melanoma).

[0294] In some embodiments, the BRAF inhibitor or dabrafenib is administered at a dose between 50 mg and 300 mg (e.g., between 100 mg and 200 mg), for example, twice a day. In certain embodiments, the BRAF inhibitor or dabrafenib is administered at a dose between 100 mg and 200 mg (e.g., a dose of about 150 mg), for example, twice a day. For example, the second dose of the BRAF inhibitor or dabrafenib can be administered about 12 hours after administration of the first dose. In some embodiments, the BRAF inhibitor or dabrafenib is administered at a total daily dose between 100 mg and 600 mg (e.g., between 200 mg and 400 mg). In certain embodiments, the BRAF inhibitor or dabrafenib is administered at a total daily dose between 200 mg and 400 mg (e.g., a total daily dose of about 300 mg). In some embodiments, the BRAF inhibitor, dabrafenib, is administered orally.

[0295] In another embodiment, the BRAF inhibitor is vemurafenib.Vemurafenib is disclosed in International Publication Nos. WO 2005 / 062795, WO 2007 / 013896, WO 2007 / 002325 and WO 2007 / 002433, the entirety of which is incorporated herein by reference.Vemurafenib is also known as PLX4032, RG7204, RO5185426 or ZELBORAF® (CAS No. 918504-65-1).As used herein, the BRAF inhibitor vemurafenib, or its pharma- ceutically acceptable salt or solvate, is a compound of formula (IV):

[0296] [ka] or a pharmaceutically acceptable salt or solvate thereof. For convenience, the group of potential compounds and salts or solvates will be referred to collectively as vemurafenib, meaning that reference to vemurafenib refers alternatively to either the compound or a pharmaceutically acceptable salt or solvate thereof.

[0297] Depending on naming conventions, vemurafenib may be properly referred to as N-[3-[[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl]-2,4-difluorophenyl]-1-propanesulfonamide.

[0298] Vemurafenib, together with its pharma- ceutically acceptable salts and also its solvates, is disclosed and claimed in WO 2007 / 002325 as being useful as an inhibitor of BRAF activity, particularly in the treatment of cancer. Vemurafenib may be prepared according to the methods in WO 2007 / 002325.

[0299] Other exemplary inhibitors of BRAF that can be used in the targeted therapies described herein include, but are not limited to, encorafenib, ABM-1310, ARQ 736, ASN003, BGB-283, BGB-3245, CEP-32496, GDC-0879, LUT014, PLX4720, PLX8394, RO5212054, or a pharma- ceutically acceptable salt thereof.

[0300] In one embodiment, the BRAF inhibitor is encorafenib or a compound disclosed in PCT Publication WO 2011 / 025927. Encorafenib is also known as LGX818 (CAS number 1269440-17-6). As used herein, the BRAF inhibitor encorafenib, or a pharma- ceutically acceptable salt or solvate thereof, is a compound of formula (IV):

[0301] [ka] or a pharma- ceutically acceptable salt or solvate thereof.

[0302] Depending on naming conventions, encorafenib may be properly referred to as N-[(1S)-2-[[4-[3-[5-chloro-2-fluoro-3-[(methylsulfonyl)amino]phenyl]-1-(1-methylethyl)-1H-pyrazol-4-yl]-2-pyrimidinyl]amino]-1-carbamic acid, methyl ester.

[0303] In one embodiment, the BRAF inhibitor or encorafenib is administered at a dose of about 200-300, 200-400, or 300-400 mg, e.g., daily. In one embodiment, the compound is administered at a dose of about 200, about 300, or about 400 mg.

[0304] In some embodiments, the BRAF inhibitor comprises ABM-1310.

[0305] In some embodiments, the BRAF inhibitor comprises ARQ 736.

[0306] In some embodiments, the BRAF inhibitor comprises ASN003.

[0307] In some embodiments, the BRAF inhibitor comprises BGB-283.

[0308] In some embodiments, the BRAF inhibitor comprises BGB-3245.

[0309] In some embodiments, the BRAF inhibitor comprises CEP-32496 (CAS number 1188910-76-0). Depending on naming conventions, CEP-32496 may be appropriately referred to as N-[3-[(6,7-dimethoxy-4-quinazolinyl)oxy]phenyl]-N'-[5-(2,2,2-trifluoro-1,1-dimethylethyl)-3-isoxazolyl]-urea.

[0310] In some embodiments, the BRAF inhibitor comprises GDC-0879 (CAS number 905281-76-7). Depending on naming conventions, GDC-0879 may be appropriately referred to as 2,3-dihydro-5-[1-(2-hydroxyethyl)-3-(4-pyridinyl)-1H-pyrazol-4-yl]-1H-inden-1-one, oxime.

[0311] In some embodiments, the BRAF inhibitor comprises LUT014.

[0312] In some embodiments, the BRAF inhibitor comprises PLX4720. PLX4720 is also known as Raf Kinase Inhibitor V (CAS number 918505-84-7). Depending on naming conventions, PLX4720 may be appropriately referred to as N-[3-[(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)carbonyl]-2,4-difluorophenyl]-1-propanesulfonamide.

[0313] In some embodiments, the BRAF inhibitor comprises PLX8394.

[0314] In some embodiments, the BRAF inhibitor comprises RO5212054. RO5212054 is also known as PLX3603.

[0315] Exemplary MEK Inhibitors In one embodiment, the targeted therapy described herein comprises an agent that targets MEK (e.g., an inhibitor of MEK). In some embodiments, the targeted therapy is used to treat cancer, such as a cancer described herein, such as a skin cancer (e.g., melanoma). Without wishing to be bound by theory, it is believed that when MEK, a member of the MAPK signaling cascade, is inhibited, cell proliferation may be blocked and apoptosis may be induced. In some embodiments, the targeted therapy is used to treat melanoma, such as unresectable or metastatic melanoma. In some embodiments, the targeted therapy is used to treat melanoma in a subject, where the melanoma has a BRAF mutation (e.g., a BRAF V600 mutation).

[0316] In some embodiments, the MEK inhibitor is trametinib. Trametinib is also known as GSK1120212, JTP-74057, TMT212, G-02442104, or MEKINIST® (CAS number 871700-17-3). Without wishing to be bound by theory, in some embodiments, trametinib is believed to be a reversible and highly selective allosteric inhibitor of MEK1 and MEK2. MEK protein is a key component of the MAPK pathway and is commonly overactivated in tumor cells, such as melanoma cells. In both BRAF and RAS, oncogenic mutations can signal through MEK1 or MEK2.

[0317] The chemical formula of trametinib is a compound of formula (I):

[0318] [ka] or a pharma- ceutically acceptable salt or solvate thereof. For convenience, the group of potential compounds and salts or solvates will be referred to collectively as trametinib, meaning that reference to trametinib refers alternatively to either the compound or a pharma- ceutically acceptable salt or solvate thereof.

[0319] Depending on naming conventions, trametinib may be properly referred to as N-{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide.

[0320] Trametinib, together with its pharma- ceutically acceptable salts and also its solvates, is disclosed and claimed in WO 2005 / 121142 as being useful as an inhibitor of MEK activity, particularly in the treatment of cancer. Trametinib is the compound of Example 4-1 and can be prepared as described in WO 2005 / 121142.

[0321] Trametinib may be in the form of a dimethylsulfoxide solvate, or in the form of a sodium salt, or in the form of a solvate selected from hydrate, acetic acid, ethanol, nitromethane, chlorobenzene, 1-pentanol, isopropyl alcohol, ethylene glycol, and 3-methyl-1-butanol. These solvates and salt forms can be prepared by one skilled in the art from the description in WO 2005 / 121142.

[0322] In some embodiments, the MEK inhibitor or trametinib is administered at a dose between 0.1 mg and 4 mg (e.g., between 0.5 mg and 3 mg, e.g., at a dose of 0.5 mg), e.g., once a day. In some embodiments, the MEK inhibitor or trametinib is administered at a dose of 0.5 mg, e.g., once a day. In certain embodiments, the MEK inhibitor or trametinib is administered at a dose between 1 mg and 3 mg (e.g., at a dose of about 2 mg), e.g., once a day. In some embodiments, the MEK inhibitor or trametinib is administered orally.

[0323] In another embodiment, the MEK inhibitor is cobimetinib. Cobimetinib is disclosed in WO 2007 / 044515, the entirety of which is incorporated herein by reference. Cobimetinib is also known as XL-518, GDC-0973, RG-7420, or COTELLIC® (CAS No. 934660-93-2). As used herein, the MEK inhibitor cobimetinib, or a pharma- ceutically acceptable salt or solvate thereof, refers to a compound of formula (III):

[0324] [ka] or a pharmaceutically acceptable salt or solvate thereof. For convenience, the group of potential compounds and salts or solvates will be referred to collectively as cobimetinib, meaning that reference to cobimetinib refers alternatively to either the compound or a pharmaceutically acceptable salt or solvate thereof.

[0325] Depending on naming conventions, cobimetinib may be properly referred to as [3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl][3-hydroxy-3-(2S)-2-piperidinyl-1-azetidinyl]-methanone.

[0326] Cobimetinib, together with its pharma- ceutically acceptable salts and also as its solvates, is disclosed and claimed in WO 2007 / 044515 as being useful as an inhibitor of MEK activity, particularly in the treatment of cancer. Cobimetinib can be prepared as described in WO 2007 / 044515.

[0327] Other exemplary MEK inhibitors that can be used in the targeted therapies described herein include, but are not limited to, binimetinib, mirdametinib, pimasertib, refametinib, selumetinib, AS703988, AZD 8330, BI 847325, BIX 02188, BIX 02189, CI-1040, CS3006, E6201, FCN-159, G-38963, GDC-0623, HL-085, PD 98059, RO4987655, RO5126766, SHR 7390, TAK-733, U0126, WX-554, or a pharma- ceutically acceptable salt thereof.

[0328] In some embodiments, the MEK inhibitor comprises binimetinib. Binimetinib is also known as ARRY-438162, MEK162, or MEKTOVI® (CAS number 606143-89-9). Depending on naming conventions, binimetinib may be appropriately named as 5-[(4-bromo-2-fluorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6-carboxamide.

[0329] In some embodiments, the MEK inhibitor comprises mirdametinib. Mirdametinib is also known as PD 0325901 (CAS number 391210-10-9). Depending on naming conventions, mirdametinib may be appropriately named as N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide. Mirdametinib is described, for example, in PCT Publication WO 2002 / 006213.

[0330] In some embodiments, the MEK inhibitor comprises pimasertib. Pimasertib is also known as AS-703206 G-02443714, or MSC1936369B (CAS number 1236699-92-5). Depending on naming conventions, pimasertib may be appropriately named as N-[(2S)-2,3-dihydroxypropyl]-3-[(2-fluoro-4-iodophenyl)amino]-4-pyridinecarboxamide.

[0331] In some embodiments, the MEK inhibitor comprises refametinib.Refametinib is also known as BAY 86-9766 or RDEA119 (CAS number 923032-37-5).Depending on naming conventions, refametinib may be appropriately referred to as N-[3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-1-[(2S)-2,3-dihydroxypropyl]-cyclopropanesulfonamide.

[0332] In some embodiments, the MEK inhibitor comprises selumetinib. Selumetinib is also known as AZD 6244, ARRY 142886, CL 1,040, G 00039805, or NSC 741O78 (CAS number 606143-52-6). Depending on naming conventions, selumetinib may be appropriately named as 5-[(4-bromo-2-chlorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6-carboxamide (CAS number 606143-52-6). Selumetinib is described, for example, in PCT Publication WO 2003 / 077914.

[0333] In some embodiments, the MEK inhibitor comprises AS703988. AS703988 is also known as MSC2015103B.

[0334] In some embodiments, the MEK inhibitor comprises AZD 8330. AZD 8330 is also known as ARRY-424704 (CAS number 869357-68-6). Depending on naming conventions, AZD 8330 may be appropriately referred to as 2-[(2-fluoro-4-iodophenyl)amino]-1,6-dihydro-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-3-pyridinecarboxamide.

[0335] In some embodiments, the MEK inhibitor comprises BI 847325 (CAS number 1207293-36-4). Depending on naming conventions, BI 847325 may be appropriately referred to as 3-[3-[[[4-[(dimethylamino)methyl]phenyl]amino]phenylmethylene]-2,3-dihydro-2-oxo-1H-indol-6-yl]-N-ethyl-2-propynamide.

[0336] In some embodiments, the MEK inhibitor comprises BIX 02188 (CAS number 334949-59-6). Depending on naming conventions, BIX 02188 may be appropriately referred to as (3Z)-3-[[[3-[(dimethylamino)methyl]phenyl]amino]phenylmethylene]-2,3-dihydro-2-oxo-1H-indole-6-carboxamide.

