Cancer therapy using il-2 prodrug

Abstract

This disclosure relates to methods and compositions for treating cancer using an inducible IL-2 prodrug, wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells prior to administration of the IL-2 prodrug.

Description

[0001] CANCER THERAPY USING IL-2 PRODRUG

[0002] [1] The present application claims the benefit of U.S. Provisional Application No. 63 / 654,605, filed on May 31, 2024, the entire contents of which is incorporated herein by reference.

[0003] 1. BACKGROUND

[0004] [2] Cancer immunotherapy has rapidly established itself as the fourth pillar of cancer treatment largely owing to the clinical success of checkpoint inhibitors. Despite the durable responses achieved by some patients using these new therapies, the proportion of responders is still relatively low and restricted to only some cancer types. Tumor mutational burden and the overall immunosuppressive microenvironment of tumors greatly influences the response to immunotherapies. Although immune checkpoint blockade can prevent the physiological stop-signal that arises in response to immune activation, other approaches can be used to positively stimulate the anti-tumor immune response. One approach involves the use of immune-activating cytokines. Numerous preclinical and clinical studies have demonstrated the promise of cytokine therapy to increase anti-tumor immunity. In fact, these were some of the first cancer immunotherapies approved for clinical use. However, systemic toxicity and poor pharmacokinetic profiles have limited their clinical application.

[0005] [3] Interleukin-2 (IU-2) has potent immunostimulatory activity and can be effective in eradicating tumors in mouse models and a recombinantIU-2 therapy (aldesleukin) was approved by the US FDA for treatment of metastatic renal cell carcinoma and metastatic melanoma. Aldesleukin has demonstrated complete cancer regression in about 10% of patients treated for metastatic melanoma and renal cancer. Unfortunately, rIU-2 has poor pharmacokinetic (PK) properties and dose-limiting systemic toxicities due to binding to the high and medium affinity IU-2 receptors (IL-2Ra / p / y and IL-2Rp / y, respectively) in the periphery. High-dose IU-2 administration results in severe hypotension and vascular leak syndrome (VUS), which has relegated its use to specialized care centers and limited its dosing to reach efficacious levels. See, Pachella LA, et al. J Adv Pract Oncol. 2015 ;6(3) :212-221 . These side effects limit the number of patients who can tolerate the recommended therapeutic regimen and, consequently, achieve the full clinical benefit from IL-2 therapy.

[0006] [4] Inducible forms of IL-2, that are conditionally activated in the tumor microenvironment through protease cleavage to release the fully active, native IL-2 cytokine within the tumor to stimulate a potent anti-tumor immune response, are described in International Publication Nos. WO2021097376 and WO2019222295. These IL-2 prodrugs include a native IL-2 molecule attached through a protease cleavable linker to a half-life extension domain (e.g., anti -human serum albumin antibody binding fragment such as a VH domain) and an IL-2 blocking element (e.g., anti-IL-2 antibody binding fragment, such as a Fab) to block binding of IL-2 to IL-2p / y receptors on nonnal tissue in the periphery. Upon cleavage of the protease cleavable linker, fully active native IL-2 is released within the tumor to stimulate a potent anti -tumor immune response.

[0007] [5] Immune checkpoint inhibitors are proteins that regulate T cell functions. T cell effector function is important for immunotherapeutic approaches to treating tumors. But immunosuppression, and decreased effector function, is often seen as tumors grow and cancer progresses. One mechanism behind this phenomenon is the activation of immune checkpoint inhibitors by cancer cells, leading to suppression of the anti-tumor immune response. This typically occurs when cancer cells express proteins on their surface that can interact with immune checkpoint proteins on the surface of T cells in the tumor microenvironment to suppress the activity of the T cells. Immune checkpoint proteins include, for example, PD-1 which binds ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (CD152) which binds B7-1 (CD80) and B7-2 (CD86). LAG 3 (CD223) which binds Galectin3, LSECtin and FGL1; TIM3 (HAVCR2) which binds ligands Ceacaml and Galectin9; TIGIT (VSTM3, WUCAM) which binds CD112 and CD155; BTLA (CD272) which binds HVEM (TNFRSF14), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55) which bind HVEM; CD134 (TNRFSR4, 0X40) which binds CD252 (OX-40L).

[0008] [6] PD-1 is recognized as an important player in immune regulation and the maintenance of peripheral tolerance. PD-1 is moderately expressed on naive T, B and NKT cells and up-regulated by T / B cell receptor signaling on lymphocytes, monocytes and myeloid cells (Sharpe et al.. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nature Immunology (2007); 8:239-245).

[0009] [7] Two known ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed in human cancers arising in various tissues. In large sample sets of e.g. ovarian, renal, colorectal, pancreatic, liver cancers and melanoma, it was shown that PD-L1 expression correlated with poor prognosis and reduced overall survival irrespective of subsequent treatment (Dong. Haidong et al., Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002 Aug;8(8):793-800; Yang, Wanhua et al., PD-1 interaction contributes to the functional suppression of T-cell responses to human uveal melanoma cells in vitro. Invest Ophthalmol Vis Sci. 2008 Jun; 49(6 (2008): 49: 2518-2525; Ghebeh, Hazem et al., Tire B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors. Neoplasia (2006) 8: 190-198; Hamanishi. Junzo et al., Programmed cell death 1 ligand 1 and tumorinfiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer. Proc. Natl. Acad. Sci. USA (2007): 104: 3360-3365; Thompson, R Houston, and Eugene D Kwon, Significance ofB7-Hl overexpression in kidney cancer. Clinical genitourin Cancer (2006): 5: 206-211; Nomi, Takeo et al., Clinical significance and therapeutic potential of the programmed death- 1 ligand / programmed death- 1 pathway in human pancreatic cancer. Clinical Cancer Research (2007); 13:2151-2157; Ohigashi, Yuichiro et al., Clinical significance of programmed death- 1 ligand- 1 and programmed death- 1 ligand 2 expression in human esophageal cancer. Clin. Cancer Research (2005): 11 : 2947-2953; Inman, Brant A et al., PD-L1 (B7-H1) expression by urothelial carcinoma of the bladder and BCG-induced granulomata: associations with localized stage progression. Cancer (2007): 109: 1499-1505; Shimauchi, Takatoshi et al., Augmented expression of programmed death- 1 in both neoplasmatic and nonneoplastic CD4+ T-cells in adult T-cell Leukemia / Lymphoma. Int. J. Cancer (2007): 121:2585-2590; Gao, Qiang et al.. Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma. Clinical Cancer Research (2009) 15: 971-979; Nakanishi, Juro et al., Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers. Cancer Immunol Immunother . (2007) 56: 1173-1182; Hino et al., Tumor cell expression of programmed cell death- 1 is a prognostic factor for malignant melanoma. Cancer (2010): 00: 1-9).

[0010] [8] Similarly, PD-1 expression on tumor infiltrating lymphocytes was found to mark dysfunctional T cells in breast cancer and melanoma (Ghebeh, Hazem et al., Foxp3+ tregs and B7-H1+ / PD-1+ T lymphocytes co-infiltrate tire tumor tissues of high-risk breast cancer patients: implication for immunotherapy. BMC Cancer. 2008 Feb 23;8:57; Ahmadzadeh, Mojgan et al., Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood (2009) 114: 1537-1544) and to correlate with poor prognosis in renal cancer (Thompson, R Houston et al., PD-1 is expressed by tumor infiltrating cells and is associated with poor outcome for patients with renal carcinoma. Clinical Cancer Research (2007) 15: 1757-1761). Tirus, it has been proposed that PD- L1 -expressing tumor cells interact with PD-1 -expressing T cells to attenuate T cell activation and evasion of immune surveillance, thereby contributing to an impaired immune response against the tumor.

[0011] [9] Immune checkpoint therapies targeting the PD-1 axis have resulted in groundbreaking improvements in clinical response in multiple human cancers (Brahmer et al., N Engl J Med 2012, 366: 2455-65; Garon et al. N Engl J Med 2015, 372: 2018-28; Hamid et al., N Engl JMed 2013, 369: 134-44; Robert et al., Lancet 2014, 384: 1109-17; Robert et al., NEngl JMed 2015, 372: 2521-32; Robert et al., N Engl JMed 2015, 372: 320-30; Topalian et al., N Engl JMed 2012, 366: 2443-54; Topalian et al., J Clin Oncol 2014, 32: 1020-30; Wolchok et al., N Engl JMed 2013, 369: 122-33).

[0012]

[0010] Therapeutic agents, such as antibodies, that bind immune checkpoint proteins and inhibit their immunosuppressive activity have been developed as anti-tumor agents. Several such agents are now commercially available for cancer therapy, including the anti-PD 1 antibodies pembrolizumab (KEYTRUDA™), Merck and Co., Inc., Railway, NJ, USA, dostarlimab (JEMPERLI), ccmiplimab-rwlc (LIBATYO), nivolumab (OPDIVO™), Bristol-Myers Squibb Company, Princeton, NJ, USA), camrelizumab, tislelizumab, toripalimab, and sintilimab (TYVYT); the anti-PD-Ll antibodies avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ); the anti-CTLA-4 antibody ipilimumab (YERVOY). While therapy with such immune checkpoint inhibitors provide advantages for cancer therapy, the overall success remains low, relapse occurs and resistance to checkpoint inhibition develops. There is an unmet medical need for improved methods for treating cancer.

[0013] 2. SUMMARY

[0014]

[0011] This disclosure relates to methods for treating cancer, comprising administering to a subject in need thereof an effective amount of an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4), wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells) prior to administration of the IL-2 prodrug. The IL-2 prodrug as disclosed herein can comprise Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the forgoing. In some instances, the method can include administering an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4) and an additional therapeutic agent as further described herein. In some instances, the method can include administering an IL-2 prodrug (e.g., Compound 1. Compound 2, Compound 3, Compound 4) as a monotherapy (e.g., without an additional therapeutic agent).

[0015]

[0012] This disclosure also relates to a method for treating cancer that comprises a) identifying or selecting a subject with a solid tumor that comprises activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells) prior to administration of the IL-2 prodrug and then b) administering to the subject an effective amount of an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4). Hie IL-2 prodrug as disclosed herein can comprise Compound 1. Compound 2, Compound 3, Compound 4. or an amino acid sequence variant of any of the forgoing. In some instances, the method can include administering an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4) as a monotherapy (e.g., without an additional therapeutic agent). In some instances, the method can include administering an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4) and an additional therapeutic agent as further described herein.

[0016]

[0013] The activated intratumoral effector T cells can be effector CD8+ T cells and can express granzyme B and be effector CD8+GrB+ T cells. For example, the tumor can contain at least about 1000 activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells) per mm2 as determined, for example, by immunofluorescence analysis. For example, the tumor can contain at least about 50 activated intratumoral effector T cells that express granzyme B (e.g., activated intratumoral effector CD8+GrB+ T cells) per mm2 as determined, for example, by immunofluorescence analysis.

[0014] The solid tumor can be characterized of further characterized by a gene expression signature indicative of activated T cells or an immune inflammatory phenotype.

[0017]

[0015] Tire gene expression signature indicative of activated T cells can include an increase or decrease in the expression of at least one gene indicative of activated T cells in comparison to a suitable standard.

[0018]

[0016] The increase or decrease in expression is preferably statistically significant to at least p< 0.05 using a suitable test such as the Wald Test. The suitable standard can comprise a corresponding gene expression signature for a solid tumor that did not respond to treatment with IL-2 prodrug. The suitable standard can comprise a gene expression signature standard compiled from gene expression signatures for tumors that did not respond to treatment with IL-2 prodrug.

[0019]

[0017] Tire gene expression signature indicative of activated T cells can include expression data for at least one gene selected from the group consisting of IL-23 A, GZMB, IL-4, IL-2RA. CCL3, TAPI, CCL2. CCL5, IL-7R. CCL11, CCL7. ITGAM, IL-2RG, LCP1, TNF-SF14. HAVCR2, TNF-SF13B, STATL IL- 6, CD3D, ITGAL, ITGB2, LCK, CD4, IL-18, LAG3, ITGAX, CD274, TNF-RSF8, PSMB10, FUT7, IL- 1B, ZAP70, TNF-SF4, CD74, MAF, SELL, IFN-B1, CD2, and IRF4, or any combination thereof, or all of the foregoing genes. In embodiments, the gene expression signature indicative of T cell activation comprises expression data for CCL11. GZMB, TNF-SF13B, IRF1, IFNG, TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2, PSEN2, and SPP1. In examples of such embodiments, the gene expression signature indicative of T cell activation can comprise increased expression of at least one gene selected from the group consisting of CCL11, GZMB, TNF-SF13B, IRF1, and IFNG and / or decreased expression of at least one gene selected from the group consisting of TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2, PSEN2, and SPP1.

[0020]

[0018] The gene expression signature indicative of activated T cells can include expression data for at least one gene selected from the group consisting of SLAMF1, IDO1, S100A8, CXCL9, GZMB, IL1B, GZMK. GZMA. PRF1, GZMH. CD3D. CCL11, SLAMF7, CCL5, CD5, CD3E. CXCR6. SH2D1A. CCR5, CD3G, CXCR3, CTLA4, CD27, and LAG3. Expression of one or more of these genes can be indicative of responsiveness to IL-2 prodrug as a monotherapy (e.g., without one or more additional therapeutic agents) or to IL-2 in combination with an immune checkpoint inhibitor.

[0021]

[0019] The gene expression signature indicative of activated T cells can include expression data for at least one gene selected from the group consisting of SLAMF1, IDO1, S100A8, IL-1B, GZMB, CCL11, CXCL9. GZMK, GZMH, IL-8, GZMA. SLAMF7, CD3G, CD3D, PRF1, CTLA4, CD5, SH2D1A, CD22, CD3E, TNFRSF17, CCR5, CXCL11, ITK, and CD247. Expression of one or more of these genes can be indicative of responsiveness to IL-2 prodrug as a monotherapy (e.g., without one or more additional therapeutic agents).

[0022]

[0020] The IL-2 prodrug is preferably administered intravenously, such as by intravenous infusion.

[0021] The effective amount of IL-2 prodrug that is administered can be 6 mg to about 28 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks. The effective amount of IL-2 prodrug that is administered can be about 12 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks. The effective amount of IL-2 prodrug that is administered can be about 18 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks. The effective amount of IL-2 prodrug that is administered can be about 23 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every tw o w eeks. The effective amount of IL-2 prodrug that is administered can be about 28 mg of Compound 1, Compound 2. Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0023]

[0022] The method of treating cancer can further comprises administering to the subject an effective amount of another therapeutic agent. For example, the another therapeutic agent is the standard of care therapy for the particular solid tumor. Hie another therapeutic agent can comprise an immune checkpoint inhibitor. The immune checkpoint inhibitor can bind to and inhibits a protein selected from the group consisting of PD-1, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (CD152). B7-1 (CD80). B7-2 (CD86), LAG 3 (CD223); TIM3 (HAVCR2): TIGIT (VSTM3, WUCAM); BTLA (CD272) which binds HVEM (TNFRSF14), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD 160 (BY55), CD 134 (TNRFSR4, 0X40) and CD252 (OX-40L) . The immune checkpoint inhibitor is an anti-PD-Ll antibody or antigen binding fragment thereof, such as avelumab, durvalumab, or atezolizumab . The immune checkpoint inhibitor can be an anti-CTLA-4 antibody or antigen binding fragment thereof, such as ipilimumab. The immune checkpoint inhibitor can be an anti-PD-1 antibody or antigen binding fragment thereof, such as. pembrolizumab, dostarlimab. cemiplimab-rwlc, nivolumab, camrelizumab, tislelizumab, toripalimab, or sintilimab.

[0024]

[0023] A preferred anti-PDl antibody or antigen binding fragment thereof for use in the methods described herein can comprise (a) light chain CDRs SEQ ID Nos: 6, 7, 8 and (b) heavy chain CDRs SEQ ID Nos: 11, 12, 13 . For example, the anti-PD-1 antibody can comprise: (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant hereof and (b) a light chain variable region comprising SEQ ID NO: 9 or a variant thereof and / or the heavy chain and light chain can comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO: 15, respectively. The anti-PD-1 antibody is pembrolizumab or a pembrolizumab variant, such as a pembrolizumab a biosimilar. About 100 mg to about 600 mg of the anti-PD-1 antibody or antigen binding fragment thereof can be administered about every three to six weeks. For example, 200 mg of the anti-PD-1 antibody or antigen binding fragment thereof can be administered about every three weeks or about 600 mg of the anti-PD-1 antibody or antigen binding fragment thereof can be administered about every six weeks.

[0025]

[0024] In some embodiments, the subject to be treated has failed to achieve a complete response to a prior treatment or to an ongoing treatment prior to administration of the IL-2 prodrug. The prior treatment or ongoing treatment can comprise treatment with an immune checkpoint inhibitor, such as

[0026]

[0025] an anti-PD-1 antibody.

[0027]

[0026] The subject to be treated can have cancer (a solid tumor) as described herein, for example, adrenocortical carcinoma, anal cancer, appendix cancer, astrocyto a, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, hypophary ngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia. malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, squamous cell carcinoma, adenocarcinoma, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasophary ngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell ly mphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor. In particular examples, the subject to be treated can have colon cancer, lung cancer, renal cell carcinoma, breast cancer, melanoma, squamous cell carcinoma, or adenocarcinoma. The subject to be treated can have cutaneous melanoma. The subject to be treated cutaneous squamous cell carcinoma. The subject to be treated gastroesophageal junction adenocarcinoma.

[0028]

[0027] This disclosure also relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4), wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells, activated intratumoral effector CD8+GrB+ T cells) prior to administration of the IL-2 prodrug, with the proviso that the method does not include administering an agent that blocks binding of PD-L1 or PD-L2 to PD-1 (e.g., pembrolizumab or a biosimilar version of pembrolizumab).

[0029]

[0028] This disclosure also relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a combination therapy comprising Compound 1, Compound 2, Compound 3, Compound 4 or an amino acid sequence variant of any of the forgoing and an anti -PD-1 antibody or antigen-binding fragment thereof comprising (a) light chain CDRs SEQ ID Nos: 6, 7, 8 and (b) heavy chain CDRs SEQ ID Nos: 11, 12, 13, wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells (e.g.. activated intratumoral effector CD8+ T cells, activated intratumoral effector CD8+GrB+ T cells) prior to administration of the IL-2 prodrug.

[0030]

[0029] This disclosure also relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4), wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells, activated intratumoral effector CD8+GrB+ T cells) prior to administration of the IL-2 prodrug, with the proviso that the method does not include administering pembrolizumab, a biosimilar version of pembrolizumab, or an anti-PD- 1 antibody or antigen-binding fragment thereof comprising (a) light chain CDRs SEQ ID Nos: 6, 7, 8 and (b) heavy chain CDRs SEQ ID Nos: 11, 12, 13.

[0031]

[0030] This disclosure also relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4). wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells, activated intratumoral effector CD8+GrB+ T cells) prior to administration of the IL-2 prodrug, with the proviso that the method does not include administering pembrolizumab or a biosimilar version of pembrolizumab.

[0032]

[0031] This disclosure also relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4). wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells, activated intratumoral effector CD8+GrB+ T cells) prior to administration of the IL-2 prodrug, with the proviso that the method does not include administering an anti-PD- 1 antibody or antigen-binding fragment thereof comprising (a) light chain CDRs SEQ ID Nos: 6, 7, 8 and (b) heavy chain CDRs SEQ ID Nos: 11, 12, 13.

[0032] This disclosure also relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a combination therapy comprising Compound 1, Compound 2, Compound 3, Compound 4 or an amino acid sequence variant of any of the forgoing and an agent that blocks binding of PD-L1 or PD-L2 to PD-1 with the proviso that agent is not pembrolizumab, a biosimilar version of pembrolizumab, or an anti -PD-1 antibody or antigen-binding fragment thereof comprising (a) light chain CDRs SEQ ID Nos: 6, 7, 8 and (b) heavy chain CDRs SEQ ID Nos: 11, 12. 13. wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells, activated intratumoral effector CD8+GrB+ T cells) prior to administration of the combination therapy.

[0033]

[0033] This disclosure also relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a combination therapy comprising Compound 1, Compound 2, Compound 3, Compound 4 or an amino acid sequence variant of any of the forgoing and an agent that blocks binding of PD-L1 or PD-L2 to PD-1 with the proviso that agent is not pembrolizumab or a biosimilar version of pembrolizumab, wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells, activated intratumoral effector CD8+GrB+ T cells) prior to administration of tire combination therapy.

[0034]

[0034] This disclosure also relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a combination therapy comprising Compound 1, Compound 2. Compound 3, Compound 4 or an amino acid sequence variant of any of the forgoing and an agent that blocks binding of PD-L1 or PD-L2 to PD-1 with the proviso that agent is not an anti-PD-1 antibody or antigen-binding fragment thereof comprising (a) light chain CDRs SEQ ID Nos: 6, 7, 8 and (b) heavy chain CDRs SEQ ID Nos: 11, 12, 13, wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells, activated intratumoral effector CD8+GrB+ T cells) prior to administration of the combination therapy.

[0035]

[0035] This disclosure also relates to a method for identifying a subject with a solid tumor that is likely to respond to therapy with an IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4), comprising analyzing a sample of a tumor obtained from the subject or the tumor in the subject for the presence of activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells, activated intratumoral effector CD8+GrB+ T cells), wherein the presence of activated intratumoral effector T cells indicates that the subject is likely to respond to therapy with an IL-2 prodrug. For example, the tumor can contain at least about 1000 activated intratumoral effector T cells (e.g., activated intratumoral effector CD8+ T cells) per mm2 as determined, for example, by immunofluorescence analysis. For example, the tumor can contain at least about 50 activated intratumoral effector T cells that express granzyme B (e.g., activated intratumoral effector CD8+GrB+ T cells) per mm2 as determined, for example, by immunofluorescence analysis.

[0036]

[0036] Tire solid tumor can be characterized of further characterized by a gene expression signature indicative of activated T cells or an immune inflammatory phenotype.

[0037]

[0037] The gene expression signature indicative of activated T cells can include an increase or decrease in the expression of at least one gene indicative of activated T cells in comparison to a suitable standard. The increase or decrease in expression is preferably statistically significant to at least p< 0.05 using a suitable test such as the Wald Test. The suitable standard can comprise a corresponding gene expression signature for a solid tumor that did not respond to treatment with IL-2 prodrag. Tire suitable standard can comprise a gene expression signature standard compiled from gene expression signatures for tumors that did not respond to treatment with IL-2 prodrug.