[0337] In some embodiments, the MEK inhibitor comprises BIX 02189 (CAS number 1265916-41-3). Depending on naming conventions, BIX 02189 may be appropriately referred to as (3Z)-3-[[[3-[(dimethylamino)methyl]phenyl]amino]phenylmethylene]-2,3-dihydro-N,N-dimethyl-2-oxo-1H-indole-6-carboxamide.

[0338] In some embodiments, the MEK inhibitor comprises CI-1040. CI-1040 is also known as PD184352 (CAS number 212631-79-3). Depending on naming conventions, selumetinib may be appropriately named as 2-[(2-chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide. CI-1040 is described, for example, in PCT Publication WO 2000 / 035436.

[0339] In some embodiments, the MEK inhibitor comprises CS3006.

[0340] In some embodiments, the MEK inhibitor comprises E6201. Depending on naming conventions, selumetinib may be appropriately named as (3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecyne-1,7(8H)-dione. E6201 is described, for example, in PCT Publication WO 2003 / 076424.

[0341] In some embodiments, the MEK inhibitor comprises FCN-159.

[0342] In some embodiments, the MEK inhibitor comprises G-38963. Depending on naming conventions, G-38963 may be appropriately referred to as 3-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)furo[3,2-c]pyridine-2-carboxamide.

[0343] In some embodiments, the MEK inhibitor comprises GDC-0623 (CAS number 1168091-68-6). Depending on naming conventions, GDC-0623 may be appropriately referred to as 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-imidazo[1,5-a]pyridine-6-carboxamide.

[0344] In some embodiments, the MEK inhibitor comprises HL-085.

[0345] In some embodiments, the MEK inhibitor comprises PD 98059 (CAS number 167869-21-8). Depending on naming conventions, PD 98059 may be appropriately referred to as 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one.

[0346] In some embodiments, the MEK inhibitor comprises RO4987655. RO4987655 is also known as CH4987655 (CAS number 874101-00-5). Depending on naming conventions, RO4987655 may be appropriately named as 3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-5-[(tetrahydro-3-oxo-2H-1,2-oxazin-2-yl)methyl]-benzamide.

[0347] In some embodiments, the MEK inhibitor comprises RO5126766. RO5126766 is also known as CH5126766 (CAS number 946128-88-7). Depending on naming conventions, RO5126766 may be appropriately referred to as N-[3-fluoro-4-[[4-methyl-2-oxo-7-(2-pyrimidinyloxy)-2H-1-benzopyran-3-yl]methyl]-2-pyridinyl]-N'-methyl-sulfamide.

[0348] In some embodiments, the MEK inhibitor comprises SHR 7390.

[0349] In some embodiments, the MEK inhibitor comprises TAK-733 (CAS number 1035555-63-5). Depending on naming conventions, TAK-733 may be appropriately referred to as 3-[(2R)-2,3-dihydroxypropyl]-6-fluoro-5-[(2-fluoro-4-iodophenyl)amino]-8-methyl-pyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione.

[0350] In some embodiments, the MEK inhibitor comprises U0126 (CAS number 109511-58-2). Depending on naming convention, U0126 may be appropriately named as 2,3-bis[amino[(2-aminophenyl)thio]methylene]-butanedinitrile. U0216 is described, for example, in U.S. Patent No. 2,779,780. U0126 is described, for example, in U.S. Patent No. 2,779,780.

[0351] In some embodiments, the MEK inhibitor comprises WX-554.

[0352] Further examples of MEK inhibitors are disclosed in WO 2013 / 019906, WO 03 / 077914, WO 2005 / 121142, WO 2007 / 04415, WO 2008 / 024725 and WO 2009 / 085983, the contents of which are incorporated herein by reference.

[0353] Combination of BRAF and MEK inhibitors In some embodiments, the targeted therapy described herein comprises an agent that targets BRAF (e.g., a BRAF inhibitor described herein) and an agent that targets MEK (e.g., a MEK inhibitor described herein). In certain embodiments, the BRAF inhibitor is dabrafenib and the MEK inhibitor is trametinib. Both molecules and their combinations are disclosed, for example, in WO2011 / 047238, which is incorporated herein by reference in its entirety. In other embodiments, the BRAF inhibitor is vemurafenib and the MEK inhibitor is cobimetinib.

[0354] Preclinical in vitro and in vivo data demonstrated increased antitumor activity with the combination of BRAF inhibitors (e.g., dabrafenib) and MEK inhibitors (e.g., trametinib). For example, the combination of dabrafenib and trametinib demonstrated enhanced antiproliferative activity against a panel of BRAF-mutated cell lines in vitro, thereby suggesting a synergistic effect of dabrafenib and trametinib in addressing primary resistance to each single agent. The combination was effective in inhibiting the growth of dabrafenib-resistant BRAF-mutated melanoma cell clones, demonstrating the potential of the combination therapy to overcome acquired resistance. This cell line data is comparable to in vitro results of other experimental BRAF and MEK inhibitor combinations (Corcoran et al. Sci Signal. 2010; 3(149):ra84; Emery et al. Proc Natl Acad Sci US A. 2009; 106(48):20411-6).

[0355] As another example, we demonstrated that the combination of dabrafenib and trametinib improved activity in a mouse xenograft model of BRAF-mutated melanoma compared to either agent alone. In a skin toxicity study performed in rats, the addition of trametinib to dabrafenib prevented the development of proliferative skin lesions observed after treatment with dabrafenib alone. These results suggest that the addition of a MEK inhibitor to a BRAF inhibitor may suppress proliferation signals in normal skin cells, leading to the development of hyperproliferative skin lesions, including keratoacanthomas and cutaneous squamous cell carcinomas, which are often observed in clinical trials involving BRAF inhibitors (Flaherty et al. CurrO pin Oncol. 2010; 22(3):178-83; Chapman et al. Expert Opin Investig Drugs. 2011; 20(2):209-20; Robert et al. CurrO pin Oncol. 2011; 23(2):177-82). Similar results have been observed with another combination of a BRAF inhibitor and a MEK inhibitor (Carnahan et al. Mol Cancer Ther. 2010; 9(8):2399-410).

[0356] Cancer immunotherapy In certain embodiments, the treatment described herein comprises cancer immunotherapy.

[0357] As used herein, "cancer immunotherapy" refers to a cancer therapy that modulates immune response. In some embodiments, the cancer immunotherapy comprises an immune checkpoint inhibitor. The term "immune checkpoint inhibitor" typically refers to a therapeutic agent that targets at least one immune checkpoint protein and modulates immune response, for example, downmodulates or inhibits immune response. Immune checkpoint proteins are known in the art and include, but are not limited to, programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), V-domain Ig inhibitor of T-cell activation (VISTA), B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, IDO, OX40, and A2aR. In some cases, immune checkpoint proteins may be expressed on the surface of activated T cells. Therapeutic agents that may act as immune checkpoint inhibitors useful in the methods of the present disclosure include, but are not limited to, therapeutic agents that target one or more of PD-1, CTLA-4, PD-L1, PD-L2, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, ICOS, HVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, IDO, OX40, and A2aR. In some cases, the immune checkpoint inhibitor enhances or suppresses the function of one or more immune checkpoint proteins. In some cases, the immune checkpoint inhibitor is a PD-L1 axis binding antagonist as described herein.

[0358] PD-1 inhibitors In certain embodiments, the cancer immunotherapy described herein includes a PD-1 binding antagonist, e.g., a PD-1 inhibitor.

[0359] In some embodiments, the PD-1 inhibitor is selected from PDR001 (Novartis), nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck & Co), MEDI0680 (Medimmune), REGN2810 / cemiplimab (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune). In some embodiments, the PD-1 inhibitor is PDR001. PDR001 is also known as spartalizumab. Nivolumab (clone 5C4) and other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,008,449 and WO 2006 / 121168, which are incorporated by reference in their entireties. Pembrolizumab and other anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134-44, U.S. Pat. No. 8,354,509, and WO 2009 / 114335, which are incorporated by reference in their entireties. MEDI0680 and other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 9,205,148 and WO 2012 / 145493, which are incorporated by reference in their entireties. Additionally, known anti-PD-1 antibodies include those described in, for example, WO 2015 / 112800, WO 2016 / 092419, WO 2015 / 085847, WO 2014 / 179664, WO 2014 / 194302, WO 2014 / 209804, WO 2015 / 200119, U.S. Patent Nos. 8,735,553, 7,488,802, 8,927,697, 8,993,731, and 9,102,727, which are incorporated by reference in their entireties.

[0360] Exemplary PD-1 Inhibitors In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule described in U.S. Patent Application Publication No. 2015 / 0210769, which is incorporated by reference in its entirety. In some embodiments, the anti-PD-1 antibody molecule is spartalizumab (also known as PDR001).

[0361] In one embodiment, the anti-PD-1 antibody molecule comprises at least one, two, three, four, five, or six complementarity determining regions (CDRs) (or collectively, all of the CDRs) derived from heavy and light chain variable regions comprising the amino acid sequences set forth in Table 1 (e.g., derived from the heavy and light chain variable region sequences of BAP049-clone-E or BAP049-clone-B disclosed in Table 1), or derived from heavy and light chain variable regions encoded by the nucleotide sequences set forth in Table 1. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set forth in Table 1). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set forth in Table 1). In some embodiments, the CDRs are according to both the Kabat and Chothia combined CDR definitions (e.g., as set forth in Table 1). In one embodiment, the Kabat and Chothia CDR combination of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO:541). In one embodiment, one or more of the CDRs (or collectively, all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to the amino acid sequence shown in Table 1, or the amino acid sequence encoded by the nucleotide sequence shown in Table 1.

[0362] In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO:501, a VHCDR2 amino acid sequence of SEQ ID NO:502, and a VHCDR3 amino acid sequence of SEQ ID NO:503; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO:510, a VLCDR2 amino acid sequence of SEQ ID NO:511, and a VLCDR3 amino acid sequence of SEQ ID NO:512, each of which are disclosed in Table 1.

[0363] In one embodiment, the antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO:524, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO:525, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO:526; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO:529, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO:530, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO:531, each of which are disclosed in Table 1.

[0364] In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:506. In one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:520, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:520. In one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:516, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:516. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506 and a VL comprising the amino acid sequence of SEQ ID NO:520. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:506 and a VL comprising the amino acid sequence of SEQ ID NO:516.

[0365] In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 507, or a nucleotide sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO: 507. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 521 or 517, or a nucleotide sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO: 521 or 517. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 507 and a VL encoded by the nucleotide sequence of SEQ ID NO: 521 or 517.

[0366] In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:508. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:522, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:522. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:518, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:518. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508 and a light chain comprising the amino acid sequence of SEQ ID NO:522. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:508 and a light chain comprising the amino acid sequence of SEQ ID NO:518.

[0367] In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 509, or a nucleotide sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO: 509. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 523 or 519, or a nucleotide sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO: 523 or 519. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 509 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 523 or 519.

[0368] The antibody molecules described herein may be produced by the vectors, host cells, and methods described in U.S. Patent Application Publication No. 2015 / 0210769, which is incorporated by reference in its entirety.

[0369] [Table 1-1]

[0370] [Table 1-2]

[0371] [Table 1-3]

[0372] [Table 1-4]

[0373] [Table 1-5]

[0374] [Table 1-6]

[0375] [Table 1-7]

[0376] [Table 1-8]

[0377] In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg to about 500 mg (e.g., about 300 mg to about 400 mg). In some embodiments, the PD-1 inhibitor is administered once every three weeks. In some embodiments, the PD-1 inhibitor is administered once every four weeks. In other embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg to about 400 mg (e.g., about 300 mg) once every three weeks. In yet other embodiments, the PD-1 inhibitor is administered at a dose of about 300 mg to about 500 mg (e.g., about 400 mg) once every four weeks.

[0378] Other Exemplary PD-1 Inhibitors Other exemplary PD-1 inhibitors include, but are not limited to, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck & Co), MEDI0680 (Medimmune), REGN2810 / cemiplimab (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 / tislelizumab (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), and AMP-224 (Amplimmune).

[0379] In one embodiment, the anti-PD-1 antibody molecule is nivolumab (Bristol-Myers Squibb), also known as MDX-1106, MDX-1106-04, ONO-4538, BMS-936558, or OPDIVO®. Nivolumab (clone 5C4) and other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,008,449 and WO 2006 / 121168, which are incorporated by reference in their entireties. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), heavy or light chain variable region sequences, or heavy or light chain sequences of nivolumab, e.g., as disclosed in Table 2.