[0038]

[0038] The gene expression signature indicative of activated T cells can include expression data for at least one gene selected from the group consisting of IL-23A, GZMB, IL-4, IL-2RA, CCL3, TAPI, CCL2, CCL5, IL-7R, CCL11, CCL7, ITGAM, IL-2RG, LCP1, TNF-SF14, HAVCR2, TNF-SF13B, STAT1, IL- 6, CD3D, ITGAL, ITGB2, LCK, CD4, IL-18, LAG3, ITGAX, CD274, TNF-RSF8, PSMB10, FUT7, IL- 1B, ZAP70, TNF-SF4, CD74, MAF, SELL, IFN-B1, CD2, and IRF4, or any combination thereof, or all of the foregoing genes. In embodiments, the gene expression signature indicative of T cell activation compnses expression data for CCL11. GZMB, TNF-SF13B, IRF1, IFNG. TRAF6, TLR4. CTSH. CXCR4, ITGAM, BCL2, PSEN2, and SPP1. In examples of such embodiments, the gene expression signature indicative of T cell activation can comprise increased expression of at least one gene selected from the group consisting of CCL11, GZMB, TNF-SF13B, IRF1, and IFNG and / or decreased expression of at least one gene selected from the group consisting of TRAF6, TLR4, CTSH, CXCR4. ITGAM, BCL2, PSEN2, and SPP1.

[0039] 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0040]

[0039] Tire drawings are not necessarily to scale or exhaustive. Instead, the emphasis is generally placed upon illustrating the principles of the inventions described herein. The accompanying drawings, which constitute part of the specification, illustrate several embodiments consistent with the disclosure and, together with the description, serve to explain the principles of tire disclosure. In the drawings:

[0041]

[0040] FIG. 1 shows the amino acid sequence of the pembrolizumab light chain (SEQ ID NO: 10) and pembrolizumab heavy chain (SEQ ID NO: 15). The light chain and heavy chain variable regions are underlined and CDRs are in bold.

[0042]

[0041] FIG. 2 is a swimmer’s plot showing clinical responses to treatment with IL-2 prodrug monotherapy or combination therapy with IL-2 prodrag and pembrolizumab of individual subjects, highlighting that anti-tumor activity is demonstrated at doses of >12 mg IV Q2W. A complete response was achieved by a subject with cutaneous squamous cell cancer at week 11 and maintained for at least 8 months. A complete response was achieved by achieved by a subject with melanoma at week 36 and maintained for at least 13 months.

[0043]

[0042] FIG. 3 shows a waterfall plot of patients treated at clinically active dose levels (12 mg IV Q2W, 18 mg IV Q2W) considered for the recommended dose for expansion (RDE) of IL-2 prodrug monotherapy either as IL-2 prodrug monotherapy or combination therapy with IL-2 prodrug and pembrolizumab. FIG. 3 shows complete regression of target lesions in tire two responding patients: one with cutaneous squamous cell carcinoma (CSCC) and one with melanoma. Six patients: five with melanoma and one with gastroesophageal junction adenocarcinoma (GEJ) had a partial response. Twelve additional patients have stable target lesions (Best Percent Change from Baseline of 0 to 20). * Metastatic lymph node target lesion normalized in size (<10 mm).

[0044]

[0043] FIGs. 4A-4D are gene expression heat maps and boxplots that show the analysis of intratumoral T cells from patients on IL-2 prodrug treatment (FIGs. 4A and 4B) and baseline prior to IL-2 prodrug treatment (FIGs. 4C and 4D). FIG. 4A is a heat map of log 2-fold change of normalized mRNA counts in on-treatment biopsies relative to baseline, showing top 40 genes that showed dose-related upregulation from 1, 3, 6. 12, 18 to 28 mg IL-2 prodrug monotherapy. The same genes are shown for combination therapy with IL-2 prodrug and pembrolizumab. The monotherapy dose of 18 mg and the combination therapy doses of 12 mg resulted in the largest changes in gene expression. FIG. 4B is a boxplot showing increased expression (NanoString) of IL2RA but not FOXP3 in tumors of patients treated with IL-2 prodrug monotherapy at doses of 1, 3, 6, 12, 18 and 28 mg. FIG. 4B also shows that the ratio IL2RA / FOXP3 increased with dose level, demonstrating that IL-2 prodrug activated effector T cells without simultaneously expanding Treg cells. FIG. 4C is a heat map of baseline transcriptional signatures obtained using DeSeq2 (NanoString) that identifies genes that are differentially expressed (p<0.05, Wald Test) in subjects treated with IL-2 prodrug who experienced anti-tumor responses, either complete response (CR) or partial response (PR), in comparison to subjects who experienced stable / progressive disease. FIG. 4D is a boxplot showing quantification of CD8+ and CD8+ GrB+ T cells / mm2in all biopsies collected at baseline demonstrating an observed increase in presence of CD8+ and CD8+ GrB+ T cells in responders (CR / PR).

[0045]

[0044] FIG. 5 shows the preliminary pharmacokinetic (PK) profile of IL-2 prodrug (dosed at 1, 3, 6. 12, 18 and 28 mg) showing the plasma concentration of IL-2 prodrug (solid lines) and free IL-2 (broken lines). The peak free IL-2 exposure following administration of high dose IL-2 is shown (High-dose IL-2 Cmax) as is the peak free IL-2 exposure after administration of 18mg of IL-2 prodrug (18mg IV Q2W Cmax). IL-2 prodrug dosed at 18 mg IV Q2W has an approximately 1.5 -fold higher Cmax than High Dose IL-2, while peak free IL-2 exposure after 18 mg IV Q2W is approximately 136-fold lower than HD-IL-2. Across all dose levels, free IL-2 levels were very low (<1.6% of prodrug exposure). IL-2 prodrug PK was approximately dose proportional up to 18 mg IV Q2W and repeat dosing did not cause accumulation of IL-2 prodrug or free IL-2.

[0046]

[0045] FIGs. 6A and 6B show the analysis of intratumoral T cells from patients at baseline. FIG. 6A is a graph showing differential gene expression in tumors at baseline. The x-axis shows fold change (log2 ratio scale) and the y-axis, the negative log 10 of p-values (higher values indicate greater significances). FIG. 6B is a heat map of baseline transcriptional signatures obtained using DeSeq2 (NanoString) that identifies the top 25 genes that were differentially expressed (p<0.05, Wald Test) in subjects at baseline who experienced an partial or complete response, stable disease or progressive disease upon therapeutic treatment. The patients were treated with either IL-2 prodrug as IL-2 monotherapy or a combination therapy with IL-2 prodrug and pembrolizumab. FIG. 6C is a boxplot showing quantification of CD8+ and CD8+ GrB+ T cells / mm2by immunofluorescence in all biopsies collected at baseline demonstrating an observed increase in presence of CD8+ and CD8+ GrB-i- T cells in responders (CR / PR).

[0047]

[0046] FIGs. 7A and 7B show the analysis of intratumoral T cells from patients at baseline. FIG. 7A is a graph showing differential gene expression of tumors at baseline. The x-axis shows fold change (log2 ratio scale) and the y-axis. the negative log 10 of p-values (higher values indicate greater significances). Subjects were treated with IL-2 prodrug as IL-2 monotherapy. FIG. 7B is a heat map of baseline transcriptional signatures obtained using DeSeq2 (NanoString) that identifies the top 25 genes that were differentially expressed (p<0.05, Wald Test) in subjects at baseline who experienced an partial or complete response, stable disease or progressive disease upon therapeutic treatment. Tire subjects were treated with IL-2 prodrug as IL-2 monotherapy.

[0048] 4. DETAILED DESCRIPTION

[0049]

[0047] This disclosure relates to methods for treating cancer comprising administering to a subject an effective amount of an IL-2 prodrug. The subject has a solid tumor that contains activated intratumoral effector T cells prior to administration of the IL-2 prodrug. The effector T cells can display certain biomarkers of activation as described further herein. For example, the solid tumor can have an immuno- inflammatory phenotype. Preferably, the intratumoral effector T cells are CD8+ T cells and more preferably CD8+ Granzyme B (GrB+) positive T cells. As described and exemplified herein, the inventors have discovered that treating this type of a solid tumor with an IL-2 prodrug, that releases IL-2 in the tumor microenvironment, can provide potent anti-tumor activity. Without wishing to be bound by any particular theory or mechanism, it was believed that cancer therapy that results in increased IL-2 in the tumor microenvironment (e.g.. using IL-2 prodrug) can be beneficial because it contributes to recruitment of effector T cells into the tumor microenvironment. As described and exemplified herein, tire inventors have surprisingly discovered through clinical research that IL-2 prodrug therapy is highly effective in tumors that contain activated intratumoral effector CD8+ T cells prior to treatment with the IL-2 prodrug and less effective in tumors that do not contain such intratumoral T cells. Without wishing to be bound by any particular theory or mechanism, this indicates that potent anti-tumor activity is due to IL-2 effects on cells present in the tumor microenvironment.

[0050] A. Patient Identification and Selection

[0051]

[0048] This disclosure relates to methods of treating a subject with a solid tumor that contains activated intratumoral effector T cells (e.g., CD8+ T cells) prior to administering therapy in accordance with this disclosure. Preferably, tire solid tumor contains activated intratumoral CD8+ effector T cells, such as CD8+GranzymeB (GrB)+ T cells prior to administering therapy in accordance with this disclosure. The method comprises administering IL-2 prodrug (e.g.. Compound 1. 2, 3 or 4) to tire subject with the solid tumor. The method can comprise administering IL-2 (e.g., Compound 1, 2, 3, or 4) to the subject with the solid tumor as a monotherapy (e.g., without one or more additional therapeutic agents). As described further herein, tire method can comprise administering one or more additional therapeutic agents in addition to IL-2 prodrug. For example, the method can comprise administering an IL-2 prodrug designed to be cleaved in the tumor microenvironment and release active IL-2 (or IL-2 mutein) as described herein. Subjects who have a solid tumor that contains activated intratumoral effector (e.g., CD8+. CD8+GrB+) T cells can be identified using any suitable methods. For example, a biopsy can be analyzed for suitable biomarkers using, for example, using immunofluorescence and other immunoassays, transcriptome analysis, expression profiling, substrate cleavage, bioassays, mass spectrometry, chemiluminescence, or other suitable methods. The tumor can also be analyzed using suitable in vivo methods such as imaging (e.g., with sensors for biomarkers).

[0052]

[0049] Solid tumors suitable for treatment in accordance with this disclosure contain activated intratumoral (e.g., CD8+, CD8+GrB+) T cell prior to the administration of an IL-2 prodrug. For example, activated intratumor effector CD8+ T cells or activated intratumor effector CD8+GrB+ T cells.

[0053]

[0050] Tire tumor microenvironment, having an immune inflamed, also known as immunoinflammatory, phenotype described herein comprises activated intratumoral effector T cells (e.g., CD8 + T cells, CD8+GrB+ and / or CD4+ T cells). The tumor microenvironment can comprise, for example, at least about 200 to about 1500 activated intratumoral effector CD8+ T cells per mm2. For example, the tumor microenvironment can comprise at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, or at least about 1500 activated intratumoral effector CD8+ T cells per mm2. Additionally, the activated intratumoral effector CD8+ T cells can also be granzyme B positive (GrB+). For example, the tumor microenvironment can comprise at least about 50 to about 1000 activated intratumoral effector CD8+ GrB+ T cells per mm2. For example, the tumor microenvironment can comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450. at least about 500, at least about 550. at least about 600, at least about 650, at least about 700, at least about 750. at least about 800, at least about 850, at least about 900, at least about 950, or at least about 1000 activated intratumoral effector CD8+ GrB+ T cells per mm2.

[0054]

[0051] The activated intratumoral T cells ( e.g., activated intratumoral CD8+ T cells or activated intratumoral CD8+GrB+ T cells) can be alternatively characterized or further characterized by a gene expression signature indicative of T cell activation and / or immuno-inflammatory phenotype. Typically, such a signature is characterized by increased or decreased expression of genes (e g., including genes associated with inflammation and T cell activation) in comparison to a suitable standard. Any suitable standard can be used, such as expression level of corresponding genes in tumors from patients that did not contain activated intratumoral effector T cells ( e.g., activated intratumoral CD8+ T cells or activated intratumoral CD8+GrB+ T cells) and did not respond to IL-2 prodrug therapy (e.g., had progressive disease or stable disease). A data set for such non-responders can be collected and used to define a standard non-responder signature based on e.g., average or median expression of the signature genes in the non-responders. The gene expression signature of activated T cells can comprise genes that show a statistically significant increase or decrease in expression of genes relative to the suitable standard, preferably with a p-value of 0.05 or less. Statistical significance and p-value can be determined using the Wald Test. If desired statistical significance and be determined using the padj value which is the false- discovery rate (FDR) adjusted p-value.

[0055]

[0052] The gene expression signature indicative of T cell activation can include gene expression of IL- 23A, GZMB, IL-4, IL-2RA. CCL3, TAPI, CCL2. CCL5, IL-7R, CCL11, CCL7, ITGAM, IL-2RG. LCP1, TNF-SF14, HAVCR2, TNF-SF13B, STAT1, IL-6, CD3D, ITGAL, ITGB2, LCK, CD4, IL-18, LAG3, ITGAX, CD274, TNFRSF8, PSMB10, FUT7, IL-1B, ZAP70, TNF-SF4, CD74, MAF, SELL, IFN-B1, CD2, IRF4, or a combination of any of the foregoing such as all of the foregoing. The gene expression signature can be indicative of responsiveness to the IL-2 prodrug as a monotherapy and / or IL- 2 prodrug in combination with one or more additional therapeutic agents.

[0056]

[0053] The gene expression signature indicative of T cell activation can include gene expression of SLAMF1, IDOL S100A8, CXCL9, GZMB, IL IB, GZMK, GZMA, PRF1, GZMH, CD3D, CCL11, SLAMF7, CCL5, CD5, CD3E, CXCR6, SH2D1A, CCR5, CD3G, CXCR3, CTLA4, CD27, LAG3, or a combination of the foregoing such as all of the foregoing. The gene expression signature can be indicative of responsiveness to the IL-2 prodrug as a monotherapy and / or IL-2 prodrug in combination with one or more additional therapeutic agents.

[0057]

[0054] Tire gene expression signature indicative of T cell activation can include gene expression of SLAMF1, IDO1, S100A8. IL-1B, GZMB, CCL11, CXCL9, GZMK, GZMH, IL-8, GZMA, SLAMF7, CD3G, CD3D, PRF1, CTLA4, CD5, SH2D1A, CD22, CD3E. TNFRSF17, CCR5, CXCL11, ITK, CD247 or a combination of the foregoing such as all of the foregoing. The gene expression signature can be indicative of responsiveness to the IL-2 prodrug as a monotherapy.

[0058]

[0055] Gene expression analysis methods are well known in the art. For example, hybridization-based analysis to quantitatively measure RNA directly from tissue or cells. For example, polymerase chain reaction (PCR)-based analysis, such as, reverse transcription quantitative PCR (RT-qPCR) or droplet digital PCR (ddPCR).

[0059]

[0056] The gene expression signature indicative of T cell activation can include an increase or decrease in expression, relative to a suitable standard, of 1 to about 40 genes selected from the group consisting of IL-23A, GZMB, IL-4, IL-2RA, CCL3, TAPI, CCL2, CCL5, IL-7R, CCL11, CCL7, ITGAM, IL-2RG, LCP1, TNF-SF14, HAVCR2, TNF-SF13B, STAT1, IL-6, CD3D, ITGAL, ITGB2, LCK, CD4, IL-18, LAG3, ITGAX, CD274. TNF activated -RSF8, PSMB10, FUT7, IL-1B, ZAP70, TNF-SF4, CD74, MAF, SELL. IFN-B1, CD2, and IRF4. For example, the gene expression signature of T cells can comprises an increase or decrease in expression of at least 1 gene, at least about 2 genes, at least about 3 genes, at least about 4 genes, at least about 5 genes, at least about 6 genes, at least about 7 genes, at least about 8 genes, at least about 9 genes, at least about 10 genes, at least about 11 genes, at least about 12 genes, at least about 13 genes, at least about 14 genes, at least about 15 genes, about 16 genes, at least about 17 genes, at least about 18 genes, at least about 19 genes, at least about 20 genes, at least about 21 genes, at least about 22 genes, at least about 23 genes, at least about 24 genes, at least about 25 genes, at least about 26 genes, at least about 27 genes, at least about 28 genes, at least about 29 genes, at least about 30 genes, at least about 31 genes, at least about 32 genes, at least about 33 genes, at least about 34 genes, at least about 35 genes, at least about 36 genes, at least about 37 genes, at least about 38 genes, at least about 39 genes, at least about 40 genes.

[0060]

[0057] The gene expression signature indicative of T cell activation can include an increase or decrease in expression, relative to a suitable standard, of 1 to about 25 genes selected from the group consisting SLAMFL 1DO1, S100A8. CXCL9, GZMB. IL IB, GZMK, GZMA. PRF1, GZMH. CD3D, CCL11. SLAMF7, CCL5, CD5, CD3E, CXCR6, SH2D1A, CCR5, CD3G, CXCR3, CTLA4, CD27, and LAG3. For example, the gene expression signature of T cells can comprises an increase or decrease in expression of at least 1 gene, at least about 2 genes, at least about 3 genes, at least about 4 genes, at least about 5 genes, at least about 6 genes, at least about 7 genes, at least about 8 genes, at least about 9 genes, at least about 10 genes, at least about 11 genes, at least about 12 genes, at least about 13 genes, at least about 14 genes, at least about 15 genes, about 16 genes, at least about 17 genes, at least about 18 genes, at least about 19 genes, at least about 20 genes, at least about 21 genes, at least about 22 genes, at least about 23 genes, at least about 24 genes, or at least about 25 genes.

[0061]

[0058] The gene expression signature indicative of T-cell activation can include an increase or decrease in expression, relative to a suitable standard of about 1 to about 25 genes selected from the group consisting SLAMF1, IDO1, S100A8, IL-1B, GZMB, CCL11, CXCL9, GZMK, GZMH, IL-8, GZMA, SLAMF7, CD3G, CD3D, PRF1, CTLA4, CD5, SH2D1A, CD22, CD3E, TNFRSF17, CCR5, CXCL11, ITK, and CD247. For example, the gene expression signature of T cells can comprises an increase or decrease in expression of at least 1 gene, at least about 2 genes, at least about 3 genes, at least about 4 genes, at least about 5 genes, at least about 6 genes, at least about 7 genes, at least about 8 genes, at least about 9 genes, at least about 10 genes, at least about 11 genes, at least about 12 genes, at least about 13 genes, at least about 14 genes, at least about 15 genes, about 16 genes, at least about 17 genes, at least about 18 genes, at least about 19 genes, at least about 20 genes, at least about 21 genes, at least about 22 genes, at least about 23 genes, at least about 24 genes, or at least about 25 genes.

[0062]

[0059] The gene expression signature indicative of T cell activation can comprise gene expression information for CCL11, GZMB. TNF-SF13B. IRF1, IFNG, TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2. PSEN2, and SPP1. In an embodiment, expression of at least 1 to at least about 5 genes selected from the group consisting of CCL11, GZMB, TNF-SF13B, IRF1, and IFNG is increased. For example, the gene expression signature indicative of T cell activation can comprise an increase in gene expression of about 1 gene, about 2 genes, about 3 genes, about 4 genes, or about 5 genes from the group consisting of CCL11, GZMB, TNF-SF13B, IRF1, and IFNG (e.g., increased expression of at least 3 genes selected from the group consisting of CCL11, GZMB, TNF-SF13B, IRF1, and IFNG). Alternatively or in addition, the gene expression signature indicative of T cell activation can comprises expression data for CCL11, GZMB, TNF-SF13B, IRF1, IFNG, TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2, PSEN2, and SPP1, wherein expression of at least about 1 to about 8 genes selected from the group consisting of TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2, PSEN2, and SPP1 is decreased. For example, the gene expression signature indicative of T cell activation can comprises a decrease in gene expression of about 1 gene, about 2 genes, about 3 genes, about 4 genes, about 5 genes, about 6 genes, about 7 genes, or about 8 genes from the group consisting of TRAF6, TLR4, CTSH, CXCR4. ITGAM, BCL2, PSEN2, and SPP1. In an embodiment, the gene expression signature indicative of T cell activation comprises expression data for CCL11, GZMB, TNF-SF13B, IRF1, IFNG, TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2, PSEN2, and SPP1, wherein there is increased expression of at least 3 genes selected from the group consisting of CCL11, GZMB, TNF-SF13B, IRF1, and IFNG, and decreased expression of at least 3 genes selected from the group consisting of TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2, PSEN2, and SPP1.

[0063]

[0060] The gene expression signature indicative of T cell activation can comprise gene expression information for SLAMF1, IDO1, S100A8, CXCL9, GZMB, IL1B, GZMK, GZMA, PRF1, GZMH, CD3D, CCL11, SLAMF7. CCL5, CD5, CD3E, CXCR6, SH2D1A, CCR5, CD3G, CXCR3, CTLA4, CD27. or LAG3. In an embodiment, expression of at least 1 to at least about 5 genes selected from the group consisting of SLAMF1. IDO1, S100A8, CXCL9, GZMB. IL1B, GZMK, GZMA. PRF1, GZMH, CD3D, CCL11, SLAMF7, CCL5, CD5, CD3E, CXCR6, SH2D1A, CCR5, CD3G, CXCR3, CTLA4, CD27, and LAG3 is increased. For example, the gene expression signature indicative of T cell activation can comprise an increase in gene expression of about 1 gene, about 2 genes, about 3 genes, about 4 genes, or about 5 genes from the group consisting of SLAMF1. IDO1, S100A8, CXCL9, GZMB, IL1B, GZMK, GZMA, PRF1, GZMH, CD3D, CCL11, SLAMF7, CCL5. CD5. CD3E, CXCR6, SH2D1A, CCR5, CD3G, CXCR3. CTLA4, CD27, and LAG3.

[0064]

[0061] In an embodiment, expression of SLAMF1 is increased. In an embodiment, expression of SLAMF1 and at least 1 or more additional genes selected from the group consisting of IDO1, S100A8, CXCL9, GZMB, IL1B, GZMK, GZMA, PRF1, GZMH, CD3D, CCL11, SLAMF7, CCL5, CD5, CD3E, CXCR6, SH2D1A, CCR5, CD3G. CXCR3, CTLA4, CD27, and LAG3 is increased. For example, the gene expression signature indicative of T cell activation can comprise an increase in SLAMF 1 and at least one of GZMB, GZMH, CD3G, CD5, PRF1. CXCL9, and IDO1.