[0380] In one embodiment, the anti-PD-1 antibody molecule is pembrolizumab (Merck & Co), also known as lambrolizumab, MK-3475, MK03475, SCH-900475, or KEYTRUDA®. Pembrolizumab and other anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134-44, U.S. Pat. No. 8,354,509, and WO 2009 / 114335, which are incorporated by reference in their entireties. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), heavy or light chain variable region sequences, or heavy or light chain sequences of pembrolizumab, e.g., as disclosed in Table 2.

[0381] In one embodiment, the anti-PD-1 antibody molecule is MEDI0680 (Medimmune), also known as AMP-514. MEDI0680 and other anti-PD-1 antibodies are disclosed in U.S. Patent No. 9,205,148 and WO 2012 / 145493, which are incorporated by reference in their entireties. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequences of MEDI0680.

[0382] In one embodiment, the anti-PD-1 antibody molecule is REGN2810 / cemiplimab (Regeneron). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequence of REGN2810.

[0383] In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (Pfizer). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequence of PF-06801591.

[0384] In one embodiment, the anti-PD-1 antibody molecule is BGB-A317 / tislelizumab or BGB-108 (Beigene). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequence of BGB-A317 / tislelizumab or BGB-108.

[0385] In one embodiment, the anti-PD-1 antibody molecule is INCSHR1210 (Incyte), also known as INCSHR01210 or SHR-1210. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequence of INCSHR1210.

[0386] In one embodiment, the anti-PD-1 antibody molecule is TSR-042 (Tesaro), also known as ANB011. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequence of TSR-042.

[0387] Additionally, known anti-PD-1 antibodies include those described in, for example, WO 2015 / 112800, WO 2016 / 092419, WO 2015 / 085847, WO 2014 / 179664, WO 2014 / 194302, WO 2014 / 209804, WO 2015 / 200119, U.S. Pat. Nos. 8,735,553, 7,488,802, 8,927,697, 8,993,731, and 9,102,727, which are incorporated by reference in their entireties.

[0388] In one embodiment, the anti-PD-1 antibody is an antibody that competes for binding with and / or binds to the same epitope of PD-1 as one of the anti-PD-1 antibodies described herein.

[0389] In one embodiment, the PD-1 inhibitor is a peptide that inhibits the PD-1 signaling pathway, e.g., as described in U.S. Patent No. 8,907,053, which is incorporated by reference in its entirety. In one embodiment, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin that includes an extracellular or PD-1 binding portion of PD-L1 or PD-L2) fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In one embodiment, the PD-1 inhibitor is AMP-224 (e.g., B7-DCIg (Amplimmune) disclosed in WO 2010 / 027827 and WO 2011 / 066342, which are incorporated by reference in their entirety).

[0390] [Table 2]

[0391] PD-L1 inhibitors In certain embodiments, the cancer immunotherapy described herein comprises a PD-L1 binding antagonist, e.g., a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule.

[0392] In some embodiments, the PD-L1 inhibitor is selected from FAZ053 (Novartis), atezolizumab (Genentech / Roche), avelumab (Merck Serono and Pfizer), durvalumab, also known as MEDI4736 (MedImmune / AstraZeneca), or BMS-936559, also known as MDX-1105 or 12A4 (Bristol-Myers Squibb).

[0393] In one embodiment, the anti-PD-1 antibody molecule is FAZ053. FAZ053 and other anti-PD-L1 antibodies are described in US Patent Application Publication No. 2016 / 0108123, which is incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequences of FAZ053.

[0394] In one embodiment, the anti-PD-L1 antibody molecule is atezolizumab (Genentech / Roche), also known as MPDL3280A, RG7446, RO5541267, YW243.55.S70, or TECENTRIQ™. Atezolizumab and other anti-PD-L1 antibodies are disclosed in U.S. Patent No. 8,217,149 and WO 2013 / 079174, which are incorporated by reference in their entireties. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequence of atezolizumab.

[0395] In one embodiment, the anti-PD-L1 antibody molecule is avelumab (Merck Serono and Pfizer), also known as MSB0010718C. Avelumab and other anti-PD-L1 antibodies are disclosed in WO 2013 / 079174, which is incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequences of avelumab.

[0396] In one embodiment, the anti-PD-L1 antibody molecule is durvalumab (MedImmune / AstraZeneca), also known as MEDI4736. Durvalumab and other anti-PD-L1 antibodies are disclosed in U.S. Patent No. 8,779,108, which is incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequences of durvalumab.

[0397] In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (Bristol-Myers Squibb), also known as MDX-1105 or 12A4. BMS-936559 and other anti-PD-L1 antibodies are disclosed in U.S. Patent No. 7,943,743 and WO 2015 / 081158, which are incorporated by reference in their entireties. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively, all of the CDR sequences), the heavy or light chain variable region sequence, or the heavy or light chain sequences of BMS-936559.

[0398] Additionally, known anti-PD-L1 antibodies include those described in, e.g., WO 2015 / 181342, WO 2014 / 100079, WO 2016 / 000619, WO 2014 / 022758, WO 2014 / 055897, WO 2015 / 061668, WO 2013 / 079 174, 2012 / 145493, 2015 / 112805, 2015 / 109124, 2015 / 195163, U.S. Pat. Nos. 8,168,179, 8,552,154, 8,460,927, and 9,175,082.

[0399] In one embodiment, the anti-PD-L1 antibody is an antibody that competes for binding with and / or binds to the same epitope of PD-L1 as one of the anti-PD-L1 antibodies described herein.

[0400] CTLA-4 inhibitors In certain embodiments, the cancer immunotherapy described herein comprises a CTLA-4 binding antagonist, e.g., a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 antibody molecule. In some embodiments, the anti-CTLA-4 antibody molecule is ipilimumab (also known as BMS-734016, MDX-010, MDX-101). In some embodiments, the anti-CTLA-4 antibody molecule is tremelimumab (also known as ticilimumab or CP-675,206).

[0401] Combining targeted therapy with cancer immunotherapy In some embodiments, the treatments described herein include targeted therapies (e.g., targeted therapies described herein) and cancer immunotherapy (e.g., cancer immunotherapy described herein).

[0402] In some embodiments, the targeted therapy includes an agent that targets BRAF (e.g., a BRAF inhibitor described herein) and / or an agent that targets MEK (e.g., a MEK inhibitor described herein), and the cancer immunotherapy includes a PD-1 inhibitor (e.g., a PD-1 inhibitor described herein). Without wishing to be bound by theory, in some embodiments, the use of an immune checkpoint inhibitor, e.g., a PD-1 inhibitor, in combination with a BRAF inhibitor, a MEK inhibitor, or both, may improve the response provided by inhibition of BRAF and / or MEK (e.g., a more rapid response, a more durable response, a higher response rate, or a more complete response).

[0403] In some embodiments, the BRAF inhibitor is dabrafenib or vemurafenib. In some embodiments, the MEK inhibitor is vemurafenib or cobimetinib. In some embodiments, the PD-1 inhibitor is spartalizumab. In certain embodiments, the treatment comprises targeted therapy comprising dabrafenib and vemurafenib and cancer immunotherapy comprising spartalizumab. In other embodiments, the treatment comprises targeted therapy comprising vemurafenib and cobimetinib and cancer immunotherapy comprising spartalizumab.

[0404] In certain embodiments, the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) is administered at a dose of between 200 mg and 600 mg, e.g., between 300 mg and 500 mg (e.g., at a dose of 400 mg), once every 3 weeks, once every 4 weeks, or once every 8 weeks.

[0405] In certain embodiments, the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) is administered at a dose between 300 mg and 500 mg (e.g., at a dose of 400 mg) once every 4 weeks, e.g., intravenously, for a period of, e.g., 15 to 120 minutes (e.g., about 30 minutes). In other embodiments, the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) is administered at a dose between 300 mg and 500 mg (e.g., at a dose of 400 mg) once every 8 weeks, e.g., intravenously, for a period of, e.g., 15 to 120 minutes (e.g., about 30 minutes). In some embodiments, administration may be discontinued for up to 12 weeks, e.g., up to 8 weeks or up to 4 weeks.

[0406] In some embodiments, the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) is administered at a dose of between 300 mg and 500 mg (e.g., at a dose of 400 mg), e.g., once every four weeks, e.g., intravenously, and (i) the BRAF targeting agent (e.g., dabrafenib) is administered at a dose of between 50 mg and 300 mg (e.g., between 100 mg and 200 mg, e.g., at a dose of 150 mg), e.g., twice a day, e.g., orally. or at a total daily dose of between 100 mg and 600 mg (e.g., between 200 mg and 400 mg, e.g., at a total daily dose of 300 mg), e.g., administered orally, (ii) an agent targeting MEK (e.g., trametinib) at a dose of between 0.5 mg and 4 mg (e.g., between 1 mg and 3 mg, e.g., at a dose of 2 mg), e.g., once a day, e.g., administered orally; or (iii) both (i) and (ii) are administered.

[0407] In some embodiments, administration of the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) begins on the same day as administration of the agent targeting BRAF (e.g., dabrafenib), the agent targeting MEK (e.g., trametinib), or both. In other embodiments, the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) is administered after the agent targeting BRAF (e.g., dabrafenib), the agent targeting MEK (e.g., trametinib), or both have been administered for about 4 weeks or longer. For example, if administration of the agent targeting BRAF (e.g., dabrafenib), the agent targeting MEK (e.g., trametinib), or both begins on day 1, administration of the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) can begin on day 29. By way of example, agents targeting BRAF (e.g., dabrafenib) can be administered twice daily per dosing regimen on days 1-28 of a 28-day cycle, and agents targeting MEK (e.g., trametinib) can be administered per dosing regimen on days 1-28 of a 28-day cycle.

[0408] In certain embodiments, the treatment includes a PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab), an agent that targets BRAF (e.g., dabrafenib), and an agent that targets MEK (e.g., trametinib).

[0409] In some embodiments, the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) is administered at a dose of between 300 mg and 500 mg (e.g., at a dose of 400 mg), e.g., once every 4 weeks, e.g., intravenously; the BRAF targeting agent (e.g., dabrafenib) is administered at a dose of between 50 mg and 300 mg (e.g., between 100 mg and 200 mg, e.g., at a dose of 150 mg), e.g., twice a day, e.g., orally, or at a total daily dose of between 100 mg and 600 mg (e.g., between 200 mg and 400 mg, e.g., at a total daily dose of 300 mg), e.g., orally; the MEK targeting agent (e.g., trametinib) is administered at a dose of between 0.5 mg and 4 mg (e.g., between 1 mg and 3 mg, e.g., at a dose of 2 mg), e.g., once a day, e.g., orally.

[0410] In some embodiments, the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) is administered at a dose of between 300 mg and 500 mg (e.g., at a dose of 400 mg), e.g., once every 8 weeks, e.g., intravenously, and (i) the BRAF targeting agent (e.g., dabrafenib) is administered at a dose of between 50 mg and 300 mg (e.g., between 100 mg and 200 mg, e.g., at a dose of 150 mg), e.g., twice a day, e.g., orally. or at a total daily dose of between 100 mg and 600 mg (e.g., between 200 mg and 400 mg, e.g., at a total daily dose of 300 mg), e.g., administered orally, (ii) an agent targeting MEK (e.g., trametinib) at a dose of between 0.5 mg and 4 mg (e.g., between 1 mg and 3 mg, e.g., at a dose of 2 mg), e.g., once a day, e.g., administered orally; or (iii) both (i) and (ii) are administered.

[0411] In some embodiments, the PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) is administered at a dose of between 300 mg and 500 mg (e.g., at a dose of 400 mg), e.g., once every 8 weeks, e.g., intravenously; the BRAF targeting agent (e.g., dabrafenib) is administered at a dose of between 50 mg and 300 mg (e.g., between 100 mg and 200 mg, e.g., at a dose of 150 mg), e.g., twice a day, e.g., orally, or at a total daily dose of between 100 mg and 600 mg (e.g., between 200 mg and 400 mg, e.g., at a total daily dose of 300 mg), e.g., orally; the MEK targeting agent (e.g., trametinib) is administered at a dose of between 0.5 mg and 4 mg (e.g., between 1 mg and 3 mg, e.g., at a dose of 2 mg), e.g., once a day, e.g., orally.

[0412] antibody molecule In one embodiment, the cancer immunotherapy comprises an antibody molecule (e.g., an anti-PD-1 antibody molecule).

[0413] As used herein, the term "antibody molecule" refers to a protein that comprises at least one immunoglobulin variable domain sequence, e.g., an immunoglobulin chain or fragment thereof. The term antibody molecule includes, for example, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region). In embodiments, an antibody molecule comprises a full-length antibody or a full-length immunoglobulin chain. In embodiments, an antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody, or a full-length immunoglobulin chain.