[0065]

[0062] The gene expression signature indicative of T cell activation can comprise gene expression information for SLAMF1, IDO1, S100A8, IL-1B, GZMB, CCL11, CXCL9, GZMK, GZMH, IL-8, GZMA, SLAMF7, CD3G, CD3D, PRF1, CTLA4, CD5, SH2D1A, CD22, CD3E, TNFRSF17, CCR5, CXCL11, ITK, or CD247. In an embodiment, expression of at least 1 to at least about 5 genes selected from tire group consisting of SLAMF1, IDOL S100A8, IL-1B, GZMB, CCL11. CXCL9, GZMK. GZMH. IL-8, GZMA. SLAMF7, CD3G, CD3D, PRF1. CTLA4, CD5, SH2D1A, CD22, CD3E. TNFRSF17, CCR5, CXCL11, ITK, and CD247 is increased. For example, the gene expression signature indicative of T cell activation can comprise an increase in gene expression of about 1 gene, about 2 genes, about 3 genes, about 4 genes, or about 5 genes from tire group consisting of SLAMF 1, IDO1, S100A8, IL-1B, GZMB, CCL11, CXCL9, GZMK, GZMH, IL-8, GZMA, SLAMF7, CD3G, CD3D, PRF1, CTLA4. CD5. SH2D1A, CD22, CD3E, TNFRSF17, CCR5. CXCL11, ITK, and CD247.

[0066]

[0063] In an embodiment, expression of SLAMF1 is increased. In an embodiment, expression of SLAMF 1 and at least 1 or more additional genes selected from the group consisting of IDO 1, S100A8, IL-1B, GZMB, CCL11, CXCL9, GZMK, GZMH, IL-8, GZMA, SLAMF7, CD3G, CD3D, PRF1, CTLA4, CD5, SH2D1A, CD22, CD3E, TNFRSF17, CCR5, CXCL11, ITK, and CD247 is increased. B. IL-2 Prodrugs

[0067]

[0064] The IL-2 prodrugs for use in the methods and compositions of this disclosure are designed to overcome the toxicity and short half-life problems that have severely limited the clinical use of cytokines in oncology. The IL-2 prodrugs can contain an IL-2 polypeptide that has receptor agonist activity of native IL-2, including binding to and activating signaling through IL-2Ra / p / y and IL-2R(3 / y. but in tire context of the prodrug, the IL-2 receptor agonist activity is attenuated, and the circulating half-life is extended. The IL-2 prodrug can contain an engineered IL-2 polypeptide or mutein, for example a mutein that is biased to bind IL-2R|3 / y, but in the context of the prodrug, the IL-2 receptor agonist activity is attenuated, and the circulating half-life is extended. See, e.g., International Publication No: WO202069398 and International Publication No: WO2021202675A1 which are incorporated herein by reference in its entirety. The IL-2 prodrugs include protease cleavage sequences, which are cleaved by proteases that are associated with, and are typically enriched or selectively present in, the tumor microenvironment. Thus, the IL-2 prodrugs are preferentially (or selectively) and efficiently cleaved in the tumor microenvironment to release active IL-2 or mutein, and to limit IL-2 activity substantially to the tumor microenvironment. Tire IL-2 or mutein that is released upon cleavage has a short half-life, which is substantially similar to the half-life of naturally occurring IL-2, further restricting IL-2 activity to the tumor microenvironment. Even though the half-life of the IL-2 prodrug is extended, toxicity is dramatically reduced or eliminated because the circulating prodrug has attenuated IL-2 activity, and active IL-2 is restricted to the tumor microenvironment.

[0068]

[0065] The IL-2 prodrugs can comprise two polypeptide chains. The first polypeptide chain comprises from amino to carboxy terminus: the IL-2 polypeptide - a protease cleavable linker - an anti-human serum albumin (HSA) binding single antibody variable domain - a linker that is preferably protease cleavable - VH and CHI of an antibody that binds IL-2. The second polypeptide chain comprises a VL and CL of an antibody that binds IL-2 and that together with the VH and CHI of the first polypeptide chain form a Fab that binds the IL-2 polypeptide. Compounds 1, 2, 3 and 4 are specific examples of preferred IL-2 prodrugs that contain native IL-2 for use according to this disclosure. Compounds 1, 2, 3, and 4 and additional details regarding their activity is disclosed in International Publication No: WO2021 / 097376.

[0069] Table 1. IL-2 prodrugs

[0070]

[0066] Amino acid sequence variants of compounds 1, 2, 3 and 4, that retain attenuated IL-2 activity in the periphery and that release active IL-2 upon protease cleavage in the tumor microenvironment can also be used in accordance with this disclosure. For example, a prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 1 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%. at least about 89%, at least about 90%, at least about 91%, at least about 92%. at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO: 5.

[0071]

[0067] A prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:2 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:5.

[0072]

[0068] A prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%. at least about 87%. at least about 88%. at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:3 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%. at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:5.

[0073]

[0069] A prodrug can comprise a first polypeptide that has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:4 and a second polypeptide that has at least about 80%, at least about 85%, at least about 86%. at least about 87%. at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:5.

[0074]

[0070] For all amino acid sequence variant prodrugs, it is preferred that the protease cleavage site contain no amino acid replacements, or only conservative amino acid replacements, so that the sequence variant prodrug is cleaved in the tumor microenvironment and releases IL-2 to substantially the same degree as the corresponding parental prodrug. Similarly, it is preferred that the complementarity determining regions of the anti-HSA single variable domain and the anti-IL2 Fab contain no amino acid replacements, or only conservative amino acid replacements, so that a) the serum half-life of the sequence variant prodrag is substantially the same as the corresponding parental prodrug, and b) the attenuation of IL-2 agonist activity of the sequence variant prodrag is substantially the same as tire corresponding parental prodrug.

[0075]

[0071] Exemplary amino acid substitutions are provided in Table 2.

[0076] Table 2. Exemplary amino acid substitutions

[0077] C. Additional Therapeutic Agents

[0078]

[0072] The methods of this disclosure can further comprising administering one or more additional therapeutic agents to treat cancer, such as chemotherapeutic agents e.g., adriamycin, cerubidine, melphalan, novantrone (mitoxantrone), cyclophosphamide, mechlorethamine, melphalan, chlorambucil, ifosfamide, busulfan, N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin, diaziquone (AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine, hexamethylmelamine, methotrexate, pemetrexed, fluorouracil (e.g. 5 -fluorouracil), capecitabine. cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine, mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, etoposide, teniposide, doxorubicin, daunorubicin, epirubicin, idarubicin, pirambicin, aclarubicin, mitoxantrone, actinomycin, bleomycin, bisantrene, gemcitabine, and the like), immuno-oncology agents and immune checkpoint inhibitors (e.g., anti-PD-Ll, anti-CTLA4. anti-PD-1, anti-CD47, anti-GD2), cellular therapies (e.g. CAR-T, TCT-T, T-cell therapy, such as tumor infiltrating lymphocyte (TIL) therapy, NK cell therapy), oncolytic viruses, T cell engagers, other cytokines including prodrugs of other cytokines (e.g., IL-2, IL-12, and IFN), angiogenesis inhibitors, antibody-drug conjugates( e.g., trastuzumab emtansine (KADCYLA), trastuzumab deruxtecan (ENHERTU), enfortumab vedotin (PADCEV), sacituzumab govitecan (TRODELVY)), radiation therapy and / or small molecules (e.g.. kinase inhibitors). Non-limiting examples of anti -cancer agents that can be used include acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin: altretamine; ambomycin: ametantrone acetate; aminoglutethimide: amsacrine; anastrozole: anthramycin: asparaginase: asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol: chlorambucil; cirolemycin: cisplatin: cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine: dactinomycin; daunorubicin hydrochloride; decitabine: dexormaplatin: dezaguanine; dezaguanine mesylate; diaziquone: docetaxel; doxombicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha- nl interferon alpha-n3; interferon bcta-I; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium: lomustine; losoxantrone hydrochloride: masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel: pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride: puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safmgol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tcgafur; teloxantrone hydrochloride; temoporfm; teniposide; teroxirone: testolactone; thiamiprine; thioguanine; thiotepa; tiazofiirin: tirapazamine; toremifene citrate: trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinzolidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. An additional therapeutic agent can be, for example, pemetrexed, a platinum chemotherapeutic agent, carboplatin, paclitaxel, protein-bound paclitaxel, fluorouracil, a fluoropyrimidine-based chemotherapeutic agent, or axitinib. The additional therapeutic agent can include any agent or combination of agents that are standard of care for the particular cancer to be treated.

[0079]

[0073] The composition of this disclosure can also include one or more additional agents to treat cancer.

[0080]

[0074] Immune checkpoint proteins regulate T cell functions. T cell effector function is important for immunotherapeutic approaches to treating tumors. But immunosuppression, and decreased effector function, is often seen as tumors grow and cancer progresses. One mechanism behind this phenomenon is the activation of immune checkpoint proteins by cancer cells, leading to suppression of the anti-tumor immune response. This typically occurs when cancer cells express proteins on their surface that can interact with immune checkpoint proteins on the surface of T cells in the tumor microenvironment to suppress the activity of the T cells. Immune checkpoint proteins include, for example, PD- 1 which binds ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (CD152) which binds B7-1 (CD80) and B7-2 (CD86). LAG 3 (CD223) which binds Galectin3, LSECtin and FGL1: TIM3 (HAVCR2) which binds ligands Ceacaml and Galectin9; T1GIT (VSTM3, WUCAM) which binds

[0081] CD112 and CD155; BTLA (CD272) which binds HVEM (TNFRSF14), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55) which bind HVEM; CD134 (TNRFSR4, 0X40) which binds CD252 (OX-40L).

[0075] Therapeutic agents, such as antibodies, which bind immune checkpoint proteins and inhibit their immunosuppressive activity or immune checkpoint inhibitors (ICIs) have been developed as anti-tumor agents. Several such agents are now commercially available for cancer therapy, including the anti-PDl antibodies pembrolizumab (KEYTRUDA), dostarlimab (JEMPERLI), cemiplimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab. tislelizumab, toripalimab, and sintilimab (TYVYT); the anti-PD- L1 antibodies avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ); the anti-CTLA-4 antibody ipilimumab (YERVOY); and the anti-LAG-3 receptor antibody relatlimab. While therapy with such immune checkpoint inhibitors provide advantages for cancer therapy, the overall success remains low, relapse occurs and resistance to checkpoint inhibition develops.

[0082]

[0076] Tire methods disclosed herein can further comprise administering, and the compositions can further comprise, any desired immune checkpoint inhibitor, such an anti-PDl. anti-CTLA-4, anti-LAG-3 receptor and combinations thereof.

[0083]

[0077] In certain preferred aspects, the methods disclosed herein comprising administering an IL-2 produg in combination with a PD-1 antagonist, such as pembrolizumab. The word “pembrolizumab’’ is an international nonproprietary name that refers to the humanized anti-PD-1 antibody marketed under the brand name “Keytruda” by Merck & Co., Inc. (Railway, NJ, USA) Pembrolizumab (formerly known as MK-3475. SCH 900475 and lambrolizumab) alternatively referred to herein as “pembro,” is a humanized IgG4 mAb with the structure described in WHO Drug Information, Vol. 27. No. 2, pages 161-162 (2013) and which comprises the heavy and light chain amino acid sequences and CDRs described in Table 3. Pembrolizumab has been approved by the U.S. FDA as described in the Prescribing Information for KEYTRUDA (Merck & Co., Inc., Rahway, NJ USA; initial U.S. approval 2014, updated March 2024).

[0084]

[0078] A “PD-1 antagonist’ or “Anti-PD-1 antibody” as used in the any of the treatment methods, means any chemical compound or biological molecule that blocks binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T cell, B cell or NKT cell) and preferably also blocks binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD- 1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7- 4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In any of the treatment methods, medicaments and uses of the present invention in which a human individual is being treated, the PD-1 antagonist blocks binding of human PD-L1 to human PD-1, and preferably blocks binding of both human PD-L1 and PD-L2 to human PD-1. Human PD-1 amino acid sequences can be found in NCBI Locus No.: NP 005009. Human PD-L1 and PD-L2 amino acid sequences can be found in NCBI Locus No.: NP 054862 and NP 079515, respectively.

[0085]

[0079] PD-1 antagonists useful in the treatment methods, medicaments and uses of the present invention include a monoclonal antibody (mAb), or antigen binding fragment thereof, which specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1.

[0086]

[0080] A specific anti -human PD-1 mAbs useful as the PD-1 antagonist in the treatment method, medicaments and uses of the present invention is pembrolizumab. Pembrolizumab is a potent humanized IgG4 monoclonal antibody with high specificity of binding to the programmed cell death 1 (PD-1) receptor, thus inhibiting its interaction with programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). PD-1 ligands are expressed at high levels in some tumors and signaling through the PD-l / PD-1 ligand pathway can contribute to inactivation of T cell immune surveillance of tumors. Pembrolizumab binds to PD-1 and blocks it interaction with PD-L1 and PD-L2, releasing PD-1 pathway-mediated inhibition of the immune response, including the anti-tumor immune response. Pembrolizumab is indicated for the treatment of patients across a number of cancer indications including melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell cancer (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability high or mismatch repair deficient cancer, microsatellite instability high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial carcinoma, tumor mutational burden high cancer, cutaneous squamous cell carcinoma (cSCC) and triple negative breast cancer (TNBC). The heavy and light chain amino acid sequences of pembrolizumab are shown in Table 3. See. U.S. Patent Nos. 8.354,509 and 8,900,587. both of which are incorporated herein by reference in their entirety.

[0087]

[0081] A variant or biosimilar of pembrolizumab may also be used in the treatment method, medicaments and uses of the present invention. As used herein, a “pembrolizumab variant” means a monoclonal antibody which comprises heavy chain and light chain sequences that are substantially identical to those in pembrolizumab, except for having three, two or one conservative amino acid substitutions at positions that are located outside of the light chain CDRs and six, five, four, three, two or one conservative amino acid substitutions that are located outside of the heavy chain CDRs, e g ., the variant positions are located in the FR regions or the constant region, and optionally has a deletion of the C -terminal lysine residue of the heavy chain. In other words, pembrolizumab and a pembrolizumab variant comprise identical CDR sequences, but differ from each other due to having a conservative amino acid substitution at no more than three or six other positions in their full-length light and heavy chain sequences, respectively. A pembrolizumab variant is substantially the same as pembrolizumab with respect to the following properties: binding affinity to PD-1 and ability to block the binding of each of PD-L1 and PD-L2 to PD-1.

[0088]

[0082] In some embodiments of the treatment methods, medicaments and uses of the present invention, the PD-1 antagonist is a monoclonal antibody, or antigen binding fragment thereof, which comprises: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13.

[0089]

[0083] In some embodiments of the treatment methods, medicaments and uses of the present invention, the PD-1 antagonist is a monoclonal antibody, or antigen binding fragment thereof, which comprises (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. A variant of a heavy chain variable region sequence is identical to the reference sequence except having up to 17 conservative amino acid substitutions in the framework region (z'.e., outside of the CDRs), and preferably has less than ten, nine, eight, seven, six or five conservative amino acid substitutions in the framework region. A variant of a light chain variable region sequence is identical to the reference sequence except having up to five conservative amino acid substitutions in the framework region (z.e., outside of the CDRs), and preferably has less than four, three or two conservative amino acid substitution in the framework region.

[0090]

[0084] In another embodiment of the treatment methods, medicaments and uses of the present invention, the PD-1 antagonist is a monoclonal antibody comprising (a) a heavy chain comprising SEQ ID NO: 15 and (b) a light chain comprising SEQ ID NO: 19.

[0091]

[0085] In all of the treatment methods, medicaments and uses of the present invention, the PD-1 antagonist inhibits the binding of PD-L1 to PD-1, and preferably also inhibits the binding of PD-L2 to PD-1. In some embodiments of the above treatment method, medicaments and uses, the PD-1 antagonist is a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds to PD-1 and blocks the binding of PD-L1 to PD-1. In one embodiment, the PD-1 antagonist is an anti -PD-1 antibody, or antigen binding fragment thereof, which comprises: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. In one embodiment, the PD-1 antagonist is an anti- PD-1 antibody, or antigen binding fragment thereof, which comprises (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. In one embodiment, tire PD-1 antagonist is an anti-PD-1 antibody which comprises a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO:5, respectively. In one embodiment, the PD-1 antagonist is pembrolizumab or a pembrolizumab variant. In one embodiment, the PD-1 antagonist is pembrolizumab.

[0092]

[0086] Table 3 below provides a list of the amino acid sequences of exemplary anti-PD-1 antibodies for use in the treatment method, medicaments and uses of the present invention. Also see FIG. 1.

[0093] Table 3. Exemplary Anti-PD-1 antibody Sequences

[0094]

[0087] The cellular therapy can comprise any desired immune cells, such as T cells, B cells, NK cells and the like and any combination of immune cells. For example, the cell therapy can be a substantially homogenous population of T cells, such as CAR-T cells. In other examples, tire cell therapy can contain one or more cell types, such as primary cells that have been expanded ex-vitro and are administered to the subject in need thereof. A cell therapy for cancer can include, for example, dendritic cells. T cells (e.g., CD3+ T cells. CD4+ T cells, CD8+ T cells, NKT cells, alpha beta T cells, gamma delta T cells, etc.), NK cells, and / or macrophages. The cell therapy can comprise immune cells that are engineered. For example, an immune cell may be engineered to express an antigen binding protein such as a Chimeric Antigen Receptor (CAR) or a T cell receptor (TCR) subunit. An “antigen binding protein” (ABP) is a protein comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. Immune cells such as T cells may be engineered to express a CAR or TCR in order to make the cell specific for an antigen of interest, such as a tumor antigen. Preferably, an antigen binding protein expressed by an immune cell directs the immune cell and its immune effector functions to cells that express a desired antigen. An immune cell may be engineered using any method, such as via viral vectors, CRISPR, TALEN, or meganucleases. Immune cells can be engineered or genetically modified using any suitable approach, in vitro or in vivo. For in vitro engineering, cells are typically cultured and genetically modified using any suitable method, such as viral transduction. Engineered cells can then be selected and if desired expanded and administered to a subject as adoptive cell therapy. For in vivo engineering, typically an engineered genetic construct is administered to the patient to transduce immune cells. A number of suitable approaches can be used such as, for example, viral vectors that infect immune cells and carry a desired transgene, or other suitable nucleic acid delivery technology. Alternatively, an immune cell may not have been engineered. For example, tumor infiltrating lymphocyte (TIL) therapy often does not involve engineering the therapeutic cells because they are capable of killing tumor cells. However, TILs may be selected and expanded, for example ex vivo, to produce a population of cells with specificity for a tumor antigen on the subject’s tumor. TILs and production of TILs are described, for example, in international patent application PCT / US2018 / 064135 and / or US patent application 17 / 041,305, each of which are incorporated by reference in their entirety. In some embodiments. TILs are selected based on their antigen specificity.

[0095]

[0088] The additional therapeutic agent can be a bispecific, trispecific or multispecific cell engager. Such cell engagers, including bispecific T-cell engagers (BiTEs), dual-affinity re-targeting molecules (DARTs), bispecific killer cell engagers (BiKEs) and trispecific killer cell engagers (TriKEs) are well- known in the art and include a binding region (typically a sdAb or two variable regions that fomi a VH / VL binding sight) that binds an antigen on an immune cell (e.g. an effector cell such as a T-cell or NK cell) and a binding region that binds a tumor-associated antigen (TAA) . See, e.g., Allen et al ., (2021), Life (Basel), 1 1 (6):465. For example, such bispecific, trispecific or multispecific cell engagers can engage T-cells and tumor cells, by binding to CD3 on the T cell and to a tumor associated antigen on a tumor cell. For example, such bispecific, trispecific or multispecific cell engagers can engage NK cells and tumor cells, by binding to CD 16 on the NK cell and to a tumor associated antigen on a tumor cell.

[0096]

[0089] Bispecific T cell engagers (BiTEs) and certain other T cell engagers frequently bind simultaneously to a TAA and CD3, consequently activating T cells irrespective of MHC, resulting in the release of perforins and granzymes. For example, tebentafiisp-tebn (KIMMTRAK) is a BiTE that is bispecific gplOO pcptidc-HLA-dircctcd T cell receptor CD3 T cell engager. D. Therapeutic Methods and Uses

[0097]

[0090] The disclosure relates to methods for treating cancer comprising administering to a subject in need thereof an effective amount of an IL-2 prodrug (e.g., compound 1, compound 2, compound 3 or compound 4). The disclosure relates to methods for treating cancer comprising administering to a subject in need thereof an effective amount of an IL-2 prodrug (e.g., compound 1. compound 2, compound 3 or compound 4) as a monotherapy. The disclosure relates to methods for treating cancer comprising administering to a subject in need thereof an effective amount of an IL-2 prodrug (e.g., compound 1, compound 2, compound 3 or compound 4) as a monotherapy or with one or more additional therapeutic agents. In embodiments tire additional therapeutic agent is not an PD-1 antagonist. Tire subject in need thereof preferably has a solid tumor that contains activated intratumoral effector T cells, preferably CD8+ T cells, prior to administration of the IL-2 prodrug. As disclosed further herein, the activated intratumoral effector T cells can be CD8+ T cells. The activated intratumoral effector T cells can be CD8+ GrB+ T cell. The activated intratumoral effector T cells (e.g., CD8+ T cells, CD8+GrB+ T cells) can alternatively be characterized by or be further characterized by a gene expression profile indicative of T cell activation. In some instances, the subject may have previously received standard of care treatment, including, for example, immune checkpoint inhibitors and been refractory to treatment or relapsed (e.g., primary or secondary resistance).

[0098]

[0091] The disclosure also relates to methods for treating cancer comprising i) identifying or selecting a subject with a solid tumor that contains activated intratumoral effector T cells (e.g., CD8+ T cells, CD8+GrB+ T cells) and ii) administering to the identified or selected subject an effective amount of an IL-2 prodrug. As disclosed further herein, the activated intratumoral effector T cells can be CD8+ T cells. The CD8+ T cells can be CD8+ GrB+ T cells. Hie activated intratumoral effector T cells (e.g., CD8+ T cells and / or CD 8+ GrB+ T cell) can be alternatively characterized or further characterized by a gene expression profile indicative of T cell activation. In some instances, the subject that is selected or identified may have previously received standard of care treatment, including, for example, immune checkpoint inhibitors and been refractory to treatment or relapsed (e.g., primary or secondary resistance).

[0099]

[0092] The actual effective amount of IL-2 prodrug that is administered will depend on the particular cancer being treated and its stage and other factors, such as tire subject’s age, gender, weight, ethnicity, prior treatments and response to those treatments and other factors.

[0100]

[0093] Suitable amounts of 1L-2 prodrug can be determined by a clinician of ordinary skill based on these and other considerations. For example, about 1 mg to about 240 mg of IL-2 prodrug can be administered about every two weeks, about every three weeks or about every four weeks.

[0101]

[0094] In preferred aspects the IL-2 prodrug is Compound 1, Compound 2, Compound 3 or Compound 4 or any combination thereof and can be administered to a subject in need thereof in the amount of about 3 mg to about 28 mg about every two weeks. For example, IL-2 prodrug (e.g.. Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14, mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg. about 27 mg, or about 28 mg about every two weeks. IL-2 prodrug (e.g.. Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14, mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, or about 28 mg about every three weeks. IL-2 prodrug (e.g., Compound 1, Compound 2. Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14, mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about

[0102] 27 mg, or about 28 mg about every four weeks.

[0103]

[0095] IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg to about 28 mg about every two weeks. IL-2 prodrug(e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg to about 28 mg about every three weeks. IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in tire amount of about 12 mg to about 28 mg about every four weeks.

[0104]

[0096] IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg, about 13 mg, about 14, mg. about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, or about 28 mg every two weeks. Typically, the IL-2 prodrug is administered to a subject in need thereof in an amount of about 12 mg every two weeks. Typically, the IL-2 prodrug is administered to a subject in need thereof in an amount of about 18 mg every two weeks. IL-2 prodrug (e.g., Compound 1. Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg, about 13 mg, about 14, mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, or about

[0105] 28 mg every three weeks. IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg, about 13 mg. about 14. mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, or about 28 mg every four weeks.

[0106]

[0097] IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg to about 23 mg about every two weeks. IL-2 prodrug (e.g.. Compound 1. Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg to about 23 mg about every three weeks. IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg to about 23 mg about every four weeks. For example, IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg, about 13 mg, about 14, mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg. about 21 mg. about 22 mg. about 23 mg every two weeks. IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg, about 13 mg, about 14, mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg every three weeks. IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) can be administered to a subject in need thereof in the amount of about 12 mg, about 13 mg, about 14, mg, about 15 mg, about 16 mg, about 17 mg. about 18 mg. about 19 mg. about 20 mg. about 21 mg, about 22 mg, about 23 mg every’ four weeks.

[0107]

[0098] The IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) typically administered systemically, for example by intravenous injection or preferably intravenous infusion. Other types of administration can be used, such as orally, parenterally, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, intrahepatically, intracranially, nebulization / inhalation, by installation via bronchoscopy, intra-articularly, or intratumorally.

[0108]

[0099] The IL-2 prodrug can be administered as a monotherapy (e.g., one or more additional agents are not administered). If desired, the methods can further comprising administering one or more additional agents to treat cancer, such as chemotherapeutic agents, immuno-oncology agents and immune checkpoint inhibitors, cellular therapies, oncolytic viruses, T cell engagers, other cytokines, angiogenesis inhibitors, antibody-drug conjugates, radiation therapy and / or small molecules (e.g.. kinase inhibitors) and the like as described further herein. When one or more additional agents are administered, they are administered in an effective amount, which will depend on the particular agent to be administered, the cancer being treated and its stage and other factors, such as the subject’s age, gender, weight, ethnicity, prior treatments and response to those treatments and other factors. Suitable amounts can be determined by a clinician of ordinary skill based on these and other considerations. The additional agent(s) can be administered in any suitable way such as locally or systemically, for example by intravenous injection, intravenous infusion, orally, parenterally, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, intrahepatically, intracranially, nebulization / inhalation, by installation via bronchoscopy, intra-articularly, or intratumorally. A clinician of ordinary skill can determine appropriate routes and administration. The IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3 or Compound 4) and additional agent(s) are administered to provide overlap in their phannacological activities in the subject under treatment. Accordingly, the IL-2 prodrug (e.g.. Compound 1, Compound 2, Compound 3 or Compound 4) can be administered before, after, concurrently with or periprocedurally with the additional agent(s).

[0109]

[0100] In exemplary embodiments, this disclosure relates to a method for treating cutaneous squamous cell carcinoma comprising administering IL-2 prodrug (e.g. Compound 1, Compound 2. Compound 3, Compound 4). and optionally an immune checkpoint inhibitor and / or standard of care therapy, wherein the tumor contains activated intratumoral T-cell effector cells as disclosed further herein. A method for treating cutaneous melanoma comprising administering IL-2 prodrug (e.g. Compound 1, Compound 2, Compound 3, Compound 4), and optionally an immune checkpoint inhibitor and / or standard of care therapy, wherein the tumor contains activated intratumoral T cell effector cells as disclosed further herein. A method for treating gastroesophageal junction adenocarcinoma comprising administering IL-2 prodrug (e.g., Compound 1, Compound 2, Compound 3, Compound 4), and optionally an immune checkpoint inhibitor and / or standard of care therapy, wherein the tumor contains activated intratumoral T cell effector cells as disclosed further herein.

[0110]

[0101] In embodiments, the method comprises administering an addition agent that is an immune checkpoint inhibitor, such as a PD-1 antagonist, a PD-L-1 antagonists, a CTLA4 antagonist, a LAG-3 antagonist or any combination thereof. In some embodiments, the immune checkpoint inhibitor is an anti- PD-L1 antibody, e.g., avelumab, durvalumab. atezolizumab. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody e.g., ipilimumab. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody e.g., pembrolizumab, dostarlimab, cemiplimab-rwlc, nivolumab, camrelizumab, tislelizumab, toripalimab, or sintilimab. In particular embodiments the immune checkpoint inhibitor is pembrolizumab.

[0111]

[0102] The disclosure relates to methods for treating cancer comprising administering to a subject in need there of a combination therapy that comprises an IL-2 prodrug (e.g.. Compound 1. Compound 2. Compound 3, Compound 4) and a PD-1 antagonist such as pembrolizumab or a pembrolizumab variant. As described herein, the subject in need thereof has a solid tumor that comprises activated intratumoral effector CD8+ T cells prior to administration of combination therapy. The IL-2 prodrug and the PD-1 antagonist, such as prembrolizumab are administered to the subject so that there is overlap of the pharmacological activities of the two therapeutic agents. Accordingly, the IL-2 prodrug can be administered before, after, concurrently, or periprocedurally with the PD-1 antagonist, such as pembrolizumab. In some practices of the methods, the PD-1 antagonist, such as pembrolizumab is administered before the IL-2 prodrug. In some practices of the methods, tire PD-1 antagonist, such as pembrolizumab, is administered after the IL-2 prodrug. In some particular practices of the methods, the PD-1 antagonist, such as pembrolizumab is administered, then about 30 minutes after the PD-1 antagonist, such as pembrolizumab administration is completed, the IL-2 prodrug is administered. For example, about 3 mg to about 18 mg of IL-2 prodrug (e.g. Compound 1, Compound 2, Compound 3, Compound 4) can be administered about every two weeks; and about 100 mg to about 600 mg of pembrolizumab or a pembrolizumab variant can be administered about every three to six weeks (e.g., 200 mg every three weeks (Q3W) or 400 mg every six weeks (Q6W)). For example, about 6 mg to about 28 mg of IL-2 prodrug (e.g. Compound 1, Compound 2, Compound 3, Compound 4) can be administered about every two weeks; and 200 mg or 400 mg of pembrolizumab or a pembrolizumab variant can be administered about every three to six weeks (e.g., 200 mg every? three weeks (Q3W) or 400 mg every six weeks (Q6W)). For example, about 6 mg to about 28 mg of IL-2 prodrug can be administered about every two weeks; and 200 mg or 400 mg of pembrolizumab or a pembrolizumab variant can be administered about every three to six weeks (e.g., 200 mg every three weeks (Q3W) or 400 mg every six weeks (Q6W)). For example, about 9 mg of IL-2 prodrug can be administered about every two weeks; and 200 mg or 400 mg of pembrolizumab or a pembrolizumab variant can be administered about every? three to six weeks (e.g., 200 mg every? three weeks (Q3W) or 400 mg every six weeks (Q6W)). For example, about 12 mg of IL-2 prodrug can be administered about every two weeks; and 200 mg or 400 mg of pembrolizumab or a pembrolizumab variant can be administered about every three to six weeks (e.g., 200 mg every three weeks (Q3W) or 400 mg every six weeks (Q6W)). For example, about 18 mg of IL-2 prodrug can be administered about every two weeks; and 200 mg or 400 mg of pembrolizumab or a pembrolizumab variant can be administered about every? three to six weeks (e.g., 200 mg every three weeks (Q3W) or 400 mg every six weeks (Q6W)). For example, about 23 mg of IL-2 prodrug can be administered about every? two weeks; and 200 mg or 400 mg of pembrolizumab or a pembrolizumab variant can be administered about every three to six weeks (e.g.. 200 mg every three weeks (Q3W) or 400 mg every six weeks (Q6W)). For example, about 28 mg of IL-2 prodrug can be administered about every two weeks; and 200 mg or 400 mg of pembrolizumab or a pembrolizumab variant can be administered about every three to six weeks (e.g., 200 mg every? three weeks (Q3W) or 400 mg every six weeks (Q6W)). Pediatric dosing can vary?. For example, for pediatric patients, pembrolizumab is typically administered at about 2 mg / kg, up to 200 mg, every three weeks.

[0112]

[0103] The methods and compositions of this disclosure can be administered to a subject in need thereof as first line therapy or subsequent to or in addition to other therapies. The subject can have a solid tumor that comprises activated intratumoral effector T cells (e.g., CD8+ T cells) prior to administration of IL-2 prodrug or combination of IL-2 prodrug and another therapeutic, for example an anti-PD-1 antibody. Tire subject in need of therapy according to this disclosure can be a subject who failed to achieve a complete response to prior treatment or ongoing treatment or failed to achieve a partial response to prior treatment or ongoing treatment. For example, the subject in need or therapy according to this disclosure can be a subject who failed to achieve a complete response, or a partial response, to prior or ongoing therapy that includes a checkpoint inhibitor. The subject in need of therapy according to this disclosure can be a subject who failed to achieve stable disease for longer than 6 months (SD>6) due to ongoing or prior therapy that included a checkpoint inhibitor. In embodiments, the subject failed to achieve a complete response, or a partial response, to prior or ongoing therapy with 1) an anti-PD-1 antibody, such as pembrolizumab (KEYTRUDA). dostarlimab (JEMPERLI), cemiplimab-rwlc (LIBATY 0), nivolumab (OPDIVO), camrelizumab, tislelizumab, toripalimab, and sintilimab (TYVYT); 2) and anti-PD-Ll antibody, such as avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ); or 3) an anti-CTLA-4 antibody, such as ipilimumab (YERVOY).

[0113]

[0104] The IL-2 prodrug and the PD-1 antagonist (e.g., pembrolizumab or a pembrolizumab variant) are typically administered systemically, for example by intravenous injection or preferably intravenous infusion. In some embodiments, the PD-1 antagonist is administered subcutaneously.

[0114]

[0105] In certain embodiments of the invention, the PD-1 antagonist in the combination therapy is an anti-PD-1 antibody, or antigen binding fragment thereof, as described herein. The anti-PD-1 antibody, or antigen binding fragment thereof, can be administered in a liquid medicament at a dose selected from the group consisting of 1 mg / kg Q2W, 2 mg / kg Q2W. 3 mg / kg Q2W, 5 mg / kg Q2W, 10 mg / kg Q2W, 1 mg / kg Q3W, 2 mg / kg Q3W, 3 mg / kg Q3W. 5 mg / kg Q3W, 10 mg / kg Q3W and flat-dose equivalents of any of these doses, i.e., such as 200 mg Q3W or 400 mg Q6W. In some embodiments. anti-PD-1 antibody, or antigen binding fragment thereof is provided as a liquid medicament which comprises 25 mg / ml of the anti-PD-1 antibody, or antigen binding fragment thereof, 7% (w / v) sucrose, 0.02% (w / v) polysorbate 80 in 10 mM histidine buffer pH 5.5. In other embodiments, the anti-PD-1 antibody, or antigen binding fragment thereof, is provided as a liquid medicament which comprises about 125 to about 200 mg / mL of the anti-PD-1 antibody, or antigen binding fragment thereof, about 10 mM histidine buffer; about 10 mM L-methionine. or a pharmaceutically acceptable salt thereof: about 7% (w / v) sucrose; and about 0.02 % (w / v) polysorbate 80. In one embodiment, the anti-PD-1 antibody, or antigen binding fragment thereof, comprises: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. In one embodiment, the anti-PD-1 antibody, or antigen binding fragment thereof, comprises (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO: 9 or a variant thereof. In one embodiment, the anti-PD-1 antibody comprises a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NOT, respectively. In one embodiment, the anti-PD- 1 antibody is pembrolizumab or a pembrolizumab variant. In one embodiment, the anti-PD-1 antibody is pembrolizumab.

[0115]

[0106] In some embodiments, the selected dose of the anti-PD- 1 antibody, or antigen binding fragment thereof, is administered by IV infusion. In one embodiment, the selected dose of the anti-PD-1 antibody, or antigen binding fragment thereof, is administered by IV infusion over a time period of between 25 and 40 minutes, or about 30 minutes. In other embodiments, the selected dose of the anti-PD-1 antibody, or antigen binding fragment thereof, is administered subcutaneously. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11. 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. Tire anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein tire heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NO:5. respectively. Tire anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab.

[0116]

[0107] The selected dose of pembrolizumab can be administered by subcutaneous injection. The selected subcutaneous dose of pembrolizumab can be about 280 mg to about 450 mg to the patient once approximately every three weeks. Typically, pembrolizumab is administered subcutaneously once every' three weeks.

[0117]

[0108] The selected subcutaneous dose of pembrolizumab can be, for example, from 340 mg to 420 mg, from 345 mg to 415 mg. from 350 mg to 410 mg, from 355 mg to 405 mg, from 360 mg to 400 mg, from 365 mg to 395 mg, from 370 mg to 390 mg. from 375 mg to 385 mg, or from 379 to 381 mg. In alternative embodiments, the dosage can be 360 mg, 365 mg, 370 mg, 375 mg, 379 mg, 379.5 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg, 415 mg, or 420 mg.

[0118]

[0109] Tire amount of pembrolizumab administered subcutaneously to the patient can be from about 280 mg to about 450 mg. Tire amount of pembrolizumab administered subcutaneously to the patient can be from about 280 mg to about 450 mg. The amount of pembrolizumab can be from about 300 mg to about 450 mg. The amount of pembrolizumab can be about 320 mg to about 450 mg. The amount of pembrolizumab can be about 340 mg to about 450 mg. The amount of pembrolizumab can be about 360 mg to about 450 mg. The amount of pembrolizumab is about 370 mg to about 450 mg. The amount of pembrolizumab can about 375 mg to about 450 mg. The amount of pembrolizumab can be about 300 mg to about 430 mg. The amount of pembrolizumab can be about 320 mg to about 430 mg. The amount of pembrolizumab can be about 340 mg to about 430 mg. The amount of pembrolizumab can about 360 mg to about 430 mg. The amount of pembrolizumab can be about 370 mg to about 430 mg. The amount of pembrolizumab can be about 375 mg to about 430 mg. The amount of pembrolizumab can about 320 mg to about 420 mg. Tire amount of pembrolizumab can be about 340 mg to about 420 mg. The amount of pembrolizumab can be about 360 mg to about 420 mg. The amount of pembrolizumab can be about 370 mg to about 420 mg. The amount of pembrolizumab can be about 345 mg to about 415 mg. The amount of pembrolizumab can be about 300 mg to about 410 mg. The amount of pembrolizumab can be about 320 mg to about 410 mg. The amount of pembrolizumab can be about 340 mg to about 410 mg. The amount of pembrolizumab can about 350 mg to about 410 mg. The amount of pembrolizumab can be about 360 mg to about 410 mg. Tire amount of pembrolizumab can be about 370 mg to about 410 mg. The amount of pembrolizumab can be about 375 mg to about 410 mg. Hie amount of pembrolizumab can be about 355 mg to about 405 mg. The amount of pembrolizumab can be about 360 mg to about 400 mg. The amount of pembrolizumab can be about 365 mg to about 395 mg. The amount of pembrolizumab can be about 300 mg to about 390 mg. The amount of pembrolizumab can be about 320 mg to about 390 mg. The amount of pembrolizumab can be about 340 mg to about 390 mg. The amount of pembrolizumab can be about 360 mg to about 390 mg. The amount of pembrolizumab can be about 370 mg to about 390 mg. The amount of pembrolizumab can be about 375 mg to about 390 mg. The amount of pembrolizumab can be about 365 mg to about 395 mg. The amount of pembrolizumab can be about 375 mg to about 385 mg. The amount of pembrolizumab can be about 379 mg to about 381 mg. The amount of pembrolizumab can be about 380 mg. In further embodiments, the amount of pembrolizumab is 380 mg.

[0119] [HO] In tire methods disclosed herein, pembrolizumab can be administered as a composition comprising pembrolizumab. For example, the composition comprising pembrolizumab can be a liquid composition as described in International Publication No. WO 2018 / 204368, the contents of which are hereby incorporated by reference. Tire composition can comprise 130 mg / ml of pembrolizumab. The composition comprises 165 mg / ml of the pembrolizumab.

[0120] [Hl] In some embodiments, the patient is treated with the combination therapy for at least 24 weeks, e.g., eight 3-week cycles. In some embodiments, treatment with the combination therapy continues until the patient exhibits evidence of PD effect, or a CR, or progressive disease.

[0121]

[0112] Hie methods and compositions disclosed herein can be used to treat any suitable cancer, in particular solid tumors, such as sarcomas and carcinomas. For examples, the methods and compositions disclosed herein can be used to treat adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, squamous cell carcinoma, adenocarcinoma, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary' gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, non-Hodgkin lymphoma, squamous carcinoma of the head and neck, malignant pleural mesothelioma, and Wilms tumor.

[0122]

[0113] Preferably, the methods and compositions disclosed herein are used to colon cancer, lung cancer, renal cell carcinoma, breast cancer, melanoma, squamous cell carcinoma, or adenocarcinoma, wherein the melanoma comprises cutaneous melanoma, wherein the squamous cell carcinoma comprises cutaneous squamous cell carcinoma, wherein the adenocarcinoma comprises gastroesophageal junction adenocarcinoma.

[0123]

[0114] In certain embodiments, the methods and compositions disclosed herein are used to treat melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell cancer (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability high or mismatch repair deficient cancer, microsatellite instability high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC). endometrial carcinoma, tumor mutational burden high cancer, cutaneous squamous cell carcinoma (cSCC), triple negative breast cancer (TNBC), urothelial carcinoma, colorectal cancer or oesophageal carcinoma.

[0124]

[0115] Any of the methods and compositions disclosed herein can be used to treat a human subject who has a cancer that tests positive for one or both of PD-L1 and PD-L2. and preferably tests positive for PD- LI expression. In some embodiments, PD-L1 expression is detected using a diagnostic anti -human PD-L1 antibody, or antigen binding fragment thereof, in an IHC assay on an FFPE or frozen tissue section of a tumor sample removed from tire patient. Typically, the subject’s physician would order a diagnostic test to determine PD-L1 expression in a tumor tissue sample removed from the patient prior to initiation of treatment with the PD-1 antagonist but it is envisioned that the physician could order the first or subsequent diagnostic tests at any time after initiation of treatment, such as for example after completion of a treatment cycle. In one embodiment, the PD-L1 expression is measured by the PD-L1 IHC 22C3 pharmDx assay. In another embodiment, the patient has a Mononuclear Inflammatory Density Score for PD-L1 expression >2. In another embodiment, the subject has a Mononuclear Inflammatory Density Score for PD-L1 expression >3. In another embodiment, the subject has a Mononuclear Inflammatory Density Score for PD-L1 expression >4. In another embodiment, tire subject has a Tumor Proportion Score for PD-L1 expression >1%. In another embodiment, the subject has a Tumor Proportion Score for PD-L1 expression >10%. In another embodiment, the subject has a Tumor Proportion Score for PD-L1 expression >20%. In another embodiment, tire subject has a Tumor Proportion Score for PD-L1 expression >30%. In another embodiment, tire subject has a Tumor Proportion Score for PD-L1 expression >40%. In another embodiment, the subject has a Tumor Proportion Score for PD-L1 expression >50%. In a further embodiment, the subject has a Combined Positive Score for PD-L1 expression >1%. In a further embodiment, the subject has a Combined Positive Score for PD-L1 expression between 1 and 20 %. In a further embodiment, the subject has a Combined Positive Score for PD-L1 expression > 2%. In a further embodiment, the subject has a Combined Positive Score for PD-L1 expression > 5%. In yet a further embodiment, the subject has a Combined Positive Score for PD-L1 expression > 10%. In a further embodiment, the subject has a Combined Positive Score for PD-L1 expression > 15%. In yet a further embodiment, the subject has a Combined Positive Score for PD-L1 expression > 20%.

[0125]

[0116] In certain embodiments, the methods and compositions disclosed herein are used to treat melanoma. As an example, the methods and compositions disclosed herein can be used to treat melanoma in subjects with unresectable or metastatic melanoma. As another example, the methods and compositions disclosed herein can be used for the adjuvant treatment of subjects with melanoma with involvement of lymph node(s) following complete resection.

[0126]

[0117] In certain embodiments, the methods and compositions disclosed herein are used to treat nonsmall cell lung cancer (NSCLC). As an example, the methods and compositions disclosed herein can be used to treat NSCLC in subjects with NSCLC expressing PD-L1 (e.g., Tumor Proportion Score (TPS) >1%) as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is: stage III where subjects are not candidates for surgical resection or definitive chemoradiation, or metastatic. As another example, the methods and compositions disclosed herein can be used to treat NSCLC in patients with metastatic NSCLC whose tumors express PD-L1 (TPS >1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. As another example, the methods and compositions disclosed herein can be used in combination with pemetrexed and platinum chemotherapy, as first-line treatment of patients with metastatic nonsquamous NSCLC, with no EGFR or ALK genomic tumor aberrations. As another example, the methods and compositions disclosed herein can be used in combination with carboplatin and either paclitaxel or paclitaxel proteinbound, as first-line treatment of patients with metastatic squamous NSCLC.

[0127]

[0118] In certain embodiments, tire methods and compositions disclosed herein are used to treat SCLC. As an example, the methods and compositions disclosed herein can be used to treat SCLC in subjects with metastatic SCLC with disease progression on or after platinum-based chemotherapy and at least one other prior line of therapy.

[0128]

[0119] In certain embodiments, the methods and compositions disclosed herein are used to treat HNSCC. As an example, the methods and compositions disclosed herein can be used to treat HNSCC in subjects with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) >1) as determined by an FDA-approved test. As another example, the methods and compositions disclosed herein can be used to treat HNSCC in subjects with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy. As another example, the methods and compositions disclosed herein can be used in combination with platinum and fluorouracil for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC.