[0414] In embodiments, antibody molecules include diabodies and single chain molecules, as well as antigen-binding fragments of antibodies (e.g., Fab, F(ab')2, and Fv). For example, an antibody molecule may include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In embodiments, an antibody molecule includes or consists of a heavy chain and a light chain (referred to herein as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by modification of a whole antibody or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be from any class of antibody, including but not limited to IgG, IgA, IgM, IgD, and IgE, and from any subclass of antibody (e.g., IgG1, IgG2, IgG3, and IgG4). The antibodies of the present disclosure can be monoclonal or polyclonal. The antibody molecule can also be a human antibody, a humanized antibody, a CDR-grafted antibody, or an in vitro generated antibody. The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably herein with the term "antibody."

[0415] Examples of antigen-binding fragments of antibody molecules include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a diabody (dAb) fragment consisting of the VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), (see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0416] The term "antibody" includes intact molecules and functional fragments thereof. The constant regions of antibodies can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function).

[0417] The antibody molecule may also be a single domain antibody. Single domain antibodies may include antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from traditional four chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. The single domain antibody may be any of the single domain antibodies in the art or any of the future single domain antibodies. The single domain antibody may be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the present disclosure, the single domain antibody is a naturally occurring single domain antibody known as a heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed, for example, in WO 9404678. For clarity, this variable domain derived from a heavy chain antibody naturally devoid of light chains is known herein as a VHH or nanobody, to distinguish it from the traditional VH of four chain immunoglobulins. Such VHH molecules may be derived from antibodies raised in Camelidae species, such as camel, llama, dromedary, alpaca and guanaco. Other non-Camelidae species may produce heavy chain antibodies naturally devoid of light chains, and such VHHs are within the scope of the present disclosure.

[0418] The VH and VL regions can be further subdivided into regions of hypervariability, called "complementarity determining regions" (CDRs), interspersed with regions that are more conserved, called "framework regions" (FR or FW).

[0419] The extent of framework regions and CDRs have been precisely defined in several ways (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definitions used in Oxford Molecular's AbM antibody modeling software). See generally, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0420] The terms "complementarity determining region" and "CDR" as used herein refer to the amino acid sequence in the antibody variable region that confers antigen specificity and binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0421] The exact amino acid sequence boundaries of a given CDR can be determined by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5 thThe CDRs may be determined using any of several well-known schemes, including those described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme). As used herein, CDRs defined according to the "Chothia" numbering scheme may also be referred to as "hypervariable loops".

[0422] For example, in Kabat, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); the amino acid residues of the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of human VL.

[0423] In general, unless otherwise indicated, the antibody molecules disclosed herein may comprise any combination of one or more Kabat CDRs and / or Chothia hypervariable loops. In one embodiment, the following definitions are used for the anti-PD-1 antibody molecules listed in Table 1: HCDR1 is a combination of both the Kabat and Chothia CDR definitions, and HCCDR2-3 and LCCDR1-3 are according to the Kabat CDR definitions. In all definitions, each VH and VL typically comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0424] As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence capable of forming the structure of an immunoglobulin variable domain. For example, this sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, this sequence may or may not include one, two, or more N-terminal or C-terminal amino acids, or may include other modifications compatible with forming a protein structure.

[0425] The term "antigen-binding site" refers to the portion of an antibody molecule that contains the determinants that form the interface that binds to a PD-1 polypeptide or an epitope thereof. For proteins (or protein mimetics), the antigen-binding site typically includes one or more loops (one or more loops of at least four amino acids or amino acid mimetics) that form the interface that binds to the PD-1 polypeptide. Typically, the antigen-binding site of an antibody molecule includes at least one or two CDRs and / or hypervariable loops, or typically at least three, four, five or six CDRs and / or hypervariable loops.

[0426] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be produced by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0427] An "effectively human" protein is one that does not induce a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. HAMA can be problematic in some situations, for example, when antibody molecules are administered repeatedly in the treatment of chronic or recurrent disease conditions. Repeated administration of antibodies can potentially be ineffective due to the HAMA response, which increases antibody clearance from serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0428] The antibody molecule may be a polyclonal or monoclonal antibody, hi other embodiments, the antibody may be recombinantly produced, for example, by phage display or combinatorial methods.

[0429] Phage display and combinatorial methods for generating antibodies are known in the art (see, e.g., U.S. Pat. No. 5,223,409 to Ladner et al.; WO 92 / 18619 to Kang et al.; WO 91 / 17271 to Dower et al.; WO 92 / 20791 to Winter et al.; WO 92 / 15679 to Markland et al.; WO 93 / 01288 to Breitling et al.; WO 92 / 01047 to McCafferty et al.; WO 92 / 09690 to Garrard et al.; WO 90 / 02809 to Ladner et al.; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. (1992), the entire contents of which are incorporated herein by reference. Hum Antibody Hybridomas 3:81-85;Huse et al. (1989) Science 246:1275-1281;Griffths et al. (1993) EMBO J 12:725-734;Hawkins et al. (1992) J Mol Biol 226:889-896;Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio / Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982).

[0430] In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, such as a rodent (mouse or rat), goat, primate (e.g., monkey), or camel antibody. Preferably, the non-human antibody is rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0431] Human monoclonal antibodies can be produced using transgenic mice carrying non-mouse human immunoglobulin genes. Spleen cells from these transgenic mice immunized with an antigen of interest are used to produce hybridomas secreting human mAbs with specific affinity for epitopes of human proteins (see, e.g., Wood et al., WO 91 / 00906; Kucherlapati et al., PCT Publication WO 91 / 10741; Lonberg et al., WO 92 / 03918; Kay et al., WO 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, LL et al. 1994 Nature Genet. 7:13-21; Morrison, SL et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 20:111-111). 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[0432] The antibody may be one whose variable region or part thereof, such as CDR, is generated in a non-human organism, such as rat or mouse.Chimeric antibodies, CDR-grafted antibodies, and humanized antibodies are within the scope of the present disclosure.Antibodies that are generated in a non-human organism, such as rat or mouse, and then modified, for example, in variable framework or constant regions to reduce antigenicity in humans, are within the scope of the present disclosure.

[0433] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (Robinson et al., International Patent Publication No. PCT / US86 / 02269; Akira et al., European Patent Publication No. 184,187; Taniguchi, M., European Patent Publication No. 171,496; Morrison et al., European Patent Publication No. 173,494; Neuberger et al., International Patent Publication No. WO 86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Publication No. 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0434] A humanized antibody or a CDR-grafted antibody will have at least one or two, but generally all three recipient CDRs (of the immunoglobulin heavy chain and / or immunoglobulin light chain) replaced with donor CDRs. The antibody may have at least a portion of a non-human CDR replaced, or only a portion of the CDR replaced with a non-human CDR. It is sufficient to replace the number of CDRs required for the binding of the humanized antibody to PD-1. Preferably, the donor is a rodent antibody, such as a rat or mouse antibody, and the recipient is a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the "donor" and the immunoglobulin providing the framework is called the "acceptor". In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework, or a consensus framework, or a sequence that is about 85% or more identical thereto, preferably 90%, 95%, 99% or more identical thereto.

[0435] As used herein, the term "consensus sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of proteins, each position of the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. "Consensus framework" refers to the framework region in a consensus immunoglobulin sequence.

[0436] Antibodies may be humanized by methods known in the art (see, e.g., Morrison, SL, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; and Queen et al., U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, the entire contents of which are incorporated herein by reference).

[0437] Humanized or CDR-grafted antibodies can be produced by CDR grafting or CDR replacement, and can replace one, two, or all of the immunoglobulin chains.See, for example, U.S. Patent No. 5,225,539;Jones et al. 1986 Nature 321:552-525;Verhoeyan et al. 1988 Science 239:1534;Beidler et al. 1988 J. Immunol. 141:4053-4060;Winter U.S. Patent No. 5,225,539, the entire contents of which are expressly incorporated herein by reference.Winter describes the CDR grafting method that can be used to prepare the humanized antibodies of the present disclosure (GB Patent Application Publication No. 2188638, filed March 26, 1987;Winter U.S. Patent No. 5,225,539), the contents of which are expressly incorporated herein by reference.

[0438] Humanized antibodies in which specific amino acids are substituted, deleted or added are also within the scope of the present disclosure. Criteria for selecting amino acids from donors are described in U.S. Patent No. 5,585,089, e.g., columns 12-16 of U.S. Patent No. 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in European Patent No. 519596, published December 23, 1992, to Padlan et al.

[0439] The antibody molecule can be a single chain antibody. Single chain antibodies (scFV) can be engineered (see, for example, Colcher, D. et al. (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). Single chain antibodies can be dimerized or multimerized to generate multivalent antibodies with specificity for different epitopes of the same target protein.

[0440] In yet other embodiments, the antibody molecule has a heavy chain constant region selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; in particular, the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4 (e.g., human). In another embodiment, the antibody molecule has a light chain constant region selected from, for example, the light chain constant regions of kappa or lambda (e.g., human). The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and can fix complement. In other embodiments, the antibody does not recruit effector cells or does not fix complement. In another embodiment, the antibody has a reduced or no ability to bind to Fc receptors. For example, the antibody can be an isotype or subtype, fragment or other mutant that does not support binding to an Fc receptor, e.g., the Fc receptor binding region has been mutagenized or deleted.

[0441] Methods for modifying antibody constant regions are known in the art.Antibodies with modified functions, such as modified affinity for effector ligands, such as FcR on cells, or C1 component of complement, can be produced by replacing at least one amino acid residue in the constant part of the antibody with a different residue (see, for example, EP 388,151, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the entire contents of which are incorporated herein by reference).Similar types of modifications can be described that reduce or eliminate these functions when applied to mouse, or other species of immunoglobulins.

[0442] An antibody molecule may be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is one that has been modified. Methods of derivatization include, but are not limited to, the addition of a fluorescent moiety, a radionuclide, a toxin, an enzyme, or an affinity ligand such as biotin. Thus, the antibody molecules of the present disclosure are intended to include derivatized and other modified forms of the antibodies described herein, including immunoadhesin molecules. For example, an antibody molecule may be functionally linked (by chemical linkage, genetic fusion, non-covalent association, etc.) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of an antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0443] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same type or different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate), which have two distinct reactive groups separated by an appropriate spacer. Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0444] Useful detection agents with which the antibody molecules of the present disclosure may be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent metal atoms such as europium (Eu) and other lanthanides, and radioactive materials (described below). Exemplary fluorescent detection agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. Antibodies may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, acetylcholinesterase, glucose oxidase, and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding an additional reagent that the enzyme uses to generate a detectable reaction product. For example, when the detection agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected. Antibody molecules may be derivatized with prosthetic groups (e.g., streptavidin / biotin and avidin / biotin). For example, an antibody may be derivatized with biotin and detected through indirect measurement of avidin or streptavidin binding. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin.

[0445] Labeled antibody molecules can be used diagnostically and / or experimentally in a number of contexts, including, for example, (i) isolating a given antigen by standard techniques such as affinity chromatography or immunoprecipitation; (ii) detecting a given antigen (e.g., in a cell lysate or cell supernatant) and assessing the abundance and pattern of protein expression; and (iii) monitoring protein levels in tissues as part of a clinical testing procedure, e.g., to determine the effectiveness of a given treatment regimen.

[0446] The antibody molecule may be conjugated to another molecular entity, typically a label or a therapeutic agent (e.g., a cytotoxic or cytostatic agent) or therapeutic moiety. Radioisotopes can be used in diagnostic or therapeutic applications. Radioisotopes that can be linked to antibodies include, but are not limited to, α-, β-, γ-emitters, or β- and γ-emitters. Such radioisotopes include, but are not limited to, iodine ( 131 I or 125 I), yttrium ( 90 Y), lutetium ( 177 Lu), Actinium ( 225 Ac), praseodymium, astatine ( 211 At), Rhenium ( 186 Re), Bismuth ( 212 Bi or 213 Bi), Indium ( 111 In), Technetium ( 99 mTc), phosphorus ( 32 P), Rhodium ( 188 Rh), Sulfur ( 35 S), carbon ( 14 C), tritium ( 3 H), Chromium ( 51 Cr), Chlorine ( 36 Cl), Cobalt ( 57 Co or 58 Co), Iron ( 59 Fe), Selenium ( 75 Se), or gallium ( 67 Radioisotopes useful as therapeutic agents include yttrium ( 90 Y), lutetium ( 177 Lu), Actinium ( 225 Ac), praseodymium, astatine ( 211 At), Rhenium ( 186 Re), Bismuth ( 212 Bi or 213 Bi), and rhodium ( 188 Radioisotopes useful as labels, for example for diagnostic use, include iodine ( 131 I or 125I), Indium ( 111 In), Technetium ( 99 mTc), phosphorus ( 32 P), carbon ( 14 C), and tritium ( 3 H), or one or more of the therapeutic isotopes listed above.

[0447] The present disclosure provides a radiolabeled antibody molecule and a method for labeling it. In one embodiment, a method for labeling an antibody molecule is disclosed. The method includes contacting an antibody molecule with a chelating agent, thereby producing a conjugated antibody. The conjugated antibody is radiolabeled with a radioisotope, such as 111 indium, 90 yttrium and 177 lutetium, thereby producing a labeled antibody molecule.

[0448] As mentioned above, the antibody molecule may be conjugated to a therapeutic agent. Therapeutically active radioisotopes have already been mentioned. Examples of other therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, such as maytansinol (see, e.g., U.S. Pat. No. 5,208,020), CC-1065 (see, e.g., U.S. Pat. Nos. 5,475,092, 5,585,499, and 5,846,545), and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepaclorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, These include cisplatin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) (cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0449] Further Therapeutic Agents The treatments described herein can be monotherapies or combination therapies. In some embodiments, the treatments described herein further comprise one or more other therapeutic agents, procedures or modalities.

[0450] In one embodiment, the therapy includes a targeted therapy as described herein and / or a cancer immunotherapy as described herein in combination with an additional therapeutic agent, procedure, or modality for treating a cancer as described herein.

[0451] In certain embodiments, the treatments described herein can be administered with one or more of other antibody molecules, chemotherapy, other anti-cancer treatments (e.g., targeted anti-cancer treatments, gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, or oncolytic drugs), cytotoxic agents, immune-based therapies (e.g., cytokines or cell-based immunotherapy), surgical procedures (e.g., lumpectomy or mastectomy) or radiation procedures, or any combination of the foregoing. The additional treatments may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional treatments are enzyme inhibitors (e.g., small molecule enzyme inhibitors) or metastasis inhibitors. Exemplary cytotoxic agents that may be administered in combination include anti-microtubule agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalating agents, agents capable of interfering with signal transduction pathways, agents that promote apoptosis, proteasome inhibitors, and radiation (e.g., local or total body irradiation (e.g., gamma irradiation)). In other embodiments, the additional treatment is surgery or radiation, or a combination thereof. In some embodiments, the additional treatment is a first-line, second-line, third-line, or fourth-line or higher treatment. In some embodiments, the additional treatment is an adjuvant or neoadjuvant treatment.

[0452] In some embodiments, the additional therapy is for treating melanoma. Exemplary additional therapies for treating melanoma include, but are not limited to, surgery, chemotherapy (e.g., dacarbazine), targeted therapy (e.g., TLR agonist or CD40 agonist), immunotherapy (e.g., cytokine (e.g., IFN-α or IL-2), adoptive cell transfer, radiation therapy, oncolytic virus therapy, or a combination thereof.

[0453] Pharmaceutical Compositions In another aspect, the disclosure provides compositions, e.g., pharma- ceutically acceptable compositions, comprising one or more of the therapeutic agents described herein formulated together with a pharma- ceutically acceptable carrier.

[0454] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient contained therein is effective and which does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0455] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, etc. Carriers can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

[0456] The compositions of this disclosure may be in various forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions. In certain embodiments, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular). In embodiments, the compositions are administered by intravenous infusion or injection. In another embodiment, the compositions are administered by intramuscular or subcutaneous injection.

[0457] The phrases "parenteral administration" and "administered parenterally", as used herein, mean modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0458] Therapeutic compositions should typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high antibody concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in a suitable solvent with one or a combination of ingredients listed above, as needed, followed by filtration sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required as listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which produce a powder of the active ingredient and any additional desired ingredients from the previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

[0459] In some embodiments, the checkpoint inhibitor, the BRAF targeting agent, the MEK targeting agent, or any combination thereof, as described herein, can be formulated into a formulation (e.g., dosage formulation or dosage form) suitable for administration to a subject.For example, a PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule, e.g., spartalizumab) can be formulated into a formulation (e.g., dosage formulation or dosage form) suitable for intravenous administration to a subject as described herein.As another example, a BRAF inhibitor (e.g., dabrafenib) and / or a MEK inhibitor (e.g., trametinib) can be formulated into a formulation (e.g., dosage formulation or dosage form) suitable for oral administration to a subject as described herein.

[0460] Therapeutic agents, e.g., inhibitors, antagonists or binding agents, can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route / mode of administration is intravenous injection or infusion. For example, antibody molecules can be administered at a dose of about 35-440 mg / m 2 , typically about 70-310 mg / m 2 , more typically about 110-130 mg / m 2 In an embodiment, the antibody molecule can be administered by intravenous infusion at a rate of greater than 20 mg / min, e.g., 20-40 mg / min, typically greater than or equal to 40 mg / min, to reach a dose of about 1-100 mg / m. 2 , preferably about 5 to 50 mg / m 2 , about 7~25mg / m 2 , more preferably about 10 mg / m 2The drug may be administered by intravenous infusion at a rate of less than 10 mg / min; preferably less than or equal to 5 mg / min, so as to reach a dose of 10 mg / min. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. In certain embodiments, the active compound may be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0461] In certain embodiments, the therapeutic agent or compound may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may be enclosed in a hard or soft gelatin capsule, compressed into a tablet, or directly incorporated into the subject's diet. For oral therapeutic administration, the compound may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer the compounds of the present disclosure by other than parenteral administration, it may be necessary to coat the compound with, or administer the compound simultaneously with, a material that prevents inactivation. The therapeutic composition may also be administered using medical devices known in the art.

[0462] Dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. Unit dosage form, as used herein, refers to a physically discrete unit suitable as a unit dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage form of the present disclosure are dictated by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of mixing such active compounds for the treatment of individual susceptibility.

[0463] An exemplary non-limiting range of a therapeutically or prophylactically effective amount of a therapeutic agent is 0.1-30 mg / kg, more preferably 1-25 mg / kg. The dosage and treatment regimen of the anti-PD-1 antibody molecule can be determined by one of skill in the art. In certain embodiments, the anti-PD-1 antibody molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1-40 mg / kg, e.g., 1-30 mg / kg, e.g., about 5-25 mg / kg, about 10-20 mg / kg, about 1-5 mg / kg, 1-10 mg / kg, 5-15 mg / kg, 10-20 mg / kg, 15-25 mg / kg, or about 3 mg / kg. The administration schedule can vary, e.g., from once a week to once every 2, 3, or 4 weeks. In one embodiment, the anti-PD-1 antibody molecule is administered every other week at a dose of about 10-20 mg / kg.

[0464] As another example, a non-limiting range for a therapeutically or prophylactically effective amount of an antibody molecule is 200-500 mg, more preferably 300-400 mg / kg. The dosage and treatment regimen of the anti-PD-1 antibody molecule can be determined by one of skill in the art. In certain embodiments, the anti-PD-1 antibody molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose (e.g., fixed dose) of about 200 mg to about 500 mg, e.g., about 250 mg to 450 mg, about 300 mg to 400 mg, about 250 mg to 350 mg, about 350 mg to 450 mg, or about 300 mg or about 400 mg. The administration schedule (e.g., fixed administration schedule) can vary, for example, from once a week to once every 2, 3, 4, 5, or 6 weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose of about 300 mg to 400 mg, once every 3 weeks or once every 4 weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose of about 300 mg once every three weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose of about 400 mg once every four weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose of about 300 mg once every four weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose of about 400 mg once every three weeks. Without wishing to be bound by theory, in some embodiments, constant or fixed dosing may be beneficial to the patient, for example, to conserve drug supplies and reduce pharmacy errors.

[0465] The antibody molecule is approximately 35-440 mg / m 2 , typically about 70-310 mg / m 2 , more typically about 110-130 mg / m 2 The drug may be administered by intravenous infusion at a rate of greater than 20 mg / min, for example 20-40 mg / min, typically greater than or equal to 40 mg / min, to reach a dose of about 110-130 mg / m. 2 In another embodiment, the antibody molecule is administered at a dose of about 1-100 mg / m 2 , preferably about 5 to 50 mg / m 2 , about 7~25mg / m 2 , or about 10 mg / m2 The antibody may be administered by intravenous infusion at a rate slower than 10 mg / min, for example slower than or equal to 5 mg / min, so as to reach a dose of 10 mg / min. In some embodiments, the antibody is infused over a period of about 30 minutes. It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated. Furthermore, for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or administering or supervising the administration of the composition, and it should be understood that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0466] The pharmaceutical composition of the present disclosure may include a "therapeutically effective amount" or a "prophylactically effective amount" of the therapeutic agent of the present disclosure. A "therapeutically effective amount" refers to an amount effective at the dosage and for the period of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the modified antibody or antibody fragment may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the modified antibody or antibody fragment are outweighed by the therapeutically beneficial effects. In the case of cancer, a therapeutically effective amount of a therapeutic agent may reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow to some extent and preferably stop) the invasion of cancer cells into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more of the symptoms associated with the disorder. To the extent that the drug may prevent growth and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic. For cancer treatment, efficacy in vivo can be measured, for example, by assessing survival, time to disease progression (TTP), time to recurrence, response rate (e.g., CR and PR), duration of response, and / or quality of life.

[0467] A "therapeutically effective dosage" preferably inhibits a measurable parameter, such as tumor growth rate, by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% relative to untreated subjects. The ability of a compound to inhibit a measurable parameter, such as cancer, can be evaluated in an animal model system that is predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit such inhibition in vitro by assays known to those skilled in the art.

[0468] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. EXAMPLES

[0469] [Example 1] Biomarker analysis of the clinical trial of the combination of spartalizumab, dabrafenib, and trametinib (the "COMBI-i" study) This example identifies exemplary biomarkers that may better define patient populations most likely to derive therapeutic benefit from treatment with a combination of spartalizumab, dabrafenib, and trametinib.

[0470] COMBI-i is a three-part, global, randomized, placebo-controlled Phase 3 study including a safety run-in (Part 1), a biomarker cohort (Part 2), and a randomized, double-blind, placebo-controlled part comparing sparta-DabTram with placebo-DabTram. A total of 532 patients were randomized across the three parts.

[0471] COMBI-i compared spartalizumab 400 mg intravenously every 4 weeks with dabrafenib 150 mg orally twice daily and trametinib 2 mg orally once daily with placebo plus dabrafenib and trametinib. Participants were aged 18 years or older and had unresectable or metastatic BRAF V600-mutated melanoma. Efficacy on baseline PD-L1 status was a secondary endpoint; other biomarker analyses were prespecified exploratory endpoints. Baseline / on-treatment pharmacodynamic markers were assessed by flow cytometry-based immunophenotyping and plasma cytokine profiling. Baseline tumor PD-L1 status, tumor mutation burden, and gene expression were assessed by immunohistochemistry and RNA / DNA sequencing.

[0472] Subgroups defined by high tumor mutation burden, regardless of PD-L1 status, benefited from spartalizumab plus dabrafenib and trametinib, whereas subgroups with immunologically cold tumors (e.g., low tumor mutation burden / PD-L1 negative, immune desert) did not. BRAF V600K mutations, baseline T-cell inflammatory phenotype, and baseline circulating tumor DNA shedding were also predictors of benefit from spartalizumab plus dabrafenib and trametinib. Spartalizumab plus dabrafenib and trametinib increased cytokine levels associated with T-cell proliferation / activation and effector T-cell activity during treatment and prolonged progression-free survival among patients with baseline CD4+ / CD8+ ratios at or above the median. Features such as BRAF V600K mutations, T-cell inflammation, high tumor mutation burden, and increased baseline circulating tumor DNA shedding characterize subgroups that may derive greater benefit from spartalizumab versus dabrafenib and trametinib. Patients with PD-L1-negative / high TMB tumors may particularly benefit.

[0473] method participants Part 3 of COMBI-I enrolled adult patients (≥18 years) with histologically confirmed unresectable or metastatic (stage IIIC / IV according to the American Joint Committee on Cancer Staging System (AJC), version 7.0) BRAF V600-mutated cutaneous melanoma. Further criteria included absence of clinically active brain metastases, Eastern Cooperative Oncology Group performance status (ECOG PS) ≤2, and no prior systemic anticancer treatment for unresectable or metastatic melanoma (e.g., chemotherapy, immunotherapy, biologic therapy, tumor vaccine therapy, targeted therapy, or any systemic investigational treatment). Prior local and / or (neo)adjuvant therapy was permitted as long as it was not administered within 6 months of initiating study treatment.