[0129]

[0120] In certain embodiments, tire methods and compositions disclosed herein are used to treat cHL. As an example, the methods and compositions disclosed herein can be used to treat cHL in subjects with relapsed or refractory cHL. As another example, the methods and compositions disclosed herein can be used to treat cHL in pediatric subjects with refractory cHL, or cHL that has relapsed after 2 or more lines of therapy.

[0130]

[0121] In certain embodiments, the methods and compositions disclosed herein are used to treat PMBCL. As an example, the methods and compositions disclosed herein can be used to treat PMBCL in subjects with refractory PMBCL, or in subjects who have relapsed after 2 or more prior lines of therapy.

[0131]

[0122] In certain embodiments, the methods and compositions disclosed herein are used to treat urothelial carcinoma. As an example, the methods and compositions disclosed herein can be used to treat urothelial carcinoma in subjects with locally advanced or metastatic urothelial carcinoma who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) >10) as determined by an FDA-approved test, or in subjects who arc not eligible for any platinum -containing chemotherapy regardless of PD-L1 status. As another example, the methods and compositions disclosed herein can be used to treat urothelial carcinoma in subjects with locally advanced or metastatic urothelial carcinoma who have disease progression during or following platinum -containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum -containing chemotherapy. As another example, the methods and compositions disclosed herein can be used to treat urothelial carcinoma in subjects with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, nonmuscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

[0132]

[0123] In certain embodiments, the methods and compositions disclosed herein are used to treat Microsatellite Instability-High (MSI-H) or Mismatch Repair Deficient (dMMR) Cancer. As an example, the methods and compositions disclosed herein can be used to treat MSI-H or dMMR cancer in subjects with unresectable or metastatic MSI-H or dMMR cancer wherein the solid tumors have progressed following prior treatment and the subject has no satisfactory alternative treatment options, or wherein the colorectal cancer has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.

[0133]

[0124] In certain embodiments, the methods and compositions disclosed herein are used to treat Microsatellite Instability-High (MSI-H) or Mismatch Repair Deficient (dMMR) Colorectal Cancer. As an example, the methods and compositions disclosed herein can be used to treat MSI-H or dMMR colorectal cancer in subjects with unresectable or metastatic MSI-H or dMMR colorectal cancer.

[0134]

[0125] In certain embodiments, the methods and compositions disclosed herein are used to treat gastric cancer. As an example, the methods and compositions disclosed herein can be used to treat gastric cancer in subjects with recurrent locally advanced or metastatic gastric or gastroesophageal junction adenocarcinoma whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) >1) as determined by an FDA-approved test, with disease progression on or after 2 or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2 / neu-targeted therapy.

[0135]

[0126] In certain embodiments, the methods and compositions disclosed herein are used to treat esophageal cancer. As an example, the methods and compositions disclosed herein can be used to treat esophageal cancer in subjects with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) (e.g., tumors with epicenter 1 to 5 centimeters above the GEJ) carcinoma that is not amenable to surgical resection or definitive chemoradiation, in combination with platinum- and fluoropyrimidine-based chemotherapy. As another example, the methods and compositions disclosed herein can be used to treat esophageal cancer in subjects with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) (e.g., tumors with epicenter 1 to 5 centimeters above the GEJ) carcinoma that is not amenable to surgical resection or definitive chemoradiation, after one or more prior lines of systemic therapy for patients with tumors of squamous cell histology that express PD-L1 (CPS >10) as determined by an FDA -approved test.

[0127] In certain embodiments, the methods and compositions disclosed herein are used to treat cervical cancer. As an example, the methods and compositions disclosed herein can be used to treat cervical cancer in subjects with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) >1) as detennined by an FDA-approved test.

[0136]

[0128] In certain embodiments, the methods and compositions disclosed herein are used to treat HCC. As an example, the methods and compositions disclosed herein can be used to treat HCC in subjects who have been previously treated with sorafenib.

[0137]

[0129] In certain embodiments, tire methods and compositions disclosed herein are used to treat MCC. As an example, the methods and compositions disclosed herein can be used to treat MCC in subjects with recurrent locally advanced or metastatic MCC.

[0138]

[0130] In certain embodiments, the methods and compositions disclosed herein are used to treat RCC. As an example, the methods and compositions disclosed herein can be used in combination with axitinib, for the first-line treatment of patients with advanced RCC.

[0139]

[0131] In certain embodiments, the methods and compositions disclosed herein are used to treat endometrial carcinoma. As an example, the methods and compositions disclosed herein can be used in combination with lenvatinib, for the treatment of subjects with advanced endometrial carcinoma that is not MSI-H or dMMR. who have disease progression following prior systemic therapy and are not candidates for curative surgery or radiation.

[0140]

[0132] In certain embodiments, tire methods and compositions disclosed herein are used to treat Tumor Mutational Burden-High (TMB-H) Cancer. As an example, the methods and compositions disclosed herein can be used to treat TMB-H cancer in subjects with unresectable or metastatic tumor mutational burden-high (e.g., >10 mutations / megabase (mut / Mb)) solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options.

[0141]

[0133] In certain embodiments, the methods and compositions disclosed herein are used to treat Cutaneous Squamous Cell Carcinoma (cSCC). As an example, the methods and compositions disclosed herein can be used to treat cSCC in subjects with recurrent or metastatic cutaneous squamous cell carcinoma that is not curable by surgery or radiation.

[0142]

[0134] In certain embodiments, the methods and compositions disclosed herein are used to treat TripleNegative Breast Cancer (TNBC). As an example, the methods and compositions disclosed herein can be used in combination with chemotherapy, for tire treatment of subjects with locally recurrent unresectable or metastatic TNBC whose tumors express PD-L1 (e.g., Combined Positive Score (CPS) >10) as determined by an FDA approved test.

[0135] The cancer to be treated using the methods and compositions of this disclosure can be metastatic cancer.

[0143]

[0136] In certain embodiments, the methods and compositions disclosed herein are used to treat metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma.

[0144]

[0137] In embodiments of the method, compound 1, 2, 3 or 4 or an amino acid sequence variant thereof is administered to the subject with cancer in an amount of about 3 mg to about 28 mg every two weeks or every three weeks, and pembrolizumab (or a pembrolizumab variant or biosimilar) is administered in an amount of 200 mg about every' three weeks (Q3W) or 400 mg about every' six weeks (Q6W). In embodiments of the method, compound 1, 2, 3 or 4 or an amino acid sequence variant thereof is administered to the subject with cancer in an amount of about 6 mg every two weeks or every three weeks, and pembrolizumab (or a pembrolizumab variant or biosimilar) is administered in an amount of 200 mg about every three weeks (Q3W) or 400 mg about every six weeks (Q6W).

[0145]

[0138] Compounds 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 9 mg every? two weeks or every' three weeks, and pembrolizumab (or a pembrolizumab variant or biosimilar) is administered in an amount of 200 mg about every three weeks (Q3W) or 400 mg about every six weeks (Q6W). Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 12 mg every two weeks or every three weeks, and pembrolizumab (or a pembrolizumab variant or biosimilar) is administered in an amount of 200 mg about every three weeks (Q3W) or 400 mg about every six weeks (Q6W). Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof is administered to the subject with cancer in an amount of about 18 mg every' two weeks or every three weeks, and pembrolizumab (or a pembrolizumab variant or biosimilar) is administered in an amount of 200 mg about every three weeks (Q3W) or 400 mg about every six weeks (Q6W). Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 23 mg every two weeks or every three weeks, and pembrolizumab (or a pembrolizumab variant or biosimilar) is administered in an amount of 200 mg about every' three weeks (Q3W) or 400 mg about every six weeks (Q6W). Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 28 mg every two weeks or every three weeks, and pembrolizumab (or a pembrolizumab variant or biosimilar) is administered in an amount of 200 mg about every three weeks (Q3W) or 400 mg about every six weeks (Q6W).

[0146]

[0139] Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 1 mg to about 28 mg every two weeks or every' three weeks, and an anti-PD-1 antibody, or antigen binding fragment thereof is administered in an amount of 200 mg about every three weeks or 400 mg about every six weeks to treat cancer or a tumor as disclosed here. such as metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NON or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NOT, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0147]

[0140] Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 1 mg to about 18 mg (e g. 1 mg, 2 mg, 3 mg, 4 mg, 5 mg. 6 mg, 7 mg, 8 mg, 9 mg. 10 mg, 11 mg, 12 mg, 13 mg, 14 mg. 15 mg, 16 mg, 17 mg, or 18 mg) every two weeks or every three weeks, and an anti-PD-1 antibody, such as pembrolizumab, or antigen binding fragment thereof, is administered in an amount of 200 mg about every three weeks (Q3W) or 400 mg about every six weeks (Q6W) to treat cancer or a tumor as disclosed here, such as metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NON or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NOT, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. Hie anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0148]

[0141] Compound 1. 2. 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 6 mg every two weeks or every three weeks, and an anti-PD-1 antibody, such as pembrolizumab, or antigen binding fragment thereof, is administered in an amount of 200 mg about every’ three weeks or 400 mg about every’ six weeks to treat cancer or a tumor as disclosed here, such as metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma. The anti- PD-1 antibody , or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody , or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NON or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NOT, respectively . The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0149]

[0142] Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 9 mg every tw o weeks or every three weeks, and an anti-PD-1 antibody, such as pembrolizumab, or antigen binding fragment thereof, is administered in an amount of 200 mg about every three weeks or 400 mg about every six weeks to treat cancer or a tumor as disclosed here, such as metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma. The anti- PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO:5, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0150]

[0143] Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 12 mg every two weeks or every three weeks, and an anti-PD-1 antibody, such as pembrolizumab. or antigen binding fragment thereof, is administered in an amount of 200 mg every about three weeks or 400 mg about every six weeks to treat cancer or a tumor as disclosed here, such as metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma. The anti- PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. Hie anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO:5, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. Tire anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0151]

[0144] Compound 1, 2. 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 18 mg every two weeks or every three weeks, and an anti-PD-1 antibody, or antigen binding fragment thereof, is administered in an amount of 200 mg about every three weeks or 400 mg about every six weeks to treat cancer or a tumor as disclosed here, such as metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NO:5, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. Hie anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0152]

[0145] Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 23 mg every two weeks or every three weeks, and an anti-PD-1 antibody, or antigen binding fragment thereof, is administered in an amount of 200 mg about every three weeks or 400 mg about every six weeks to treat cancer or a tumor as disclosed here, such as metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma. The anti-PD- 1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NOT. respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0153]

[0146] Compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 28 mg every two weeks or every three weeks, and an anti-PD-1 antibody, or antigen binding fragment thereof, is administered in an amount of 200 mg about every three weeks or 400 mg about every six weeks to treat cancer or a tumor as disclosed here, such as metastatic renal clear cell carcinoma or metastatic cutaneous malignant melanoma. The anti-PD- 1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NOT, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0154]

[0147] For example, a method for treating metastatic nonsquamous non-small cell lung cancer can comprise administering an IL-2 prodrug as disclosed herein (e.g., Compound 1, Compound 2, Compound 3, Compound 4), an anti-PD-1 antibody (or antigen binding fragment thereof), pemetrexed and a platinum chemotherapeutic agent. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. Tire anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO:5, respectively. Tire anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti- PD-1 antibody can be pembrolizumab (or a biosimilar).

[0155]

[0148] In another example, a method for treating metastatic squamous non-small cell lung cancer can comprise administering IL-2 prodrug as disclosed herein (e.g., Compound 1, Compound 2, Compound 3, Compound 4), an anti-PD-1 antibody (or antigen binding fragment thereof), and paclitaxel. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NOT, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0156]

[0149] In another example, a method for treating metastatic squamous non-small cell lung cancer can comprise administering IL-2 prodrug as disclosed herein (e.g., Compound 1, Compounds 2, Compound 3. Compound 4). an anti-PD-1 antibody (or antigen binding fragment thereof), and protein bound paclitaxel. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD- 1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NOT. respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0157]

[0150] In another example, a method for treating metastatic or with unrcscctablc, recurrent head and neck squamous cell cancer can comprise administering IL-2 prodrug as disclosed herein (e.g., Compound 1, Compound 2, Compound 3, Compound 4), an anti-PD-1 antibody (or antigen binding fragment thereof), and fluorouracil. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. Tire anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO:5, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti- PD-1 antibody can be pembrolizumab (or a biosimilar).

[0158]

[0151] In another example, a method for treating locally advanced or metastatic esophageal or gastroesophageal junction carcinoma that is not amenable to surgical resection or definitive chemoradiation comprises administering IL-2 prodrug as disclosed herein (e.g. Compound 1, Compound

[0159] 2, Compound 3, Compound 4), an anti-PD-1 antibody (or antigen binding fragment thereof), and a platinum- and fluoropyrimidine-based chemotherapeutic agent. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11. 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NO:5, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0160]

[0152] In another example, a method for treating gastroesophageal junction adenocarcinoma comprises administering IL-2 prodrug as disclosed herein (e.g., Compound 1, Compound 2, Compound 3, Compound 4), an anti-PD-1 antibody (or antigen binding fragment thereof), and a platinum- and fluoropyrimidine-based chemotherapeutic agent. The anti-PD- 1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. Hie anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO: 9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NOT, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0161]

[0153] In another example, a method for treating advanced or metastatic melanoma for example, cutaneous melanoma, comprises administering IL-2 prodrug as disclosed herein (e.g., Compound 1, Compound 2, Compound 3, Compound 4), an anti-PD-1 antibody (or antigen binding fragment thereof), and optionally nivolumab. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. Tire anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO:5, respectively. Tire anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti- PD-1 antibody can be pembrolizumab (or a biosimilar).

[0162]

[0154] In another example, a method for treating advanced renal cell carcinoma comprises administering IL-2 prodrug as disclosed herein (e.g.. Compound 1, Compound 2, Compound 3, Compound 4). an anti- PD-1 antibody (or antigen binding fragment thereof), and axitinib. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. Tire anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO: 9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO:5, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0163]

[0155] In another example, a method for treating renal cell carcinoma comprises administering compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 6 mg to about 28 mg (e.g.. about 9 mg, about 12 mg, about 18 mg. about 23 mg, 28 mg) every two weeks or every three weeks, and an anti-PD-1 antibody, or antigen binding fragment thereof, is administered in an amount of 200 mg about every three weeks or 400 mg about every six weeks, and axitinib administered in an amount of 5 mg orally twice daily. Tire anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NO:5, respectively. Tire anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. Tire anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0164]

[0156] In another example, a method for treating advanced endometrial carcinoma that is not microsatellite instability -high or mismatch repair deficient, in subjects who have disease progression following prior systemic therapy and are not candidates for curative surgery or radiation, comprises administering IL-2 prodrug as disclosed herein (e.g. Compound 1. Compound 2, Compound 3, Compound 4). an anti-PD-1 antibody (or antigen binding fragment thereof), and lenvatinib. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. Tire anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NOTO and SEQ ID NOT, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. Tire anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0165]

[0157] In another example, a method for treating endometrial carcinoma or renal cell carcinoma comprises administering compound 1, 2, 3 or 4 or an amino acid sequence variant thereof can be administered to the subject with cancer in an amount of about 6 mg to about 28 mg (e g., about 9 mg, about 12 mg, about 18 mg, about 23 mg, 28 mg) even’ two weeks or every three weeks, and an anti-PD-1 antibody, or antigen binding fragment thereof, is administered in an amount of 200 mg about every three weeks or 400 mg about every six weeks, and lenvatinib administered in an amount of 20 mg orally once daily. Tire anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD- 1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NOT, respectively. Tire anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0166]

[0158] In another example, a method for treating locally recurrent unresectable or metastatic triplenegative breast cancer whose tumors express PD-L1 comprises administering IL-2 prodrug as disclosed herein (e.g., Compound 1, Compound 2, Compound 3, Compound 4), an anti-PD-1 antibody (or antigen binding fragment thereof), and a chemotherapeutic agent. Tire anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO:9 or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein the heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO:5, respectively. Tire anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. Hie anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0167] E. Pharmaceutical Compositions

[0168]

[0159] This disclosure also relates to pharmaceutical compositions for use in such methods, compositions which may also be referred to as medicaments. This disclosure provides pharmaceutical compositions comprising a PD-1 antagonist for use in combination with an IL-2 prodrug (e.g. Compound

[0169] 1, Compound 2, Compound 3. Compound 4), pharmaceutical compositions comprising an IL-2 prodrug (e.g. Compound 1, Compound 2, Compound 3, Compound 4) for use in combination with a PD-1 antagonist, and pharmaceutical compositions that contain an IL-2 prodrug (e.g. Compound 1, Compound

[0170] 2, Compound 3, Compound 4) in combination with the PD-1 antagonist, preferably prembrolizumab or a prembrolizumab variant.

[0171]

[0160] The pharmaceutical compositions can take a variety of forms, e g., liquid, lyophilized, and typically contain a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers (or excipients) are the non-active ingredient components of the pharmaceutical composition and are not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical formulation or composition in which it is contained. Carriers are frequently selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.

[0172]

[0161] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy. 21stEdition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Examples of the pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides. Matrices are in tire form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of the chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides to humans or other subjects.

[0173]

[0162] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and tire like. Preservatives and other additives are optionally present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic, although the formulation can be hypertonic or hypotonic if desired. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5.

[0174]

[0163] Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like are optionally necessary or desirable.

[0175]

[0164] Compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders are optionally desirable.

[0176]

[0165] This disclosure also relates to a kit that includes a) a pharmaceutical composition that contains an IL-2 prodrug composition , for example as a liquid composition or a lyophilized composition, in a suitable container (e.g., a vial, bag or the like), and b) a composition comprising an anti-PD-1 antibody (or antigen binding fragment thereof), for example as a liquid composition or a lyophilized composition, in a suitable container (e.g., a vial, bag or the like). The kit can further include other components, such as sterile water or saline for reconstitution of lyophilized compositions. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise: (a) light chain CDRs SEQ ID NOs: 6, 7 and 8 and (b) heavy chain CDRs SEQ ID NOs: 11. 12 and 13. The anti-PD-1 antibody, or antigen binding fragment thereof, can comprise (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NON or a variant thereof. The anti-PD-1 antibody can comprise a heavy chain and a light chain, and wherein tire heavy and light chains comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO:5, respectively. The anti-PD-1 antibody can be pembrolizumab or a pembrolizumab variant. The anti-PD-1 antibody can be pembrolizumab (or a biosimilar).

[0177] F. Definitions and Abbreviations

[0178]

[0166] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein arc intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted. To the extent any material incorporated herein by reference is inconsistent with the express content of this disclosure, the express content controls. In this application, the use of tire singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless the context requires otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0179]

[0167] Unless otherwise indicated, the terms "at least," "less than," and "about." or similar terms preceding a series of elements, or a range are to be understood to refer to every element in the series or range. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0180]

[0168] Reference in the specification to “some embodiments”, “an embodiment”, “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the inventions. “Comprising” or variations such as “comprise”, “comprises” or “comprised of’ are used throughout the specification and claims in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features that may materially enhance the operation or utility of any of the embodiments of the invention, unless the conte xt requires otherwise due to express language or necessary implication.

[0181]

[0169] ‘ ’Attenuated” activity, means that biological activity and typically IL-2 receptor agonist activity is decreased as compared to the activity of natural IL-2. The IL-2 prodrugs disclosed herein have attenuated IL-2 receptor agonists activity, which is at least about 10X, at least about 50X, at least about 100X, at least about 250X, at least about 500X, at least about 1000X or less agonist activity as compared to natural IL-2. Upon cleavage in the tumor microenvironment, IL-2 is release that is active. Typically, tire IL-2 that is released has IL-2 receptor agonist activity that is at least about 10X, at least about 50X, at least about 100X, at least about 250X, at least about 500X, or at least about lOOOx greater than the IL-2 receptor activating activity of the prodrug.

[0182]

[0170] ‘‘Cytokine” is a well-known term of art that refers to any of a class of immunoregulatory proteins (such as interleukin or interferon) that are secreted by cells especially of the immune system and that are modulators of the immune system. Cytokine polypeptides that can be used in the fusion proteins disclosed herein include, but are not limited to transforming growth factors, such as TGF-a and TGF-|3 (e.g., TGFbetal, TGFbeta2, TGFbeta3); interferons, such as interferon-a, interferon-|3, interferon-y, interferon- kappa and interferon-omega; interleukins, such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-L5, IL-16, IL-17, IL-18. IL-21 and IL-25; tumor necrosis factors, such as tumor necrosis factor alpha and lymphotoxin; chemokines (e.g., C-X-C motif chemokine

[0183] 10 (CXCL10). CCL19, CCL20, CCL21), and granulocyte macrophage-colony stimulating factor (GM- CS), as well as fragments of such polypeptides that active the cognate receptors for the cytokine (i.e., functional fragments of the foregoing). ‘'Chemokine” is a term of art that refers to any of a family of small cytokines with the ability to induce directed chemotaxis in nearby responsive cells.

[0184]

[0171] As used herein, references to “decreasing”, “reducing”, or “inhibiting” include a change of at least about 10%. of at least about 20%, of at least about 30%, of at least about 40%, of at least about 50%. of at least about 60%, of at least about 70%. of at least about 80%, of at least about 90% or greater as compared to a suitable control level. Such terms can include but do not necessarily include complete elimination of a function or property, such as agonist activity.

[0185]

[0172] Tire term “effective amount,” as used herein, refers to the amount of an agent (e.g., an IL-2 prodrug) that is administered to achieve the desired effect under the conditions of administration, such an amount that reduces tumor size, reduces tumor burden, extends progression free survival or extends overall survival. The actual effective amount selected will depend on the particular cancer being treated and its stage and other factors, such as the subject’s age, gender, weight, ethnicity, prior treatments and response to those treatments and other factors. Suitable amounts of IL-2 prodrug to be administered, and dosage schedules for a particular patient can be determined by a clinician of ordinary' skill based on these and other considerations.

[0186]

[0173] The terms “immune-inflamed” and “inflamed immune” are terms of art that are understood bypersons of ordinary skill in the art to refer to tumor microenvironments that are characterized by intratumoral lymphocyte infdtration, typically? CD8+ T cells that display biomarkers (protein, expression) of activation. See, e.g., Sahu et al. Nat Commun 13, 5312 (2022); Zhao, et al. Sig Transduct Target Thcr., 6, 263 (2021); and Chcrkasscky et al.. Am J Cancer Res. 12(7):3099- 3110 (2022).

[0187]

[0174] As used herein, the tenns “peptide”, “poly peptide”, or “protein” are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.

[0188]

[0175] As used herein, the terms “prevent”, “preventing”, and “prevention” of a disease or disorder refers to an action, for example, administration of the chimeric polypeptide or nucleic acid sequence encoding the chimeric polypeptide, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder.

[0189]

[0176] Tire term “sequence variant” refers to an amino acid sequence of a polypeptide that has substantially similar biological activity as a reference polypeptide but differs in amino acid sequence or to the nucleotide sequence of a nucleic acid that has substantially similar biological activity (e.g., encodes a protein with substantially similar activity) as a reference sequence but differs in nucleotide sequence. Typically, the amino acid or nucleotide sequence of a “sequence variant” is highly similar (e.g. at least about 80% similar) to that of a reference sequence. Those of skill in the art readily understand how to determine the identity of two polypeptides or two nucleic acids. For example, the identity can be calculated after aligning the two sequences so that the identity is at its highest level over a defined number of nucleotides or amino acids. Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr.. Madison. Wis.), or by inspection.

[0190]

[0177] As used throughout, “subject” can be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. In some embodiments, the mammal is a human. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, “patient” or “subject” may be used interchangeably and can refer to a subject with a disease or disorder (e.g., cancer). The term patient or subject includes human and veterinary subjects. The term “subject in need thereof’ as used herein refers to a subject diagnosed with or suspected of having cancer or an infectious disease as defined herein.

[0191]

[0178] As used herein the terms “treatment”, “treat”, or “treating” refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus, in the disclosed methods, treatment can refer to at least about 10%, at least about 20%, at least about 30%, at least about 40%. at least about 50%. at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially complete reduction in the severity of an established disease or condition or symptom of the disease or condition, such as reduction in tumor volume, reduction in tumor burden, reduction in death. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%. 60%. 70%. 80%. 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0192]

[0179] Tire term “conservative amino acid substitution” is a term of art that refers to the replacement of an amino acid in a polypeptide with another amino acid that has similar biochemical properties, such as size, charge and hydrophobicity as a reference amino acid. It is well-known that conservative amino acid replacements in the amino acid sequence of a polypeptide frequently do not significantly alter the overall structure or function of the polypeptide. Exemplary conservative substitutions are set forth in Table 4 below.

[0193] Table 4. Exemplary Conservative Amino Acid Substitutions

[0194] 5. NUMBERED EMBODIMENTS

[0180] 1. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of an IL-2 prodrug, wherein the subject in need thereof has a solid tumor that comprises activated intratumoral effector CD8+ T cells prior to administration of the IL-2 prodrug.

[0195]

[0181] 2. Tire method of numbered embodiment 1, wherein the activated intratumoral effector CD 8+ T cells express granzyme B.

[0196]

[0182] 3. The method of numbered embodiment 1, wherein at least about 1000 activated intratumoral effector CD8+ T cells that express per mm2are present in the solid tumor as determined by immunofluorescence analysis.

[0197]

[0183] 4. Tire method of numbered embodiment 2, wherein at least about 50 activated intratumoral effector CD8+ T cells that express granzyme B per mm2are present in the solid tumor, e.g., as determined by immunofluorescence analysis.

[0198]

[0184]

[0199]

[0185] 5. The method of any one of numbered embodiments 1-4, wherein the solid tumor is further characterized by a gene expression signature indicative of activated T cells.

[0200]

[0186] 6. Tire method of numbered embodiment 4, wherein the gene expression signature indicative of activated T cells comprises an increase or decrease in the expression of at least one gene indicative of activated T cells in comparison to a suitable standard.

[0201]

[0187] 7. The method of numbered embodiment 6, wherein the increase or decrease in expression is statistically significant to at least p< 0.05 using Wald Test.

[0202]

[0188] 8. Tire method of numbered embodiments 6 or 7, wherein the suitable standard comprises a corresponding gene expression signature for a solid tumor that did not respond to treatment with IL-2 prodrug.

[0203]

[0189] 9. The method of any one of numbered embodiments 6-7, wherein the suitable standard comprises a gene expression signature standard compiled from gene expression signatures for tumors that did not respond to treatment with IL-2 prodrug.

[0204]

[0190] 10. Tire method of any one of numbered embodiments 5-9, wherein the gene expression signature indicative of activated T cells comprises expression data for at least one gene selected from the group consisting of IL-23A, GZMB, IL-4, IL-2RA, CCL3, TAPI, CCL2, CCL5, IL-7R, CCL11, CCL7. ITGAM, IL-2RG, LCP1, TNF-SF14, HAVCR2. TNF-SF13B. STAT1, IL-6, CD3D, ITGAL, ITGB2, LCK, CD4, IL-18. LAG3. ITGAX, CD274, TNF-RSF8. PSMB10, FUT7. IL-1B, ZAP70. TNF-SF4, CD74, MAF, SELL, IFN-B1, CD2, and IRF4.

[0205]

[0191] 11. Tire method of any one of numbered embodiments 5-10, wherein the gene expression signature indicative of T cell activation comprises expression data for CCL11, GZMB, TNF-SF13B, IRF1, IFNG, TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2, PSEN2, and SPP1.

[0192] 12. The method of any one of numbered embodiments 5-11, wherein the gene expression signature indicative of T cell activation comprises increased expression of at least one gene selected from the group consisting of CCL11, GZMB, TNF-SF13B, IRF1, and IFNG.

[0206]

[0193] 13. Tire method of any one of numbered embodiments 5-12, wherein the gene expression signature indicative of T cell activation comprises decreased expression of at least one gene selected from the group consisting of TRAF6, TLR4, CTSH, CXCR4. ITGAM, BCL2, PSEN2, and SPP1.

[0207]

[0194] 14. The method of any one of numbered embodiments 1-13, wherein the IL-2 prodrug compnses Compound 1 (SEQ ID NO: 1 / SEQ ID NO:5), Compound 2 (SEQ ID NO:2 / SEQ ID NO:5), Compound 3 (SEQ ID NO:3 / SEQ ID NO:5), Compound 4 (SEQ ID NO:4 / SEQ ID NO:5) or an amino acid sequence variant of any of the foregoing.

[0208]

[0195] 15. The method of any one of numbered embodiments 1-14, wherein the effective amount of the IL-2 prodrug is administered intravenously.

[0209]

[0196] 16. The method of numbered embodiment 15, wherein the effective amount of the IL-2 prodrug is administered by intravenous infusion.

[0210]

[0197] 17. Tire method of any one of numbered embodiments 1-16, wherein about 6 mg to about 28 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0211]

[0198] 18. The method of numbered embodiment 17, wherein about 12 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0212]

[0199] 19. Tire method of numbered embodiment 17, wherein about 18 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0213]

[0200] 20. The method of numbered embodiment 17, wherein about 23 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0214]

[0201] 21. Tire method of numbered embodiment 17, wherein about 28 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0215]

[0202] 22. The method of any one of numbered embodiments 1-21, further comprising administering to the subject an effective amount of another therapeutic agent.

[0216]

[0203] 23. Tire method of numbered embodiment 22, wherein the another therapeutic agent is the standard of care therapy for the solid tumor.

[0204] 24. The method of numbered embodiment 23, wherein the another therapeutic agent comprises an immune checkpoint inhibitor.

[0217]

[0205] 25. Tire method of numbered embodiment 24, wherein the immune checkpoint inhibitor binds to and inhibits a protein selected from the group consisting of PD-1, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (CD 152), B7-1 (CD80), B7-2 (CD86), LAG 3 (CD223); TIM3 (HAVCR2); TIGIT (VSTM3, WUCAM); BTLA (CD272) which binds HVEM (TNFRSF14), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55), CD134 (TNRFSR4, 0X40) and CD252 (OX-40L).

[0218]

[0206] 26. Tire method of numbered embodiment 24, wherein the immune checkpoint inhibitor is an anti-PD-Ll antibody.

[0219]

[0207] 27. The method of numbered embodiment 26, wherein the anti-PD-Ll antibody comprises avelumab. durvalumab, or atezolizumab.

[0220]

[0208] 28. The method of numbered embodiment 24, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[0221]

[0209] 29. Tire method of numbered embodiment 28, wherein the anti-CTLA-4 antibody comprises ipilimumab.

[0222]

[0210] 30. The method of numbered embodiment 24, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or antigen binding fragment thereof.

[0223]

[0211] 31. The method of numbered embodiment 30, wherein the anti-PD- 1 antibody comprises pembrolizumab, dostarlimab, cemiplimab-rwlc, nivolumab, camrelizumab, tislelizumab, toripalimab, or sintilimab.

[0224]

[0212] 32. The method of numbered embodiment 30, wherein tire anti-PD-1 antibody or antigen binding fragment thereof comprises: (a) light chain CDRs SEQ ID Nos: 6, 7, 8 and (b) heavy chain CDRs SEQ ID Nos: 11, 12, 13.

[0225]

[0213] 33. The method of numbered embodiment 30, wherein the anti-PD-1 antibody comprises: (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant hereof and (b) a light chain variable region comprising SEQ ID NO: 9 or a variant thereof.

[0226]

[0214] 34. The method of numbered embodiment 30, wherein the anti-PD-1 antibody comprises a heavy chain and a light chain, and wherein the heavy chain and light chain comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO: 15, respectively.

[0227]

[0215] 35. The method of numbered embodiment 30, wherein the anti-PD-1 antibody is pembrolizumab or a pembrolizumab variant.

[0228]

[0216] 36. Tire method of numbered embodiment 30, wherein the anti-PD-1 antibody is pembrolizumab (or a biosimilar).

[0217] 37. The method of any one of numbered embodiments 24-36, wherein about 100 mg to about 600 mg of the anti-PD-1 antibody or antigen binding fragment thereof, is administered about every three to six weeks.

[0229]

[0218] 38. Tire method of numbered embodiment 37, wherein 200 mg of tire anti-PD-1 antibody, or antigen binding fragment thereof, is administered about every three weeks.

[0230]

[0219] 39. The method of numbered embodiment 37, wherein 600 mg of the anti-PD-1 antibody, or antigen binding fragment thereof, is administered about every six weeks.

[0231]

[0220] 40. Tire method of any one of numbered embodiments 1-39, wherein the subject has failed to achieve a complete response to a prior treatment or to an ongoing treatment prior to administration of the IL-2 prodrug.

[0232]

[0221] 41. The method of numbered embodiment 40, wherein the prior treatment or ongoing treatment comprises treatment with an immune checkpoint inhibitor.

[0233]

[0222] 42. The method of numbered embodiment 41, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0234]

[0223] 43. Tire method of any one of numbered embodiments 1-42, wherein the cancer is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, squamous cell carcinoma, adenocarcinoma, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fiingoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary’ gland cancer, Sezary’ syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0235]

[0224] 44. Tire method of any one of numbered embodiments 1-42, wherein the cancer is colon cancer, lung cancer, renal cell carcinoma, breast cancer, melanoma, squamous cell carcinoma, or adenocarcinoma.

[0236]

[0225] 45. The method of numbered embodiment 44, wherein the melanoma comprises cutaneous melanoma.

[0237]

[0226] 46. Tire method of numbered embodiment 44, wherein the squamous cell carcinoma comprises cutaneous squamous cell carcinoma.

[0238]

[0227] 47. The method of numbered embodiment 44, wherein the adenocarcinoma comprises gastroesophageal junction adenocarcinoma.

[0239]

[0228] 48. A method for treating cancer, comprising a) identifying or selecting a subject with a solid tumor that comprises activated intratumoral effector CD8+ T cells prior to administration of the IL-2 prodrug, and b) administering to the subject an effective amount of an IL-2 prodrug.

[0240]

[0229] 49. The method of numbered embodiment 48, wherein tire activated intratumoral effector CD 8+ T cells express granzyme B.

[0241]

[0230] 50. The method of numbered embodiment 48, wherein at least about 1000 activated intratumoral effector CD8+ T cells that express per mm2are present in the solid tumor as determined by immunofluorescence analysis.

[0242]

[0231] 51. Tire method of numbered embodiment 49, wherein at least about 50 activated intratumoral effector CD8+ T cells that express granzyme B per mm2are present in the solid tumor, e g., as determined by immunofluorescence analysis.

[0243]

[0232] 52. The method of any one of numbered embodiment 48-51, wherein the solid tumor is further characterized by a gene expression signature indicative of activated T cells.

[0244]

[0233] 53. Tire method of numbered embodiment 51, wherein the gene expression signature indicative of activated T cells comprises an increase or decrease in the expression of at least one gene indicative of activated T cells in comparison to a suitable standard.

[0245]

[0234] 54. The method of numbered embodiment 53, wherein the increase or decrease in expression is statistically significant to at least p< 0.05 using Wald Test.

[0246]

[0235] 55. The method of numbered embodiments 53 or 54, wherein the suitable standard comprises a corresponding gene expression signature for a solid tumor that did not respond to treatment with IL-2 prodrug.

[0236] 56. The method of any one of numbered embodiments 53-54, wherein the suitable standard comprises a gene expression signature standard compiled from gene expression signatures for tumors that did not respond to treatment with IL-2 prodrug.

[0247]

[0237] 57. Tire method of any one of numbered embodiments 52-56, wherein the gene expression signature indicative of activated T cells comprises expression data for at least one gene selected from the group consisting of IL-23A. GZMB, IL-4. IL-2RA, CCL3, TAPI. CCL2, CCL5, IL-7R. CCL11, CCL7. ITGAM, IL-2RG, LCP1, TNF-SF14, HAVCR2, TNF-SF13B, STAT1, IL-6, CD3D, ITGAL, ITGB2, LCK, CD4, IL-18, LAG3, ITGAX, CD274, TNF-RSF8, PSMB10, FUT7, IL-1B, ZAP70, TNF-SF4, CD74, MAF, SELL, IFN-B1, CD2, and IRF4.

[0248]

[0238] 58. Tire method of any one of numbered embodiments 52-57, wherein the gene expression signature indicative of T cell activation comprises expression data for CCL11. GZMB, TNF-SF13B, IRF1, IFNG, TRAF6, TLR4, CTSH, CXCR4. ITGAM, BCL2, PSEN2, and SPP1.

[0249]

[0239] 59. The method of claim any one of numbered embodiments 52-58, wherein the gene expression signature indicative of T cell activation comprises increased expression of at least one gene selected from the group consisting of CCL11, GZMB, TNF-SF13B, IRF1, and IFNG.

[0250]

[0240] 60. The method of any one of numbered embodiments 52-59, wherein the gene expression signature indicative of T cell activation comprises decreased expression of at least one gene selected from the group consisting of TRAF6. TLR4, CTSH, CXCR4. ITGAM, BCL2, PSEN2, and SPP1.

[0251]

[0241] 61. The method of any one of numbered embodiments 48 - 60, wherein the IL-2 prodrug compnses Compound 1 (SEQ ID NO: 1 / SEQ ID NO:5), Compound 2 (SEQ ID NO:2 / SEQ ID NO:5), Compound 3 (SEQ ID NO:3 / SEQ ID NO:5), Compound 4 (SEQ ID NO:4 / SEQ ID NO:5) or an amino acid sequence variant of any of the foregoing.

[0252]

[0242] 62. The method of any one of numbered embodiments 48-61. wherein the effective amount of the IL-2 prodrug is administered intravenously.

[0253]

[0243] 63. The method of numbered embodiments 62, wherein the effective amount of the IL-2 prodrug is administered by intravenous infusion.

[0254]

[0244] 64. Tire method of any one of numbered embodiments 48-63, wherein about 6 mg to about 28 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0255]

[0245] 65. The method of numbered embodiment 64, wherein about 12 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0246] 66. The method of numbered embodiment 64, wherein about 18 mg of Compound 1,

[0256] Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0257]

[0247] 67. Tire method of numbered embodiment 64, w herein about 23 mg of Compound 1,

[0258] Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0259]

[0248] 68. The method of numbered embodiment 64, wherein about 28 mg of Compound 1,

[0260] Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

[0261]

[0249] 69. Tire method of any one of numbered embodiments 48-68, further comprising administering to the subject an effective amount of another therapeutic agent.

[0262]

[0250] 70. The method of numbered embodiment 69, wherein the another therapeutic agent is the standard of care therapy for the solid tumor.

[0263]

[0251] 71. Tire method of numbered embodiment 69, wherein the another therapeutic agent comprises an immune checkpoint inhibitor.

[0264]

[0252] 72. The method of numbered embodiment 71, wherein tire immune checkpoint inhibitor binds to and inhibits a protein selected from the group consisting of PD-1, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (CD152), B7-1 (CD80), B7-2 (CD86), LAG 3 (CD223); TIM3 (HAVCR2); TIGIT (VSTM3, WUCAM); BTLA (CD272) which binds HVEM (TNFRSF 14), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55), CD134 (TNRFSR4, 0X40) and CD252 (OX-40L).

[0265]

[0253] 73. The method of numbered embodiment 71, w herein tire immune checkpoint inhibitor is an anti-PD-Ll antibody.

[0266]

[0254] 74. The method of numbered embodiment 73, wherein the anti-PD-Ll antibody comprises avelumab, durvalumab, or atezolizumab.

[0267]

[0255] 75. Tire method of numbered embodiment 71, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[0268]

[0256] 76. The method of numbered embodiment 75, wherein the anti-CTLA-4 antibody comprises ipilimumab.

[0269]

[0257] 77. The method of numbered embodiment 71, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or antigen binding fragment thereof.

[0270]

[0258] 78. Tire method of numbered embodiment 77 wherein the anti-PD-1 antibody comprises pembrolizumab, dostarlimab, cemiplimab-rwlc, nivolumab, camrelizumab, tislelizumab, toripalimab, or sintilimab.

[0259] 79. The method of numbered embodiment 77, wherein the anti-PD-1 antibody or antigen binding fragment thereof comprises: (a) light chain CDRs SEQ ID Nos: 6, 7, 8 and (b) heavy chain CDRs SEQ ID Nos: 11, 12, 13.

[0271]

[0260] 80. Tire method of numbered embodiment 77 wherein the anti-PD-1 antibody comprises: (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant hereof and (b) a light chain variable region comprising SEQ ID NO: 9 or a variant thereof.

[0272]

[0261] 81. The method of numbered embodiment 77, wherein the anti-PD- 1 antibody comprises a heavy chain and a light chain, and wherein the heavy chain and light chain comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO: 15, respectively.

[0273]

[0262] 82. Tire method of numbered embodiment 77, wherein the anti-PD-1 antibody is pembrolizumab or a pembrolizumab variant.

[0274]

[0263] 83. The method of numbered embodiment 77, wherein the anti-PD-1 antibody is pembrolizumab (or a biosimilar).

[0275]

[0264] 84. Tire method of any one of numbered embodiments 71-83, wherein about 100 mg to about 600 mg of the anti-PD-1 antibody or antigen binding fragment thereof, is administered about every three to six weeks.

[0276]

[0265] 85. The method of numbered embodiment 84 wherein 200 mg of the anti-PD-1 antibody, or antigen binding fragment thereof, is administered about every three weeks.

[0277]

[0266] 86. The method of numbered embodiment 84, wherein 600 mg of the anti-PD-1 antibody, or antigen binding fragment thereof, is administered about every six weeks.

[0278]

[0267] 87. Tire method of any one of numbered embodiments 48-86, wherein the subject has failed to achieve a complete response to a prior treatment or to an ongoing treatment prior to administration of the IL-2 prodrug.

[0279]

[0268] 88. The method of numbered embodiment 87, wherein the prior treatment or ongoing treatment comprises treatment with an immune checkpoint inhibitor.

[0280]

[0269] 89. Tire method of numbered embodiment 88, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0281]

[0270] 90. The method of any one of numbered embodiments 48-89, wherein tire cancer is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, csthcsioncuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, hypophan ngcal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, lary ngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, squamous cell carcinoma, adenocarcinoma, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, orophary ngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[0282]

[0271] 91. The method of any one of numbered embodiments 48-49. wherein the cancer is colon cancer, lung cancer, renal cell carcinoma, breast cancer, melanoma, squamous cell carcinoma, or adenocarcinoma.

[0283]

[0272] 92. Tire method of numbered embodiment 91, wherein the melanoma comprises cutaneous melanoma.

[0284]

[0273] 93. The method of numbered embodiment 91, wherein the squamous cell carcinoma comprises cutaneous squamous cell carcinoma.

[0285]

[0274] 94. The method of numbered embodiment 91, wherein the adenocarcinoma comprises gastroesophageal junction adenocarcinoma.

[0286]

[0275] 95. A method for identifying a subject with a solid tumor that is likely to respond to therapy with an IL-2 prodrug, comprising analyzing a sample of a tumor obtained from the subject or the tumor in the subject for the presence of activated intratumoral effector CD8+ T cells, wherein the presence of activated intratumoral effector CD8+ T cells indicates that the subject is likely to respond to therapy with an IL-2 prodrug.

[0287]

[0276] 96. The method of numbered embodiment 95, wherein the activated intratumoral effector CD8+ T cells also express granzyme B.

[0277] 97. The method of numbered embodiment 95, wherein at least about 1000 activated intratumoral effector CD8+ T cells that express per mm2are present in the solid tumor as determined by immunofluorescence analysis.

[0288]

[0278] 98. Tire method of numbered embodiment 96, wherein at least about 50 activated intratumoral effector CD8+ T cells that express granzyme B per mm2are present in the solid tumor, e g., as determined by immunofluorescence analysis.

[0289]

[0279] 99. The method of any one of numbered embodiment 95-98, wherein the solid tumor is further characterized by a gene expression signature indicative of activated T cells.

[0290]

[0280] 100. Tire method of numbered embodiment 98, wherein the gene expression signature indicative of activated T cells comprises an increase or decrease in the expression of at least one gene indicative of activated T cells in comparison to a suitable standard.

[0291]

[0281] 101. The method of numbered embodiment 100. wherein the increase or decrease in expression is statistically significant to at least p< 0.05 using Wald Test.

[0292]

[0282] 102. Tire method of numbered embodiment 100 or 101, wherein the suitable standard comprises a corresponding gene expression signature for a solid tumor that did not respond to treatment with IL-2 prodrug.

[0293]

[0283] 103. The method of any one of numbered embodiments 100-101, wherein the suitable standard comprises a gene expression signature standard compiled from gene expression signatures for tumors that did not respond to treatment with IL-2 prodrug.

[0294]

[0284] 104. Tire method of any one of numbered embodiments 99-103, wherein the gene expression signature indicative of activated T cells comprises expression data for at least one gene selected from the group consisting of IL-23A, GZMB, IL-4, IL-2RA, CCL3, TAPI, CCL2, CCL5, IL-7R, CCL11, CCL7. ITGAM, IL-2RG, LCP1, TNF-SF14, HAVCR2. TNF-SF13B. STAT1, IL-6, CD3D, ITGAL, ITGB2, LCK, CD4, IL-18. LAG3. ITGAX, CD274. TNF-RSF8. PSMB10, FUT7. IL-1B, ZAP70. TNF-SF4, CD74, MAF, SELL, IFN-B1, CD2, and IRF4.