[0474] Randomization and Masking Patients were randomized 1:1 to a treatment arm receiving spartalizumab in combination with dabrafenib and trametinib or a control arm receiving spartalizumab-matched placebo in combination with dabrafenib and trametinib. Dabrafenib and trametinib are approved as first-line treatment for patients with BRAF V600-mutated unresectable or metastatic melanoma and other solid tumors. Randomization followed a random permutation block scheme and was performed using an Interactive Response Technology provider with a validated system that automates the random assignment of randomization numbers to patient numbers. Patients were stratified by Eastern Cooperative Oncology Group performance status (0 vs. 1 vs. 2) and lactate dehydrogenase level (<1 × upper limit of normal [ULN] vs. ≥1 to <2 × ULN vs. ≥2 × ULN).

[0475] Patients were blinded to treatment from the time of randomization until database lock for the primary analysis. Individual patients could be unblinded to manage emergency situations for regulatory reporting purposes or to determine next treatment after disease progression, if necessary.

[0476] Outcome The primary endpoint was investigator-assessed progression-free survival using the Response Evaluation Criteria in Solid Tumors version 1-1, defined as the time from the date of randomization to the date of first documented progression or death from any cause. Overall survival was a key secondary endpoint, defined as the time from the date of randomization to the date of death from any cause. In some biomarker subgroups, next-line progression-free survival (PFS2) was evaluated as an exploratory endpoint, defined as the time from randomization to the first documented investigator-assessed disease progression on the first new systemic antitumor therapy initiated after discontinuation of study treatment, or to death from any cause.

[0477] Tumor assessments using RECIST 1·1 were performed at baseline, 12 weeks, then every 8 weeks for 18 months after initiation of treatment, and every 3 months thereafter until disease progression, death, loss to follow-up, or study discontinuation. Additional confirmatory tumor assessments were required ≥4 weeks after the first response criterion was met. Tumor response was assessed by blinded independent central review based on the original imaging scans.

[0478] Efficacy by baseline PD-L1 status was a secondary endpoint, and efficacy by tumor mutation burden (TMB) alone or in combination with PD-L1 status was a key exploratory endpoint. All other biomarker analyses were exploratory endpoints. Biomarker analyses were performed using tumor tissue and blood samples obtained from consenting patients. Collection of a baseline tumor tissue sample, either freshly obtained (preferred) or archived (at or after diagnosis, preferably within 3 months prior to study treatment), during screening was mandatory. Further tumor sampling during treatment (at 2–3 weeks, 8–12 weeks, or at disease progression) was at the investigator's discretion. Only core, excision, or incisional biopsies from tissues other than the central nervous system and bone were permitted. Collection of blood samples for circulating biomarker analysis (e.g., tumor DNA, cytokine profiling, and flow cytometry) was mandatory at baseline, 4 weeks, 8 weeks, and disease progression.

[0479] Immunophenotyping by flow cytometry analysis To determine the effect of treatment on the maturation, activation, and proliferation status of T cell subsets in the periphery of patients with melanoma, multiparametric flow cytometry analysis was performed on peripheral blood mononuclear cells (PBMCs) at baseline and on day 28 (week 4) of treatment. PBMCs were isolated by Ficoll density gradient centrifugation and frozen live in dimethyl sulfoxide / fetal bovine serum (DMSO [10%] / FBS) freezing buffer. For baseline and day 28 (week 4) paired samples, fluorochrome-conjugated monoclonal antibodies were used to detect CD45 BUV395 (clone HI30), CD3 FITC (clone SK7), CD4 BUV737 (SK3), CD8 BVy5.521 (clone RPA-TA), TCRvd2 PerCP-Cy5.5 (clone B6), CD45RA BV711 (clone HI100), CCR7 PE (clone 150503), HLA-DR BV786 (clone G46-6), CD38 APC-eF780 (clone HIT2), PD-1 PE-Cy7 (clone EH12·1), LAG-3 PE-eF610 (clone 3DS223H), and TIM-3 BV650 (7D3) (BD Biosciences; SanBiosciences). PBMCs were immunophenotyped for cell surface expression of CD38+ / HLA-DR+ / CD8+ (activated / cytotoxic CD8+ T cells), Ki67+ / CD8+ (proliferating CD8+ T cells), Ki67+ / PD-1+ / CD8+ and PD-1+ / Ki67+ / CD8+ (activated / proliferating CD8+ T cells) and intracellular expression of Ki67 AF647 (clone B56). Cell viability was measured using Viability Dye BV510 (Thermo Fisher Scientific; Waltham, MA). Pharmacodynamic biomarkers and T cell phenotypic and functional characteristics were defined by co-expression of CD38+ / HLA-DR+ / CD8+ (activated / cytotoxic CD8+ T cells), Ki67+ / PD-1+ / CD8+ and PD-1+ / Ki67+ / CD8+ (activated / proliferating CD8+ T cells). A minimum of 5000 leukocytes per sample were acquired using a BD LSRFortessa™ X-20 and analyzed by FlowJo (v10·2) software (BD Biosciences) at Navigate Biopharma.For visualization purposes, the logistic transformation log(p / [1-p]) was applied to the proportions positive for the plotted analytes and the proportions were converted to "logit". Because proportions that are 0·0 or 1·0 are indefinite, all proportions were scaled towards 0·5 using the transformation ([100 p+1 / 2] / [100+1]) before applying the logistic transformation.

[0480] Cytokine profiling Profiling of human cytokines (interferon (IFN)-γ, IL-6, IL-8, TNF-α, IL-12p40, IL-15, IL-16, IL-17a, TNF-β, eotaxin, IP-10, MDC, MIP1a, PLGF, VEGFC, VEGFR1, CRP, ICAM-1, SAA1, VCAM-1, and IL-18) was performed using a multiplex sandwich electrochemiluminescence immunoassay (BioAgilytix; Durham, NC) that was validated by Meso Scale Diagnostics (MSD; Rockville, MD) and at a clinical research site selected by the study sponsor. Briefly, plasma samples were diluted 2-fold with diluent 2 as recommended by the assay kit manufacturer (Meso Scale Diagnostics [MSD]; Rockville, MD), with 50 microliters of calibrator and diluted sample used for each replicate. Three levels of controls were included in each run. Standards, controls, and samples were tested in duplicate. The assay signal, proportional to the amount of analyte present in the sample, was read on the MSD instrument. A four parameter logistic curve fit was used to construct a standard curve from which cytokine levels in the test samples were determined. Control results were checked before accepting sample results. Those below the lower limit of detection were analyzed and plotted as limit / 2, and those above the upper limit of detection were analyzed and plotted as 1·1×limit. Limits were batch specific and the data set contained multiple lower and upper limits for a given analyte.

[0481] Immunohistochemistry (IHC) For IHC staining, formalin-fixed paraffin-embedded (FFPE) tissue blocks or unstained slides were used. Blocks were cut into 4-μm-thick FFPE slides in a nuclease-free oven and baked at 60° ± 2°C (30 min to 2 h depending on the antibody) at HistoGeneX (now CellCarta; Antwerp, Belgium). Tumor evaluation was performed by a certified pathologist.

[0482] PD-L1 expression <1% or ≥1% was assessed using the IHC 28-8 PharmDx assay (Dako; Carpinteria, CA) on an Autostainer Link 48 (Agilent Technologies; Santa Clarita, CA) as performed and extensively validated with HistoGeneX to follow FDA-approved guidelines. The percentage of viable tumor cells expressing PD-L1 was scored according to the Melanoma Interpretation Manual provided by Dako. Discernible membranous staining of any intensity was included, but cytoplasmic staining, immune cells, and necrotic cells were excluded. Negative and positive controls were scrutinized to determine the presence of any interfering variables.

[0483] Dual-plex IHC assay and digital image analysis (IA) with HALO To assess the levels of CD8+ immune cells within melanoma tumor nests and stromal compartments, a specific dualplex IHC assay (CD8 / Melanoma Triple Cocktail [MTC]) consisting of an anti-CD8 rabbit monoclonal primary antibody (SP57, Ventana: Roche Diagnostics; Basel, Switzerland) and MTC (HMB45, A103, and T311 antibodies) was performed on a Benchmark XT platform (Ventana) and quantification, including filtration analysis, was performed using HALO® software v2-3 (Indica Labs; Albuquerque, NM).

[0484] Stained slides were scanned at 20x magnification using an Aperio AT2, Leica digital whole slide scanner (Leica Biosystems; Wetzlar, Germany). Digital IA was then performed using the Multiplex IHC module of the HALO v2.3 software platform (Indica Labs; Albuquerque, NM). At Novartis Precision Medicine, a specific IA algorithm was developed in-house to evaluate CD8+ cells within melanoma lesions that were annotated by pathologists in close collaboration with imaging researchers, and the residential lymphoid tissue was excluded from the IA. Using this algorithm and a tissue classifier, the percentage of CD8+ cells within the melanoma tumor and stromal compartments was quantitatively evaluated. Finally, invasion analysis was performed within a region of interest defined using the tumor border as the invasion margin by the melanoma lesion classifier. Five bands (30 μm each) were analyzed inside and outside the tumor margin, respectively, for a total distance of 150 μm. CD8+ cell density (cells / mm 2 ) was reported for each invaded band.

[0485] Detailed methods and descriptions of predefined CD8+ T cell phenotypes are described herein. Assessment of antigen-presenting cells within defined melanoma tumor compartments was performed using multiplex fluorescent immunohistochemistry (FIHC) by Automated Quantitative Analysis (AQUA®) from Navigate Biopharma (Carlsbad, CA).

[0486] NanoString Testing and TIS Sections of 4 μm (± 1 μm) thickness were cut from all received tissue blocks. A pathologist visually inspected archived FFPE and freshly cut slides and determined the region of interest (ROI) and total tumor area (mm 2The approximate percentage of tumor content in the ROI was recorded. Depending on the tumor content, 4–8 slides were microdissected for RNA and DNA isolation. If the tumor content of the ROI was less than 10%, further processing was stopped. RNA and DNA were co-extracted from all available samples using the AllPrep RNA / DNA Extraction from FFPE Tissue Kit (Qiagen; Hilden, Germany). Samples with a resulting RNA concentration less than 5 ng / μL were not processed further. After RNA and DNA isolation, up to 200 ng of RNA was combined with the capture and reporter probes of the NanoString PanCancer IO 360™ panel (NanoString Technologies; Seattle, WA) at 65 °C overnight. After hybridization, the target-probe complex was purified, conjugated to streptavidin-coated cartridges, and quantified using the nCounter® Analysis System (NanoString).

[0487] T cell inflammatory signature scores (TIS) were calculated as previously described. For each sample and control, the 18 target gene counts comprising the signature were divided by the geometric mean of 10 internal housekeeping genes (HKGs) to generate HKG-normalized expression data. The HKG-normalized data for each sample were then scaled by the HKG-normalized expression of the control samples to obtain reference sample-corrected, HKG-normalized results. These were then log2 transformed and a target-specific factor was applied before summing the normalized expression values ​​and generating the TIS. The cutoff log2 value for HKG-normalized counts defining low and high TIS was 6·29, derived from the distribution of values ​​excluding the lowest 25%.

[0488] CD8 phenotype for infiltration analysis Using the infiltration analysis, three CD8 phenotypes (T cell exclusion / infiltration / immune desert) were determined based on five 30 μm inner bands (150-0 μm) and two 30 μm outer bands (0-60 μm) of CD8 density around the tumor-stroma infiltration margin. Samples were ranked by the mean tumor CD8 density, with the top third of tumors defined as inflammatory. Similarly, stroma and other samples with values ​​ranked in the bottom third were defined as desert. Samples below the tumor threshold for inflammatory and above the stroma cutoff for desert were defined as exclusion. A minority of cases had high CD8 density in the tumor area but low CD8 density in the stroma, usually due to small stromal areas, and were defined as inflammatory tumors.

[0489] CD11b / CD14 / CD80 / CD86 / HLA-DR / DAPI multiplex fluorescent IHC (FIHC) and IA by Automated Quantitative Analysis (AQUA®) Formalin-fixed paraffin-embedded (FFPE) tissue samples were dewaxed and rehydrated in distilled water through a series of xylene-alcohol washes. Heat-induced antigen retrieval was then performed using the NxGen Decloaking Chamber in Diva buffer (Biocare Medical; Pacheco, CA) and transferred to tromethamine-buffered saline. All subsequent staining steps were performed at room temperature. Endogenous peroxidase was blocked using Peroxidazed 1 (Biocare) and incubated in protein blocking solution (Background Sniper; Biocare) to reduce nonspecific antibody staining. Slides were stained according to the procedure described below:

[0490] [Table 3]

[0491] Fluorescent images were acquired on a Vectra 2 Intelligent Slide Analysis System (Akoya Biosciences; Menlo Park, CA) at 4x magnification to initially identify tissue areas based on DAPI signal. These 4x magnified images were processed using the automated INFORM software enrichment algorithm (INFORM GmbH; Aachen, Germany) to identify and rank 20x high-power fields (HPFs) according to the high co-expression of CD11b and HLA-DR. All raw images were reviewed for acceptability by a pathologist. Images lacking tumor or highly necrotic cells were rejected prior to analysis by Automated Quantitative Analysis (AQUA® [v3·2·4]; HistoRx; New Haven, CT) in a fully automated process. AQUA® has been widely validated in clinical settings to objectively quantify biomarkers in tissues. The AQUA assay applied in these analyses was validated at the vendor (Navigate Biopharma Services), a subsidiary of Novartis, using Novartis in-house guidance and standard operating procedures for discovery assay development. The DAPI signal in each received image was used to identify cell nuclei, which were then expanded to the approximate size of whole cells. An overlap approach was used to create a binary mask of CD11b and HLA-DR to identify the CD11b+ / HLA-DR- population, representing myeloid-derived suppressor cells (MDSCs).

[0492] Targeted and CfDNA sequencing Samples were submitted to Foundation Medicine, Inc. (Cambridge, MA) for next-generation sequencing (NGS) of tumor and cell-free DNA using FoundationOne CDx™ assays.

[0493] After sample qualification of FFPE tissues, FMI performed DNA extraction and QC check of extracted DNA. If DNA samples were sufficient, library construction was performed to check the quality of the resulting libraries. After successful library construction, FMI performed hybrid capture using the Dx1 bait set, which included a total of 324 genes that are commonly rearranged and altered in cancer. These included coding sequences of 309 cancer-related genes, one promoter region, one non-coding RNA, and intronic regions selected from 34 commonly rearranged genes (21 of which also contained coding exons). If the hybrid capture passed the quality check, FMI performed comprehensive genomic profiling using the company's Solid Tumor (Dx1) assay, interpreted the resulting NGS data, and identified genetic alterations using proprietary software. The sequencing data was then mapped to the human genome (build hg19). Variant calling, including base substitutions, insertions / deletions (indels), copy number alterations, and genomic rearrangements, was performed on the targeted genomic regions. Libraries were sequenced using Illumina v4 chemistry and paired-end 100-bp reads (HiSeq; Illumina; San Diego, CA).

[0494] Tumor mutation burden (TMB) was determined by counting all synonymous and nonsynonymous variants present at an allele frequency of 5% or higher and filtering potential germline variants. Known and potential driver mutations were filtered to eliminate bias in the dataset. The resulting mutation counts were then divided by the coding region equivalent of the total variant counts, i.e., 793 kilobases (kb), and reported as mutations per megabase (Mb) (mut / Mb).

[0495] Processing and analysis of cfDNA sequencing data For cfDNA-seq data, unique molecular identifiers (UMIs) were trimmed from reads using UMI-Toolkit v.1, and reads were then aligned to the human reference genome (build hg38) using BWA-MEM. Alignments were then locally realigned and base quality scores recalibrated (Genome Analysis Toolkit [GATK]). To remove PCR duplicate reads and sequencing artifacts, consensus reads were created using UMIs and alignment positions (UMI-Toolkit). Single nucleotide variants (SNVs) were identified using MuTect v-1-1-7. Indels were identified using Pindel v-1-0. Structural variants were identified using PureCN v-1-8-1. Chromosomal rearrangements were called using Socrates v-1.

[0496] cfDNA libraries were included in downstream analyses if they had coverage ≥ 500× and GC / AT dropout < 20%. Potential sequencing artifacts and germline genetic variants were removed from downstream analyses. Position-specific error rates were calculated based on sequencing of plasma from 24 healthy controls, and mutations were retained only if they had support significantly greater than the position-specific error rate. Further potential artifacts were removed based on low allele fraction (< 0·005 unless known or likely to be oncogenic), poor alignment support (> 50 MQ0 reads), poor base quality (< 20), low coverage (< 100×), or being in a repetitive region. Likely germline SNVs and indels were identified by their presence at significant frequency (ESP minor allele frequency >0·001 or ExAC count >3, unless known hotspot mutations) in the dbSNP 147, Exome Sequencing Project (ESP6500SI-V2-SSA137-GRCh38-liftover) and Exome Aggregation Consortium (releases 0-3; now part of gnomAD) databases. SNVs and indels were given functional significance based on their presence and functional effect in the Catalog of Somatic Mutations in Cancer (COSMIC v-83), where mutations reported in ≥5 tumors in COSMIC were considered “known” oncogenic, mutations with COSMIC counts <5 but predicted to lead to premature protein truncation were considered “likely” oncogenic, and all others were considered of “unknown” oncogenic status. Copy number alterations were considered as amplifications if the estimated copy number was ≥7 and homozygous deletions if the estimated copy number was ≤0·5. PureCN uses a combination of the allele frequencies of single nucleotide polymorphisms in copy number alterations and somatic point mutations to determine the proportion of cfDNA derived from the tumor.

[0497] RNA sequencing Ribosomal RNA (rRNA) was depleted from extracted total RNA using RNAseH (Sigma-Aldrich; St. Louis, MO). The rRNA-depleted samples were then fragmented, converted to cDNA, and the remaining steps of next-generation sequencing library construction were carried out using the TruSeq RNA Library Prep Kit v2 (Illumina; San Diego, CA): end repair, A-tailing, index adapter ligation, and PCR amplification. The captured libraries were pooled with other libraries, each with a unique adapter index sequence, and applied to a sequencing flow cell for cluster amplification by synthesis and massively parallel sequencing using Illumina v4 chemistry and paired-end 100 bp reads (Illumina).

[0498] Sequence data were aligned to the reference human genome (build hg19) using STAR. Mapped reads were then used to quantitate transcripts using HTSeq and Refseq GRCh38 v82 gene annotation. Data were normalized using trimmed mean M-value normalization implemented in the edgeR R / Bioconductor package. Pathway / gene set expression was derived using the geometric mean expression of all genes in each set. The analysis included 1329 gene sets from MSigDB C2 Canonical Pathways, as well as in-house and published gene sets. Pathways were ranked in an unbiased analysis using a two-tailed Wilcoxon rank sum test.

[0499] statistical analysis For each assay, baseline clinical covariates were compared between cohorts with and without biomarker data; available Pearson χ analyses were used to assess potential case selection bias. 2, proportional odds likelihood ratios, and unadjusted p-values ​​for multiplicity based on Wilcoxon rank-sum tests were applied to assess the null hypothesis of "no difference" for unordered categorical, ordinal categorical, and continuous covariates, respectively. The contribution of biomarkers and covariates to progression-free and overall survival was estimated using univariate or multivariate Cox proportional hazards models, as appropriate. Between-group comparisons were addressed by Wald tests or Wilcoxon rank-sum tests with descriptive p-values ​​without adjustment for multiple comparisons.

[0500] All biomarker analyses were performed using R 3·6·1 and Bioconductor 3·9. Kaplan-Meier curves and Cox proportional hazards models for the biomarker cohort were generated using the R survival(3·1-7) and survminer(0·4·6) packages. Population comparisons were assessed using the R Hmisc(4·3·0) package.

[0501] result Baseline characteristics In part 3 of COMBI-i, a total of 532 patients were randomized to receive sparta-DabTram (n = 267) or placebo-DabTram (n = 265) ( Fig. 1 ), and baseline characteristics were well balanced between treatment arms ( Table A ).

[0502] [Table 4]

[0503] Table B summarizes the availability for each of the planned analyses.

[0504] [Table 5]

[0505] Most patients were included, with available specimens for approximately 64%–90% (339–481 of 532) of the intention-to-treat population. Key clinical and demographic variables were comparable between most biomarker cohorts and each subset where biomarker results were unavailable (Table C). Cohorts lacking flow cytometry data at baseline were enriched for features of high tumor burden (e.g., disease stage or sum of lesion diameters).

[0506] [Table 6-1]

[0507] [Table 6-2]

[0508] [Table 6-3]

[0509] Pharmacodynamic markers Immunophenotyping and cytokine profiling of peripheral blood mononuclear cells (PBMCs) using preselected pharmacodynamic markers was performed at baseline and after 4 weeks of treatment to assess T cell activation, proliferation, and cytotoxicity (Figure 2). An increase in proliferating CD8+ / PD-1+ T cells was observed from baseline to week 4 in patient samples from the sparta-DabTram arm (median change, 2·49) compared with the placebo-DabTram arm (median change, 1·09). CD8+ T cells with an effector / cytotoxic phenotype (CD38+ / HLA-DR+) also increased from baseline to week 4 in sparta-DabTram (median change, 3·79) versus placebo-DabTram (median change, 1·57). Total T cell counts (CD3+) were within normal limits, with increased CD8+ and CD4+ proliferation and activation in the sparta-DabTram arm (Figure 3). In both arms, treatment enhanced modulation of cytotoxic effector T cells, reflected by elevated plasma IFN-γ levels (Figure 2C). Statistical analysis and modeling approaches suggest that more robust T cell modulation was observed in samples from the sparta-DabTram arm (data not shown). Additional cytokines indicative of effector T cell immune activity showed similar results (Figure 4).

[0510] Analysis of CD4+ / CD8+ T cell ratio In addition to pharmacodynamic biomarkers, systemic T cell-mediated immune activity was assessed at baseline by determining the peripheral blood helper T cell / cytotoxic T cell ratio (CD4+ / CD8+) by flow cytometric immunophenotyping. The baseline CD4+ / CD8+ ratio was associated with progression-free survival in the placebo-DabTram arm (Figure 5); patients with higher CD4+ / CD8+ ratios typically experienced shorter progression-free survival. However, no such association was observed in the sparta-DabTram arm.

[0511] Analysis of CD4+ / CD8+ ratios across treatment arms suggested that the addition of spartalizumab to dabrafenib and trametinib may improve progression-free survival in patients with a baseline CD4+ / CD8+ ratio ≥ 2·9. In these patients, treatment with sparta-DabTram improved progression-free survival compared with placebo-DabTram (HR, 0·58 [95% CI, 0·40–0·84]; Fig. 6). Further cut-points for selection of patients with baseline CD4+ / CD8+ ratios above the median were systematically evaluated by testing all possible values ​​between the 10th and 90th percentiles of the ratio values ​​(Fig. 6C). This data-driven approach suggested a favorable range of cut-off values ​​between 2·01 and 3·34, which would select between 73·8% and 40·8% of patients, respectively.

[0512] Analysis of PD-L1 and TMB subgroups At data cutoff, a total of 147 patients (55%) in the sparta-DabTram arm had a progression-free survival event compared with 165 patients (62%) in the placebo-DabTram arm; median progression-free survival was 16·2 months (95% CI, 12·7 to 23·9 months) versus 12·0 months (95% CI, 10·2 to 15·4 months), respectively.

[0513] The primary endpoint was not met (HR, 0 82 [95% CI, 0 66 to 1 03]; one-sided; p = 0 042). Preplanned subgroup analyses demonstrated that the benefit of sparta-DabTram was similar regardless of PD-L1 status, with a numerically lower HR in the PD-L1-positive (≥ 1%) subgroup (positive HR, 0 76 [95% CI, 0 54 to 1 07] vs. negative HR, 0 84 [95% CI, 0 60 to 1 18]). Meanwhile, patients with high TMB (≥ 10 mut / Mb) appeared to derive greater benefit than those with low TMB (HR, 0 70 [95% CI, 0 47 to 1 06] vs. HR, 0 91 [95% CI, 0 65 to 1 26], respectively).

[0514] As a prespecified key exploratory endpoint, we further evaluated outcomes in subgroups based on the combination of PD-L1 status and TMB (Figure 7). Patients with “cold” tumors lacking immunogenic potential, characterized by negative PD-L1 expression and low TMB, did not obtain a progression-free survival benefit with sparta-DabTram (HR, 1·11 [95% CI, 0·71 to 1·75]; Figure 7A). In patients with tumors characterized by high TMB, sparta-DabTram was associated with a numerically longer progression-free survival, independent of PD-L1 status (PD-L1 negative / high TMB: HR, 0·71 [95% CI, 0·38 to 1·32]; PD-L1 positive / high TMB: HR, 0·73 [95% CI, 0·44 to 1·23]; Figure 7A). Treatment with sparta-DabTram was also associated with improved overall survival in the PD-L1-negative / TMB-high subgroup (HR, 0 33 [95% CI, 0 13 to 0 79]; Figure 7B), but not in other PD-L1 / TMB-defined subgroups, which also had a progression-free survival benefit after next-line treatment in the sparta-DabTram arm (Figure 8)....

Claims

1. 1. A method for identifying a subject having cancer likely to benefit from a treatment, comprising: obtaining a value for the level or activity of CD4+ immune effector cells relative to the level or activity of CD8+ immune effector cells in a sample from the subject; and (a) a value greater than or equal to the reference value identifies the subject as likely to benefit from the treatment, wherein the treatment comprises a targeted therapy in combination with a cancer immunotherapy; or (b) a value less than the reference value identifies the subject as likely to benefit from the treatment, wherein the treatment comprises a targeted therapy that is not a cancer immunotherapy. method.