[0295]

[0285] 105. Tire method of any one of numbered embodiments 99-104, wherein the gene expression signature indicative of T cell activation comprises expression data for CCL11, GZMB, TNF-SF13B, IRF1, IFNG, TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2, PSEN2, and SPP1.

[0296]

[0286] 106. The method of claim any one of numbered embodiments 99-105, wherein the gene expression signature indicative of T cell activation comprises increased expression of at least one gene selected from the group consisting of CCL11, GZMB, TNF-SF13B, IRF1, and IFNG.

[0297]

[0287] 107. Tire method of any one of numbered embodiments 99-106, wherein the gene expression signature indicative of T cell activation comprises decreased expression of at least one gene selected from the group consisting of TRAF6, TLR4, CTSH, CXCR4, ITGAM, BCL2, PSEN2, and SPP1.

[0288] 108. The method of any one of numbered embodiments 95 - 107, wherein the IL-2 prodrug comprises Compound 1 (SEQ ID NO: 1 / SEQ ID NO:5), Compound 2 (SEQ ID NO:2 / SEQ ID NO:5), Compound 3 (SEQ ID NO:3 / SEQ ID NO:5), Compound 4 (SEQ ID NO:4 / SEQ ID NO:5) or an amino acid sequence variant of any of the foregoing.

[0298] 6. EQUIVALENTS

[0299]

[0289] It will be readily apparent to those skilled in the art that other suitable modifications and adaptions of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments. Having now described certain compounds and methods in detail, the same will be more clearly understood by reference to the following examples, which are introduced for illustration only and not intended to be limiting.

[0300] 7. EXAMPLES

[0301]

[0290] The present invention is further described by the following examples, w hich are not intended to be limiting in any way.

[0302] Example 1. A Multicenter Phase I / Ib Dose Escalation Study of IL-2 Prodrug as Monotherapy and in Combination with Pembrolizumab in Patients with Selected Advanced or Metastatic Solid Tumors

[0303] Table 5. List of Abbreviations and Definition of Terms

[0304]

[0305] 1.1 Introduction and Background

[0306] Interleukin-2

[0307]

[0291] Interleukin (IL)-2 is a pro-inflammatory cytokine which can drive the immune-mediated killing of cancer cells through tire proliferation and activation of T cells and natural killer (NK) cells and inducing the differentiation of cluster of differentiation (CD)8 cells into effector and memory cells. The IL-2 receptor (IL-2R) is composed of 3 subunits named IL 2Ra (CD25). IL-2RP (CD122), and IL-2 Ry (CD132). Binding to monomeric IL-2Ra does not induce signaling, while binding to the medium affinity dimeric receptor comprised of a complex of the and y subunits will induce signaling. The trimeric receptor composed of all 3 subunits is a high affinity receptor for IL-2, with binding affinity approximately 100-fold higher than the medium affinity receptor. Binding to the medium or high affinity IL-2R activates the Janus kinase / signal transducer and activator of transcription (STAT), mitogen- activated protein kinase (MAPK), and phosphoinositide 3 -kinase signaling pathways in target immune cells resulting in immune cell activation and proliferation.

[0308]

[0292] The medium affinity IL-2R0 / y is expressed on NK cells, monocytes, macrophages, and resting CD4+ and CD8+ T cells, while the high affinity IL-2Ra / 0 / y is transiently induced on activated T and NK cells and is constitutively expressed on CD4+FoxP3+ regulatory T cells (Tregs). Basal levels of IL-2 bind predominantly to high affinity IL-2Ra / 0 / y on Tregs to maintain immune homeostasis. IL-2 production during an immune response results in levels of IL-2 which can activate both the medium and high affinity receptors, resulting in the activation and proliferation of effector lymphocyte populations.

[0309]

[0293] Numerous preclinical studies have demonstrated that administration of IL-2 can be effective in eradicating tumors in mouse models. This concept has also been clinically validated with the approval in 1992 of recombinant IL-2 (rIL-2) therapy (aldesleukin) for renal cell carcinoma and in 1998 for metastatic melanoma. Aldesleukin has demonstrated complete cancer regression in about 10% of patients treated for metastatic melanoma and renal cancer. Unfortunately, rIL-2 has poor pharmacokinetic (PK) properties and dose limiting systemic toxicities due to binding to its high and medium affinity receptors in the periphery. High dose IL-2 administration results in severe hypotension and vascular leak syndrome (VLS), which has relegated its use to specialized care centers and limited its dosing to reach efficacious levels. Uiese side effects limit the number of patients who can tolerate the recommended therapeutic regimen and, consequently, achieve the full clinical benefit from IL-2 therapy. It has been postulated that another contributing factor limiting the clinical benefit of IL-2 is that by binding to the high-affinity IL- 2Ra / 0 / y, it induces the expansion of immunosuppressive Tregs, which can counteract anti-tumor immune responses.

[0310] IL-2 Prodrug

[0311]

[0294] The IL-2 prodrug is a systemically delivered, conditionally activated, form of interleukin 2 designed to minimize the severe toxicities observed with rIL-2 therapy and maximize clinical benefit when administered as monotherapy or in combination with immune checkpoint inhibitors in advanced or metastatic tumors.

[0312]

[0295] Tire IL-2 prodrug is engineered with a native IL-2 molecule attached via protease -clcavablc linkers to both an IL-2R0 / / blockade element to eliminate binding of tire IL-2 to IL-2R0 / y-expressing normal tissues in the periphen' and a half-life extension domain. The prodrug is conditionally activated in the tumor microenvironment through protease cleavage to release the fully active, native IL-2 cytokine within tire tumor to stimulate a potent anti -tumor immune response.

[0313]

[0296] Several companies are also developing IL-2 therapies designed to address the limitations of rIL-2 by engineering molecules that bind only to tire medium affinity receptor IL-2Rp / y and avoid binding to the high affinity receptor IL-2Ra / (3 / y, so called "non -alpha" molecules, in the hope of alleviating toxicities and reducing activation of Tregs. However, many of these molecules activate IL-2Rp / y receptors in the periphery (due to lack of an IL-2Rp / y blockade element) and are also attenuated in inducing newly primed T cell proliferation in the tumor microenvironment (TME) due to their reduced IL- 2Ra binding, which may limit their ability to reach efficacious exposures. Binding to the high affinity receptor IL- 2Ra / p / y may be necessary for optimal anti-tumor activity as recent published work shows. First, analysis of T cell clonal content and activation state in patients with basal cell carcinoma after positive response to anti -programmed cell death 1 (PD-1) therapy showed that those tumors go through a process of clonal replacement where most of the activated / exhausted T cells found after treatment are newly activated clones that express the high affinity receptor IL-2Ra / |3 / y during expansion. These cells will benefit from the presence of exogenous fully active IL-2 during this process. Second, signaling through the high affinity receptor IL-2Ra / p / y plays a critical role directing tire generation of effective memory formation and secondary responses.

[0314]

[0297] The specific design features of the IL-2 prodrug will address these challenges by inhibiting the interaction of IL-2 with the IL-2Rp / y in the periphery to minimize systemic toxicity while delivering a native IL-2 into the TME with IL-2Ra / / y binding to realize the full pharmacology of IL-2 in driving anti -tumor immune responses. In certain embodiments, the IL-2 prodrug used in this Example 1 is Compound 1. In certain embodiments, the IL-2 prodrug used in this Example 1 is Compound 2. In certain embodiments, the IL-2 prodrug used in this Example 1 is Compound 3. In certain embodiments, the IL-2 prodrug used in this Example 1 is Compound 4.

[0315] Pembrolizumab

[0316]

[0298] Pembrolizumab is a potent humanized immunoglobulin (Ig) G4 (IgG4) monoclonal antibody with high specificity of binding to the programmed cell death 1 (PD-1) receptor, thus inhibiting its interaction with programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). Based on preclinical in vitro data, pembrolizumab has high affinity and potent receptor blocking activity for PD- 1. Pembrolizumab has an acceptable preclinical safety profile and is in clinical development as an intravenous (IV) immunotherapy for advanced malignancies. KEYTRUDA® (pembrolizumab) is indicated for the treatment of patients across a number of indications.

[0299] Refer to the approved labeling for detailed background information on pembrolizumab.

[0317] Pharmaceutical and Therapeutic Background

[0318]

[0300] Tire importance of intact immune surveillance function in controlling outgrowth of neoplastic transformations has been known for decades. Accumulating evidence shows a correlation between tumorinfiltrating lymphocytes in cancer tissue and favorable prognosis in various malignancies. In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector T cells / FoxP3+ Tregs correlates with improved prognosis and long-term survival in solid malignancies, such as ovarian, colorectal, and pancreatic cancer, hepatocellular carcinoma, malignant melanoma, and renal cell carcinoma. Tumorinfiltrating lymphocytes can be expanded ex vivo and reinfused, inducing durable objective tumor responses in cancers such as melanoma.

[0319]

[0301] The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1, expressed on the cell surface of activated T cells under healthy conditions, is to down-modulate unwanted or excessive immune responses, including autoimmune reactions. PD-1 (encoded by the gene PDCD1) is an Ig superfamily member related to CD28 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) that has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and / or PD L2).

[0320]

[0302] The structure of murine PD-1 has been resolved. PD-1 and its family members are type 1 transmembrane glycoproteins containing an Ig-variable (IgV)-type domain responsible for ligand binding and a cytoplasmic tail responsible for the binding of signaling molecules. The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an immunoreceptor tyrosine based inhibition motif, and an immunoreceptor tyrosine-based switch motif. Following T cell stimulation, PD-1 recruits the tyrosine phosphatases, src homology 2 domain-containing protein tyrosine phosphatase (SHP)-l and SHP-2. to the immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, leading to the dephosphorylation of effector molecules such as CD3^, protein kinase CO, and zeta-chain-associated protein kinase 70, which are involved in the CD3 T cell signaling cascade. The mechanism by which PD-1 down-modulate s T cell responses is similar to, but distinct from, that of CTLA-4 because both molecules regulate an overlapping set of signaling proteins. As a consequence, the PD-1 / PD-L1 pathway is an attractive target for therapeutic intervention in combination with a pro-inflammatory T cell directed mechanism, such as that generated by the IL-2 prodrugs disclosed herein.

[0321] 1.2 Study Rationale

[0322]

[0303] Immunotherapy, which includes proinflammatory cytokines like rIL-2, has become a well- established treatment modality for multiple cancer indications. rIL-2 therapy demonstrated dramatic clinical activity with durable complete responses in metastatic renal cell carcinoma (mRCC) and cutaneous malignant melanoma, leading to its Food and Drug Administration (FDA) approval forthose indications in 1992 and 1998, respectively. However, administration of high dose IL-2 therapy has been limited due to its short half-life, poor pharmaceutical properties, and severe toxicities requiring it to be administered to patients under monitored conditions in tire Intensive Care Unit. Therefore, it has been replaced overtime by other more tolerable therapies, including checkpoint inhibitors such as PD-L1 antagonists and targeted therapies. Data from the Proleukin® Observational Study to Evaluate the Treatment Patterns and Clinical Response in Malignancy registry, a national observational database established to document and study the current treatment outcomes with high-dose (HD) IL-2, have shown that overall response rates and survival benefit of HD IL-2 in metastatic melanoma and mRCC sequenced after or in combination with Standard of Care therapies, including prior checkpoint inhibitor therapy, are consistent with previously reported data showing durable, long-term responses.

[0323]

[0304] Despite advances in cancer care and the demonstration that numerous tumor types beyond malignant melanoma and mRCC are sensitive to checkpoint blockade, there remains an unmet medical need for a next-generation, full-potency IL-2 that can be safely administered. Hie IL-2 prodrug is a systemically delivered, conditionally activated IL-2 INDUKINE molecule that is being developed to minimize the severe toxicities observed with rIL-2 therapy and maximize clinical benefit when administered as monotherapy or in combination with an immune checkpoint inhibitor (CPI) in immunosensitive advanced or metastatic solid tumors.

[0324]

[0305] The IL-2 prodrug as a monotherapy or in combination with CPI in tumor types for which CPI therapy has demonstrated activity has the potential to safely deliver the full biological potency of IL-2 and result in a powerful anti-tumor immune response.

[0325]

[0306] Data from cynomolgus monkeys have demonstrated a wide therapeutic index with tire safe delivery of the IL-2 prodrug at doses associated with efficacious exposures in mouse tumor models. The combination of the IL-2 prodrug and anti-PD-1 in anti-PD-1 resistant models has shown synergistic antitumor activity.

[0326]

[0307] This is a Phase 1 / lb dose escalation and expansion study to detennine the safety, tolerability, PK, and preliminary anti-tumor activity of the systemic administration of the IL-2 prodrug alone or in combination with pembrolizumab in selected advanced or metastatic solid tumors and to identify the doses and schedule appropriate for further study.

[0327] 1.3 Rationale for IL-2 Prodrug Starting Dose

[0328]

[0308] Hie starting dose for the IL-2 prodrug will be selected based on IND enabling study data or GLP toxicology data and will be delivered systemically every 2 weeks (Q2W). Tire current proposed doses of the IL-2 prodrug are: 1, 3, 10, 30, 60, 120, and 240 mg. The predicted Cmaxand AUC for the 3 mg dose is expected to have a 65-fold and 31 -fold exposure margin, respectively, when compared with the top dose administered in the DRF NHP toxicology study. The 3 mg dose (0.542 nmol / kg for a 70 kg patient) is also lower than the recommended dose of rIL-2 (2.387 nmol / kg) given even’ 8 hours for 5 consecutive days, and, for all dose levels of the IL-2 prodrug, systemic exposure to free IL-2 is expected to be below the maximum tolerated exposure for rIL 2 given as a continuous IV infusion. Confirmation of the IL-2 prodrug first in human (FIH) starting dose and rationale will be finalized following the conduct of the GLP toxicology study.

[0329] 1.4 Rationale for Pembrolizumab Dose

[0330]

[0309] The dose of pembrolizumab for this study is 400 mg even 6 weeks (Q6W).

[0331] 1.5 Risk / Benefit

[0332]

[0310] Patients enrolled in this study will be those with metastatic malignancies who have limited treatment options. Appropriate eligibility criteria and specific dose-limiting toxicity (DLT) definitions, as well as specific monitoring guidelines and dose modification and individual patient stopping rules are included in this protocol.

[0333] 1.6 Study Objectives

[0334] Primary Objectives

[0335] [3H] The pri ary objectives of the Dose Escalation Phase of this study are the following:

[0336] • To determine the MTD and / or recommended dose for expansion (RDE) and evaluate the safety and tolerability of the IL-2 prodrug; and

[0337] • To determine the MTD and / or RDE and evaluate the safety and tolerability of the IL-2 prodrug in combination with pembrolizumab.

[0338]

[0312] The primary7objectives of the Expansion Phase of this study are the following:

[0339] • To further characterize the safety of the IL-2 prodrug when administered as monotherapy and in combination with pembrolizumab; and

[0340] • To evaluate the anti-tumor activity of the IL-2 prodrug (as monotherapy and in combination with pembrolizumab) in advanced or metastatic renal clear cell carcinoma and advanced or metastatic cutaneous malignant melanoma, as measured by overall response rate (ORR) (complete response [CR] + partial response [PR]) by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 and immune ORR (iORR) (iORR = immune-CR + immune-PR) by immune-RECIST (iRECIST).

[0341] Secondary Objectives

[0342]

[0313] The secondary objectives of this study are the following:

[0343] • To characterize the PK profile of the IL-2 prodrug (parent compound and free IL-2); To evaluate the anti-tumor activity of the IL-2 prodrug, as measured by ORR (CR + PR), DOR, and PFS by RECIST 1.1 and iRECIST;

[0344] • To evaluate the anti -tumor activity of the IL-2 prodrug in combination with pembrolizumab, as measured by ORR (CR + PR), DOR, and PFS by RECIST 1.1 and iORR (iORR = immune complete response [iCR] + immune-partial response [iPR]), iDOR, and iPFS by iRECIST;

[0345] • To evaluate the changes in certain immunological biomarkers in blood and in baseline and posttreatment tumor biopsies in response to the IL-2 prodrug as monotherapy or in combination with pembrolizumab;

[0346] • To evaluate the anti-tumor activity of the IL-2 prodrug (as monotherapy and in combination with pembrolizumab) in advanced or metastatic renal cell carcinoma and advanced or metastatic cutaneous malignant melanoma, as measured by duration of response (DOR) and progression-free survival (PFS) by RECIST 1.1 and immune-RECIST (iRECIST); and

[0347] • To evaluate immunogenicity of the IL-2 prodrug, including the potential to generate an anti-drug antibody (ADA) response.

[0348] Exploratory Objectives

[0349]

[0314] The exploratory objectives of this study are the following:

[0350] • To evaluate pharmacodynamic (PD) activities of the IL-2 prodrug (alone and in combination with pembrolizumab);

[0351] • To investigate immunological biomarkers in peripheral blood and tumor that may correlate with the treatment outcome of the IL-2 prodrug as monotherapy or in combination with pembrolizumab; and

[0352] • To assess tumor biopsies for potential biomarkers of target engagement and immune pathway activation.

[0353] Study Description

[0354]

[0315] This is a FIH, open-label, multicenter study to determine tire safety profile and MTD and / or RDE of the IL-2 prodrug alone or in combination with pembrolizumab.

[0355]

[0316] The study will enroll patients in the monotherapy and combination arms of the Dose Escalation Phase with rclapscd / rcfractory locally advanced or metastatic immunotherapy-sensitive solid tumors, defined as specific indications for which CPIs are approved, who have progressed on or are intolerant to standard therapy, or for whom no standard therapy with proven benefit exists. Patients will have received prior CPI therapy, including prior anti-PD-1 or -(L) 1 inhibitors alone or in combination with other agents. Patients in the combination arm should have discontinued that therapy due to either disease progression or reasons other than toxicity. Patients in all phases of the study may not have received prior IL 2 directed therapy.

[0356]

[0317] Patients will be enrolled in the monotherapy and combination therapy amis of the Dose Expansion Phase who have advanced or metastatic renal clear cell carcinoma, advanced or metastatic cutaneous malignant melanoma, or advanced or metastatic cutaneous squamous cell carcinoma. For monotherapy dose expansion, patients with mRCC may have received up to 3 prior lines of therapy and must have received prior anti-angiogenic therapy (VEGFi) and a CPI. Patients with v-raf murine sarcoma viral oncogene homolog B 1 (BRAF) wild-type cutaneous malignant melanoma must have received anti- PD-(L)1 inhibitor therapy alone or in combination with a CTLA-4 inhibitor or other therapy. Patients with BRAF mutant melanoma may have received 2 prior lines of therapy, which included a CPI and a BRAF inhibitor with or without a MEK inhibitor.

[0357]

[0318] For the mRCC patients in the combination part of the Dose Expansion Phase specifically, patients must have had no more than 2 prior lines of therapy, only 1 of which included an anti-PD-(L) 1 inhibitor therapy alone or in combination. Cutaneous malignant melanoma patients enrolled in the combination part of the Dose Expansion Phase may have received 0-2 prior lines of therapy depending upon BRAF mutation status. Patients in tire combination therapy arms of the dose expansion phase should not have discontinued prior CPI therapy for immune-related toxicities.

[0358]

[0319] The assignment of a patient to a particular dose cohort during dose escalation and to a particular arm during dose expansion will be coordinated by Sponsor. When monotherapy and combination therapy arms are open in parallel, eligible patients will be enrolled sequentially into available slots.

[0359]

[0320] The overall sample size for this study is approximately 150 patients between the Dose Escalation Phase and Dose Expansion Phase but will depend on the observed DLT profiles of the IL-2 prodrug monotherapy and the IL-2 prodrug in combination with pembrolizumab.

[0360] Dose Escalation Phase

[0361]

[0321] Dose escalation will utilize a modified Toxicity Probability Interval (mTPI)-2 design. The starting dose of the IL-2 prodrug may be a 1 mg or 3 mg flat dose administered IV every 2 weeks (Q2W), and the proposed doses, if supported by mTPI-2, may be 1, 3, 10, 30, 60, 120, and 240 mg IV Q2W. Intermediate doses may be evaluated. Modifications in the dosing regimen to Q3W may be made based on the PK and safety profiles observed and will be implemented by amendment. A stagger of at least 2 days is required between dosing of the first and second patients at each new untested dose level, and a stagger of 1 day is required between dosing of the second and third patients.

[0362]

[0322] Dose confirmation using an mTPI-2 design

[0363]

[0323] An mTPI-2 design with a target DLT rate of approximately 30% and an acceptable DLT range of 25% to 35% (el = E2 = 0.05) will be applied for determining the MTD and / or RDE for the IL-2 prodrug.

[0364]

[0324] The current proposed doses of the IL-2 prodrug are: 1, 3, 10, 30, 60, 120, and 240 mg. The predicted Cmax and AUC for the 3 mg dose is expected to have a 65 fold and 31 fold exposure margin, respectively, when compared with the top dose administered in the GLP NHP toxicology study (FIH starting dose will be finalized after completion of GLP toxicology study). Hie 3 mg dose (0.542 nmol / kg for a 70 kg patient) is also lower than the recommended dose of rIL 2 (2.387 nmol / kg) given every 8 hours for 5 consecutive days and for all dose levels of the IL-2 prodrug, and systemic exposure to free IL- 2 is expected to be below the maximum tolerated exposure for rIL-2 given as a continuous IV infusion. Both total parent drug (the IL-2 prodrug) and free IL-2 measurements will be performed and may be considered when selecting the dose for the next cohort based on the PK and the safety profiles observed.

[0365]

[0325] Escalation or de-escalation decisions will be based on the occurrence of DLTs and adverse events, particularly severe immune-related toxicities, at a given dose for the first 28-day period (Cycle 1) and will be made by the Dose Escalation Committee (DEC).

[0366]

[0326] During the continuous safety assessment phase, a minimum of 3 patients are required at each dose. Depending on accrual rate and occurrence of DLTs, 3, 4, 5, or 6 patients may be enrolled at each new dose until the last of those patients completes the 28-day DLT assessment period. For example, if 1 out of the first 3 patients at a given dose level develops a DLT, no more than an additional 3 patients should be enrolled at this dose level until additional DLT data are available since this dose will be deescalated if 2 of the additional patients experience a DLT (i.e., 3 out of 5 patients). If 2 out of the first 3 patients at a given dose level develop a DLT, the dose will be de-escalated to the next lower level. If 3 out of the first 3 patients at a given dose level develop a DLT, this dose will be considered unacceptably toxic (i.e.. the dose will be de-escalated and never re-escalated to that dose again). If a lower dose is used, and 0 out of the next 3 patients at a given dose level develops a DLT, then the dose can be re-escalated back to the next level. The same principle will be applied whether 3, 4, 5, or 6 patients are enrolled in the same dose cohort.