2. (a) the subject identified as having a value greater than or equal to a reference value is more likely to have an increased benefit from the treatment that includes the targeted therapy in combination with a cancer immunotherapy compared to the treatment that includes the targeted therapy without a cancer immunotherapy; or (b) the subject identified as having a value less than the reference value is unlikely to have a substantial increase in benefit from a treatment comprising the targeted therapy in combination with a cancer immunotherapy; The method of claim 1.

3. 1. A method of selecting a treatment for a subject having cancer, comprising: obtaining a value for the level or activity of CD4+ immune effector cells relative to the level or activity of CD8+ immune effector cells in a sample from the subject; and (a) if the value is greater than or equal to a reference value, selecting a treatment for the subject comprising a targeted therapy in combination with a cancer immunotherapy; or (b) if the value is less than a reference value, selecting a treatment for the subject comprising a targeted treatment that does not use cancer immunotherapy; The method includes:

4. The object is (a) administering an initial or subsequent administration of an effective amount of said treatment; (b) administering an altered dosing regimen of said treatment; (c) discontinuing the administration of a different treatment; (d) administration of an additional treatment; or (e) administering a pretreatment, wherein the pretreatment increases the value of the level or activity of CD4+ immune effector cells relative to the level or activity of CD8+ immune effector cells in a sample from the subject; The method according to any one of claims 1 to 3, wherein the method is selected for

5. 1. A pharmaceutical composition comprising a targeted therapeutic agent for use in a therapy, including a method of treating a subject with cancer, the method comprising: receiving a value for the level or activity of CD4+ immune effector cells relative to the level or activity of CD8+ immune effector cells in a sample from the subject; (a) administering an effective amount of said pharmaceutical composition in combination with a cancer immunotherapy to said subject having a value greater than or equal to a reference value; or (b) administering the pharmaceutical composition without cancer immunotherapy to a subject having a value smaller than the reference value. The method includes:

6. 6. The method of any one of claims 1 to 4, or the pharmaceutical composition of claim 5, wherein the value for the level or activity of CD4+ immune effector cells relative to the level or activity of CD8+ immune effector cells comprises a ratio of the amount of CD4+ immune effector cells to the amount of CD8+ immune effector cells.

7. The value is (a) Greater than or equal to: (i) 2, (ii) 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10; (iii) 3.3, or (iv) 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10, or (b) is less than: (i) 3.3, (ii) 3, 2.5, 2, 1.5, 1, or 0.5 (iii) 2, or (iv) 1.5, 1, or 0.5 7. The method or pharmaceutical composition of claim 6.

8. 8. The method of any one of claims 1 to 4, 6, or 7, wherein the step of obtaining the value comprises determining the level or activity of CD4+ immune effector cells relative to the level or activity of CD8+ immune effector cells in a sample from the subject. (a) the sample from the subject comprises a blood sample or a tumor sample. (b) the value is obtained before administration of the treatment begins; and / or (c) the value is obtained after administration of the treatment has begun; A method according to any one of claims 1 to 4, or 6 to 8, or a pharmaceutical composition according to any one of claims 5 to 7.

10. 10. The method or pharmaceutical composition of claim 9, wherein the value is obtained 1, 2, 3, 4, 8, 10, 12, 20, 30, 40 weeks or more or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 months or more after administration of the treatment has begun.

11. A method according to any one of claims 1 to 4 or 6 to 10, or a pharmaceutical composition according to any one of claims 5 to 7, 9, or 10, wherein the method further comprises the step of obtaining a level or activity value of CD8+ tumor infiltrating lymphocytes (TIL) in a sample from the subject, and an increase in the level or activity value of CD8+ TIL compared to a reference value further identifies that the subject is more likely to benefit from a treatment comprising the targeted therapy in combination with cancer immunotherapy.

12. The method according to claim 1, further comprising obtaining a value of tumor mutation burden (TMB) in a sample from the subject, (a) an increase in the value of TMB compared to a reference value further identifies the subject as being more likely to benefit from said treatment, including a targeted therapy in combination with a cancer immunotherapy; (b) a decrease in the value of TMB compared to the reference value further identifies the subject as likely to benefit from said treatment without cancer immunotherapy; or (c) a decrease in the value of TMB compared to the reference value further identifies the subject as unlikely to have a substantial increase in benefit from a treatment comprising a targeted therapy in combination with a cancer immunotherapy; A method according to any one of claims 1 to 4 or 6 to 11, or a pharmaceutical composition according to any one of claims 5 to 7, or claims 9 to 11.

13. The value of TMB is (a) greater than or equal to 10 mut / Mb; or (b) less than 10 mut / Mb, e.g., less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 mut / Mb, or less; 13. The method or pharmaceutical composition of claim 12.

14. The method according to claim 1, further comprising obtaining a value for a level and / or activity of PD-L1 in a sample from the subject, (a) a decrease in said level and / or activity value of PD-L1 compared to a reference value further identifies said subject as likely to benefit from said treatment, including a targeted therapy in combination with a cancer immunotherapy; (b) further identifying that a decrease in PD-L1 level and / or activity, e.g., in conjunction with a decrease in TMB, indicates that the subject is more likely to benefit from the treatment without cancer immunotherapy; (c) further identifying that a decrease in the value of PD-L1 level and / or activity, together with a decrease in the value of TMB, makes the subject unlikely to have a substantial increase in benefit from a treatment comprising a targeted therapy in combination with a cancer immunotherapy; A method according to any one of claims 1 to 4 or 6 to 13, or a pharmaceutical composition according to any one of claims 5 to 7, or claims 9 to 13.

15. A method according to any one of claims 1 to 4 or 6 to 14, or a pharmaceutical composition according to any one of claims 5 to 7, or claims 9 to 14, wherein the method further comprises the step of obtaining a value of circulating tumor DNA (ctDNA) in a sample from the subject, wherein an increase in the value of ctDNA compared to a reference value further identifies that the subject is more likely to benefit from the treatment.

16. A subject who is likely to benefit from said treatment, or who is likely to have an increased benefit from said treatment, (a) has improved progression-free survival (PFS), duration of objective response (DOR), and / or overall survival (OS) compared to subjects unlikely to benefit from said treatment or subjects unlikely to have an increased benefit from said treatment, or (b) having improved PFS, DOR, and / or OS compared to a subject who did not receive said treatment, or a subject who received only the targeted therapy but not cancer immunotherapy. (a) the PFS, DOR, and / or OS is improved for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60 months or longer; or 17. The method of claim 16, wherein (b) the PFS, DOR, and / or OS is improved by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 fold or more.

18. (a) the subject has been or is being treated with a targeted therapy. (b) the subject has not been treated or has not been treated with a targeted therapy; (c) the subject has undergone or is undergoing cancer immunotherapy; (d) the subject has not received or has not received cancer immunotherapy; (e) the subject has received a treatment comprising a targeted therapy in combination with cancer immunotherapy; (f) the subject has not received or has not received a treatment involving a targeted therapy in combination with cancer immunotherapy; (g) the subject has received or has received a targeted therapy and the cancer has recurred; (h) the subject is a non-responder to a targeted therapy or has been identified as a non-responder to a targeted therapy; or (i) the subject is a partial responder to a targeted therapy or has been identified as a partial responder to a targeted therapy; A method according to any one of claims 1 to 4 or 6 to 17, or a pharmaceutical composition according to any one of claims 5 to 7, or claims 9 to 15.

19. the targeted therapy comprises an agent that targets BRAF and / or an agent that targets MEK; (a) the agent that targets BRAF inhibits wild-type BRAF and / or BRAF with a V600 mutation; (b) the agent targeting BRAF is dabrafenib, vemurafenib, encorafenib, ABM-1310, ARQ 736, ASN003, BGB-283, BGB-3245, CEP-32496, GDC-0879, LUT014, PLX4720, PLX8394, RO5212054, or a pharma- ceutically acceptable salt thereof; (c) the agent targeting BRAF is administered at a dose between 25 mg and 300 mg; (d) the agent targeting MEK is trametinib, cobimetinib, binimetinib, mirdametinib, pumasertib, refametinib, selumetinib, AS703988, AZD 8330, BI 847325, BIX 02188, BIX 02189, CI-1040, CS3006, E6201, FCN-159, G-38963, GDC-0623, HL-085, PD 98059, RO4987655, RO5126766, SHR 7390, TAK-733, U0126, WX-554, or a pharma- ceutically acceptable salt thereof; (e) the agent targeting MEK is administered at a dose between 0.1 mg and 5 mg; (f) the agent that targets MEK is trametinib, and / or (g) The agent that targets MEK is cobimetinib.

19. The method of any one of claims 1 to 4 or 6 to 18, or the pharmaceutical composition of any one of claims 5 to 7, 9 to 15, or 18.

20. 20. The method of any one of claims 1 to 4 or 6 to 19, or the pharmaceutical composition of any one of claims 5 to 7, 9 to 15, 18 or 19, wherein the cancer immunotherapy comprises a PD-1 or PD-L1 binding antagonist.

21. (a) The cancer immunotherapy comprises an anti-PD-1 antibody molecule, the anti-PD-1 antibody molecule comprising: (i) a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO:501, a VHCDR2 amino acid sequence of SEQ ID NO:502, and a VHCDR3 amino acid sequence of SEQ ID NO:503; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO:510, a VLCDR2 amino acid sequence of SEQ ID NO:511, and a VLCDR3 amino acid sequence of SEQ ID NO:512; (ii) a VH comprising an amino acid sequence of SEQ ID NO: 506, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO: 506, or a VL comprising an amino acid sequence of SEQ ID NO: 520, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO: 520; (iii) a VH comprising an amino acid sequence of SEQ ID NO:506, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:506, and a VL comprising an amino acid sequence of SEQ ID NO:516, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:516; (iv) a heavy chain comprising an amino acid sequence of SEQ ID NO:508, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:508, and a light chain comprising an amino acid sequence of SEQ ID NO:522, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:522; or (v) a heavy chain comprising an amino acid sequence of SEQ ID NO:508, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:508, and a light chain comprising an amino acid sequence of SEQ ID NO:518, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more identical to SEQ ID NO:

518. Including, (b) the cancer immunotherapy comprises an anti-PD-1 antibody molecule, and the anti-PD-1 antibody molecule is administered to the subject at a dose of about 300 mg to 400 mg once every three weeks or once every four weeks; and / or (c) the cancer immunotherapy comprises a second cancer immunotherapeutic agent; A method according to any one of claims 1 to 4 or 6 to 20, or a pharmaceutical composition according to any one of claims 5 to 7, 9 to 15, or 18 to 20.

22. The cancer is (a) the tumor is a solid tumor, a blood cancer, or a metastasis thereof; (b) the cancer is melanoma or a metastasis thereof; (c) the cancer is melanoma, and the melanoma is stage I melanoma, stage II melanoma, stage III melanoma, or stage IV melanoma. (d) the cancer is a cancer other than melanoma. (e) the cancer is lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, or colorectal cancer, or a metastasis thereof; (f) the cancer is refractory to agents targeting BRAF and / or MEK. (g) the cancer comprises a BRAF mutation.

22. The method of any one of claims 1 to 4 or 6 to 21, or the pharmaceutical composition of any one of claims 5 to 7, 9 to 15, or 18 to 21.

23. 23. The method of any one of claims 1 to 4, or 6 to 22, or the pharmaceutical composition of any one of claims 5 to 7, 9 to 15, or 18 to 22, wherein the subject is further selected for administration of a further treatment.

24. (a) The treatment is a first-line, second-line, third-line, or fourth-line or beyond procedure. (b) the treatment is an adjuvant treatment, or (c) the treatment is a neoadjuvant treatment; 24. The method or pharmaceutical composition of claim 23.

25. The method of any one of claims 1 to 4 or 6 to 24, or the pharmaceutical composition of any one of claims 5 to 7, 9 to 15, or 18 to 24, wherein the method further comprises the step of obtaining a value for a level and / or activity of immune activation comprising TIL, PD-L1, CD8, IFNγ, or a T cell inflammatory gene expression signature.

26. 1. A method of stratifying a subject having cancer into a first group and a second group, comprising: obtaining a value for the level or activity of CD4+ immune effector cells relative to the level or activity of CD8+ immune effector cells in a sample from the subject; assigning subjects having a value less than the reference value to said first group likely to benefit from a treatment comprising a targeted therapy without cancer immunotherapy; assigning subjects having a value greater than or equal to the reference value to said second group likely to benefit from a treatment comprising a targeted therapy in combination with a cancer immunotherapy; The method includes:

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