[0367]

[0327] Based on the mTPI-2 design, the number of patients who are enrolled at a dose, but are not yet fully evaluable for DLT assessment, may not exceed the number of remaining patients who are at risk of developing a DLT before the dose would be considered unacceptably toxic. In total. 3 to 12 patients may be enrolled at a given dose level for the continuous safety assessment phase.

[0368]

[0328] Dose escalation and confirmation of the RDE will generally end after up to 12 patients have been treated at any of the selected doses found to be acceptable. The MTD is defined as the dose level that can be given such that the estimated DLT probability is closest to approximately 30%. Estimation of the MTD will be based on the estimation of the incidence rate of DLTs. Hie totality of the data will be considered before a dose is selected to carry forward to further enrollment and the escalation schedule may be adjusted based on PK and safety data emerging throughout the study to determine the RDE.

[0369]

[0329] Note that while 30% was the target toxicity rate used to generate the guidelines, the observed rates of patients with DLTs at the MTD may be slightly above or below 30%.

[0370] Dose Escalation Phase with pembrolizumab

[0330] Dose escalation for the first combination cohort will begin for the IL-2 prodrug in combination with pembrolizumab following completion of the third monotherapy dose cohort (i.e., in parallel to the fourth monotherapy dose cohort). Tire dose escalation for the IL-2 prodrug in combination with pembrolizumab cohorts will follow an mTPI 2 design using the same rules as described above for monotherapy with the starting dose level for the IL-2 prodrug at the dose level that is 1 dose level below the highest dose determined to be safe in the monotherapy dose escalation at that time. For each new dose level, the the IL-2 prodrug dose level to be tested in combination with pembrolizumab must have been shown to be safe and tolerable as monotherapy. Tire pembrolizumab dose will be administered at the approved dose of 400 mg Q6W and will not be escalated or de-escalated.

[0371] Dose-Limiting Toxicity Criteria

[0372]

[0331] Toxicity will be evaluated according to the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0. The DLT observation period for both monotherapy the IL-2 prodrug and pembrolizumab combination dose escalation is 28 days. A DLT is defined as an adverse event that is at least possibly related to study drug and not reasonably attributed to the patient’s underlying disease, other medical conditions, or concomitant medications or procedures.

[0373] Study Stopping Rules

[0374]

[0332] Beyond Cycle 1 in the escalation part and during the expansion part, the study will use the DLT criteria, and if observed toxicities meeting these DLT criteria in >33% of subjects at any point in time, the DEC will be convened to review the available safety data and detennine appropriate subsequent steps (e.g. refinement of dose, modification of study assessments, study closure, etc.). Enrollment may be paused while such a review is undertaken. Any death that is considered possibly related to the IL-2 prodrug alone or in combination with pembrolizumab. occurring within 28 days of receiving the first dose(s) of study treatment, will result in a study enrollment pause and cessation of dosing at the current dose level, to allow for an expedited ad hoc evaluation by the DEC prior to further enrollment in the study.

[0375] Dose Expansion Phase

[0376]

[0333] Once the respective MTD(s) and / or RDE(s) are determined for the IL-2 prodrug as monotherapy or in combination with pembrolizumab in the Dose Escalation Phase, patients will be enrolled into the respective expansion arms for the tumor types specified below, 2 monotherapy expansion arms and 2 combination expansion arms, with up to 20 patients enrolled in each. Expansion arms for monotherapy and combination therapy may open and begin enrollment independently. Based on evolving safety and efficacy data, additional patients may be enrolled by amendment. The 4 expansion cohorts will include the following:

[0377] 1. Arm A: the IL-2 prodrug as a monotherapy at the RDE in advanced or mRCC;

[0378] 2. Amr B: the IL-2 prodrug as a monotherapy at tire RDE in advanced or metastatic cutaneous malignant melanoma;

[0379] 3. Arm C: the IL-2 prodrug as a monotherapy at the RDE in advanced or metastatic cutaneous squamous cell carcinoma

[0380] 4. Arm D: the IL-2 prodrug at the RDE in combination with pembrolizumab in advanced or mRCC; and

[0381] 5. Amr E: the IL-2 prodrug at the RDE in combination with pembrolizumab in advanced or metastatic cutaneous malignant melanoma.

[0382] Selection of Study Population

[0383]

[0334] The study will enroll patients in the monotherapy and combination arms of the Dose Escalation Phase with relapsed / refractory locally advanced or metastatic immunotherapy-sensitive solid tumors, defined as specific indications for which CPIs are approved, who have progressed on or are intolerant to standard therapy, or for whom no standard therapy with proven benefit exists. Patients will have received prior CPI therapy, including prior anti -programmed death 1 (PD-1) or -(L)l inhibitors alone or in combination with other agents. Patients enrolled in the combination arm of the Dose Escalation phase should have discontinued that therapy due to either disease progression or reasons other than immune- related toxicity. Patients in all phases of the study may not have received prior IL 2 directed therapy.

[0384]

[0335] Patients will be enrolled in the monotherapy and combination therapy arms of the Dose Expansion Phase who have locally advanced or metastatic renal clear cell carcinoma or locally advanced or metastatic cutaneous malignant melanoma. For monotherapy dose expansion, patients with mRCC may have received up to 3 prior lines of therapy and must have received prior anti-angiogenic therapy (vascular endothelial growth factor inhibitor [VEGFi]) and a CPI. Patients with BRAF wild-type cutaneous malignant melanoma may have received 1 prior line of therapy and must have received a CPI, anti-PD-(L) 1 inhibitor therapy alone or in combination with a cytotoxic T lymphocyte associated protein 4 (CTLA-4) inhibitor or other therapy. Patients with v-raf murine sarcoma viral oncogene homolog Bl (BRAF) mutant melanoma may have received 2 prior lines of therapy, which included a CPI and a BRAF inhibitor with or without a mitogen-activated protein kinase kinase (MEK) inhibitor. For the mRCC patients in the combination part of the Dose Expansion Phase specifically, patients must have had no more than 2 prior lines of therapy, only 1 of which included an anti-PD-(L) 1 inhibitor therapy alone or in combination and a VEGFi. Cutaneous malignant melanoma patients enrolled in the combination part of the Dose Expansion Phase may have received 0-2 prior lines of therapy depending upon BRAF mutation status. Patients in the combination therapy arms of the dose expansion phase should not have discontinued prior CPI therapy for immune-related toxicities.

[0385] Inclusion Criteria

[0386]

[0336] Each patient must meet all the following criteria to participate in the study:

[0387] 1. Able to understand and voluntarily sign a written informed consent form (ICF) and is willing and able to comply with protocol requirements;

[0388] 2. Has histological or cytological documentation of the solid tumor indication for which an anti PD- (L) 1 is indicated (eg, melanoma, non-small cell lung cancer, small cell lung cancer, head and neck squamous cell cancer, urothelial cancer, microsatellite instability-high or mismatch repair deficient cancer, microsatellite instability high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal clear cell carcinoma, endometrial carcinoma, tumor mutational burden-high cancer, cutaneous squamous cell carcinoma, advanced basal cell carcinoma) for all parts of the clinical study;

[0389] 3. Monotherapy Dose Escalation: Patients with relapsed / refractory locally advanced or metastatic solid tumors for which immunotherapy is approved, who have progressed on or are intolerant to standard therapy, including CPIs, or for whom no standard therapy with proven benefit exists;

[0390]

[0337] Combination Dose Escalation: Patients with relapsed / refractory locally advanced or metastatic solid tumors for which immunotherapy is approved, who have progressed on or are intolerant to standard therapy, including CPIs, or for whom no standard therapy with proven benefit exists. Patients must have progressed on prior CPI as defined by RECIST 1.1 or iRECIST or discontinued for reasons other than toxicity. Patients in either dose escalation ann may have received no more than 3 prior lines of therapy.

[0391]

[0338] Monotherapy Dose Expansion: Patients with relapsed advanced / mRCC or cutaneous malignant melanoma who received prior CPI alone or in combination.

[0392] • Arm A: Patients with relapsed advanced or mRCC: may have received no more than 3 prior lines of therapy, only 1 of which included CPI, and must have received an anti-angiogenic agent (VEGFi);

[0393] • Ann B: Patients with relapsed advanced or metastatic cutaneous malignant melanoma: may have received no more than 1 prior line of therapy for BRAF V600 wild type (WT), no more than 2 prior lines of therapy for BRAF V600 mutant and may have received BRAF inhibitor with or without mitogen-activated protein kinase kinase (MEK) inhibitor. Adjuvant therapy is excluded as 1 of the lines of therapy if there were >6 months to relapse. T-VEC therapy is allowed, but treated lesions cannot be used as target lesions for biopsies. • Arm C: Patients with relapsed advanced or metastatic cutaneous squamous cell carcinoma: may have received no more than 1 prior line of therapy and that line had to have been an immune checkpoint inhibitor.

[0394]

[0339] Combination Dose Expansion: Patients with relapsed advanced / mRCC or metastatic cutaneous malignant melanoma.

[0395] • Arm D: Patients with relapsed advanced or mRCC: may have received no more than 2 prior lines of therapy, only 1 of which included CPI, and must have received an anti-angiogenic agent (VEGFi).

[0396] • Arm E: Patients with relapsed advanced or metastatic cutaneous malignant melanoma: may have received 0-2 prior lines of therapy depending upon BRAF status, 0-1 prior lines of therapy for BRAF V600 WT and 0-2 prior lines of therapy for VRAF V600 mutant, which could include a CPI and BRAF inhibitor with or without a MEK inhibitor. Adjuvant therapy is excluded as 1 of the lines of therapy if there were >6 months to relapse. T-VEC therapy is allowed, but treated lesions cannot be used as target lesions or for biopsies.

[0397] 4. >18 years of age;

[0398] 5. Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1 ;

[0399] 6. Has at least 1 measurable lesion per RECIST 1.1 (lesions situated in a previously irradiated area are considered measurable if progression has been demonstrated in such lesions);

[0400] 7. Agrees to undergo a pre-treatment and post-treatment biopsy of a primary or metastatic solid tumor lesion;

[0401] 8. Has adequate organ and bone marrow function defined by: a. Absolute neutrophil count >1.5 x 109 / L (>1500 / mm3): b. Hemoglobin >9.0 g / dL or equivalent. Criteria must be met without packed red blood cell transfusion within the prior 2 weeks; c. Platelet count >100 x 109 / L (>100,000 / mm3); d. Total bilirubin <1.5 * ULN in the absence of Gilbert’s syndrome and <3 x ULN if the patient has Gilbert’s syndrome; e. Measured or calculated creatinine clearance (estimated glomerular filtration rate) >30 mL / min / 1.73 m2; and f. ALT and AST <2.5 x ULN or <5 x ULN for patients with hepatic metastases.

[0402] 9. Willingness of men and women of reproductive potential to observe highly effective birth control for the duration of treatment and for 4 months following the last dose of study drug;

[0403]

[0340] Male study participants should refrain from sperm donation during study treatment and up to 6 months following the last dose of study drug.

[0404]

[0341] A woman of childbearing potential is a woman who is fertile following menarche and until becoming post-menopausal unless permanently sterile. Permanent sterilization methods include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy.

[0405]

[0342] A man is considered fertile after puberty unless permanently sterile by bilateral orchidectomy.

[0406] Exclusion Criteria

[0407]

[0343] Patients who meet any of the following criteria will be excluded from participating in the study:

[0408] 1. Have a history of another active malignancy (a second cancer) within the previous 2 years except for localized cancers that are not related to the current cancer being treated, are considered cured, and, in the opinion of the Investigator, presents a low risk of recurrence. These exceptions include, but are not limited to, basal or squamous cell skin cancer, superficial bladder cancer, or carcinoma in situ of the prostate, cervix, or breast;

[0409] 2. Has a history of (non-infectious) pneumonitis / interstitial lung disease that required steroids or has current pneumonitis / interstitial lung disease.

[0410] 3. Have a diagnosis of uveal or mucosal melanoma in Dose Expansion Phase arms;

[0411] 4. Have received prior IL-2 -directed therapy;

[0412] 5. Have had an allogeneic tissue / solid organ transplant;

[0413] 6. Have known symptomatic brain metastases requiring steroids. Patients with previously diagnosed brain metastases are eligible if they have completed their treatment, have recovered from the acute effects of radiation therapy or surgery7prior to enrollment, and are neurologically stable and asymptomatic;

[0414] 7. Have significant cardiovascular disease, including myocardial infarction, arterial thromboembolism, or cerebrovascular thromboembolism, within 6 months prior to the first dose of study drug; symptomatic dysrhythmias or unstable dysrhythmias requiring medical therapy; angina requiring therapy; symptomatic peripheral vascular disease; New York Heart Association Class 3 or 4 congestive heart failure; or history of congenital prolonged QT syndrome;

[0415] 8. Have significant electrocardiogram (ECG) abnormalities at Screening, including unstable cardiac arrhythmia requiring medication, left bundle branch block, second-degree atrioventricular (AV) block type II, third-degree AV block, >Grade 2 bradycardia, or QT interval corrected for heart rate using Fridericia's formula (QTcF) >470 msec:

[0416] 9. Have an active autoimmune disease that required systemic treatment in the past 2 years (i.e., with use of disease-modifying antirheumatic agents or immunosuppressive drugs):

[0417] Note: Replacement therapy (e g., thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal, thyroid, or pituitary7insufficiency) is permitted.

[0418] 10. Diagnosis of immunodeficiency, is on immunosuppressive therapy, or is receiving chronic systemic or enteric steroid therapy (dose >10 mg / day of prednisone or equivalent) or any other form of immunosuppressive therapy within 7 days prior to the first dose of study drug;

[0419] Note: At Screening and during study participation, patients may be using systemic corticosteroids (dose <10 mg / day of prednisone or equivalent) or topical, intraocular, intra articular, or inhaled corticosteroids.

[0420] 11. Major surgery (excluding placement of vascular access) within 2 weeks prior to tire first dose of study drug;

[0421] 12. Investigational agent or anti cancer therapy (including chemotherapy, biologic therapy, immunotherapy, anticancer Chinese medicine, or other anticancer herbal remedy) within 5 half-lives or 4 weeks (whichever is shorter) prior to the first dose of study drag. In addition, no concurrent investigational anticancer therapy is permitted;

[0422] 13. Has received prior radiotherapy within 2 weeks of start of study treatment. Participants must have recovered from all radiation-related toxicities, not require corticosteroids, and not have had radiation pneumonitis. A 1-week washout is permitted for palliative radiation (<2 weeks of radiotherapy) to non- CNS disease;

[0423] 14. Any unresolved toxicities from prior therapy greater than National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0 Grade 1 at the time of starting study drag with the exception of alopecia and Grade 2 prior platinum therapy related neuropathy;

[0424] 15. Use of a strong inhibitor or inducer of cytochrome P450 (CYP)3A4 prior to starting study drug and during study participation;

[0425] 16. Use of sensitive substrates to major CYP450 isozymes;

[0426] 17. Have any illness, medical condition, organ system dysfunction, or social situation, including mental illness or substance abuse, deemed by the Investigator to be likely to interfere with a patient's ability to sign the ICF. adversely affect the patient’s ability to cooperate and participate in the study, or compromise the interpretation of study results;

[0427] 18. Received a live or live-attenuated vaccine within 30 days of the first dose of study drug;

[0428] Note: Administration of killed vaccines or other formats are allowed.

[0429] 19. Active, uncontrolled systemic bacterial, viral, or fungal infection;

[0430] 20. Known human immunodeficiency virus (HIV) antibody;

[0431] 21. Active infection as determined by hepatitis B surface antigen and hepatitis B core antibody, or hepatitis B virus DNA by quantitative polymerase chain reaction (qPCR) testing;

[0432] 22. Active infection as determined by hepatitis C virus (HCV) antibody or HCV RNA by qPCR testing;

[0433] 23. Pregnant or lactating;

[0434] Note: Defined as a WOCBP who has a positive urine pregnancy test (within 72 hours) prior to treatment. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required. 24. History of hypersensitivity to any of the study drug components; or

[0435] 25. Patients will be excluded from the combination arms of the IL-2 prodrug with pembrolizumab if they discontinued prior anti-PD-(L)-! therapy due to any grade immune-related adverse events (irAEs) except for thyroid abnormalities or adverse events on replacement therapy.

[0436] Study Treatments

[0437] Investigational Product

[0438]

[0344] The IL-2 prodrug has been developed as a lyophilized product supplied in 20R glass vials with flip caps. Upon reconstitution with sterile water, each vial will contain 6 mL nominal volume at 5mg / mL or lOmg / mL. Tire lyophilized IL-2 prodrug will be stored at 2 degrees C to 8 degrees C.

[0439] Study Drug Administration

[0440] General Dosing Instructions

[0441]

[0345] IL-2 prodrug:

[0442]

[0346] Tire IL-2 prodrug monotherapy will be administered as a 15-minute IV infusion via syringe pump Q2W in 28 day cycles until progressive disease by RECIST, unacceptable toxicity, withdrawal of consent by the patient, discontinuation of the patient by the Investigator. Sponsor decision to terminate the study or treatment, or death. Infusion duration may be prolonged in the event of an infusion-related reaction.

[0443]

[0347] During the Dose Escalation Phase, a stagger of at least 2 days is required between dosing of the first and second patients at each new untested dose level, and a stagger of 1 day is required between dosing of the second and third patients.

[0444]

[0348] Pembrolizumab :

[0445]

[0349] Pembrolizumab will be administered using IV infusion on Day 1 of each 6-week (42-day) treatment cycle after all procedures and assessments have been completed.

[0446]

[0350] Pembrolizumab will be administered as a dose of 400 mg using a 30-minute IV infusion. Sites should make every effort to target infusion timing to be as close to 30 minutes as possible; however, given the variability of infusion pumps from site to site, a window between -5 minutes and +10 minutes is permitted (i.e., infusion time is 30 minutes [-5 minutes / +10 minutes]).

[0447]

[0351] For the IL-2 prodrug + pembrolizumab combination arms of tire Dose Escalation Phase or Dose Expansion Phase, pembrolizumab will be administered first. Patients can receive IL-2 prodrug infusion approximately 30 minutes after they have received the entire infusion of pembrolizumab.

[0448]

[0352] Pembrolizumab dosing will be capped at 18 cycles (~2 years). However, the IL-2 prodrug may be continued as a monotherapy for as long as the patient is deriving clinical benefit and per criteria in the IL- 2 prodrug section above. Dose Modification Guidelines

[0449]

[0353] If a patient experiences treatment-emergent adverse events (TEAEs) that may be related to the IL- 2 prodrug, pembrolizumab, or both, including irAEs, further doses might be modified, halted, or permanently discontinued. During Cycle 1, dose modification should be avoided if a patient has not experienced a >Grade 2 TEAE related to study drug. In subsequent cycles, dose interruptions and / or modifications are permissible.

[0450] Study Procedures

[0451] Tests and Evaluations

[0452] Vital Signs and Physical Examinations

[0453]

[0354] Vital signs will be measured and will include measurements of systolic and diastolic blood pressure, heart rate, and body temperature.

[0454]

[0355] A physical examination and review of relevant systems, body weight, and height will occur at screening. Height will be measured at the Screening Visit only but may be measured later if missed. An abbreviated physical examination that is directed by disease site and symptoms will be performed on Day I of subsequent cycles after Cycle 1.

[0455] Pharmacokinetics and Electrocardiograms

[0456] Pharmacokinetics

[0457]

[0356] Plasma samples for IL-2 prodrug PK assessment of the parent compound and free IL-2 will be obtained. Additional PK assessments may be conducted when considered necessary by the Investigator to understand exposure in relationship to possible safety or anti-tumor activity findings. Samples will be collected, and concentrations of the IL-2 prodrug and free IL-2 will be detennined with a validated bioanalytical method.

[0458] Electrocardiograms

[0459]

[0357] Twelve-lead ECGs will be performed. Serial triplicate 12-lead ECGs (separated by >1 minute) will be performed throughout tire DLT period during the Dose Escalation Phase. Single 12-lead ECGs will be performed after the DLT period during the Dose Escalation Phase and throughout the Dose Expansion Phase. Single ECGs should be repeated if an anomaly or abnormality is observed. When the ECG measurements coincide with blood sample draws, the ECG assessment should be timed sufficiently prior to blood sample collection to not impact the PK sample collection time. ECGs will be recorded after the patient has been in a resting (semi recumbent or scmi-supinc) position breathing quietly for 5 minutes. All pre-dose ECGs will be performed 15 to 30 minutes prior to study drug administration. Hie evaluation of ECGs will include assessment of changes in the following ECG parameters: heart rate, PR, QRS, QT, and QTcF intervals. Medpace Core Laboratory will provide equipment for the Dose Escalation Phase of the study and ECG images will be stored centrally. Local evaluation and equipment for ECGs will be utilized during the Expansion Phase of the study, and ECG images will be stored locally.

[0460] Pharmacodynamics

[0461]

[0358] Whole blood samples will be collected for pharmacodynamic assessments and biomarker assessments, as specified in Table 6 below.

[0462]

[0359] While tire goal of the biomarker assessments is to provide supportive data for the clinical study, there may be circumstances when a decision is made to stop a collection, not perform, or discontinue an analysis due to either practical or strategic reasons (e.g., inadequate sample number, issues related to the quality of the sample or issues related to the assay that preclude analysis, impossibility to perform correlative analyses, etc.). Therefore, depending on the results obtained during the study, sample collection analysis may be omitted at the discretion of the Sponsor.

[0463] Table 6. Biomarker Sample Collection Plan for the IL-2 prodrug

[0464] Tumor Measurements per RECIST and iRECIST

[0465]

[0360] Tumors will be assessed based on RECIST 1. 1 (see Tumor Measurements and Assessment of Disease Response using RECIST 1.1) and iRECIST (see Modified Response Evaluation Criteria in Solid Tumors 1.1 for Immune-Based Therapeutics (IRECIST) Quick Reference). Baseline disease assessment will include radiographic tumor measurements using computed tomography (CT) or magnetic resonance (MR) imaging of the chest, abdomen, pelvis, or any other areas with suspected disease involvement at Screening within 28 days of Cycle 1 Day 1. Patients with CNS metastases must have brain imaging (MR imaging preferred, CT with contrast is acceptable if MR imaging contraindicated) during Screening. For each modality, intravenous (IV) and oral contrast should be utilized (chest CT does not require IV contrast) unless there is a clear contraindication (eg, decreased renal function or allergy that cannot be addressed with standard prophylactic treatments). On-study scans should be performed every 8 weeks (±7 days) from Cycle 1 Day 1 for the first 6 cycles and every 12 weeks (±7 days) thereafter, including imaging of the chest, abdomen, pelvis, or any other areas of known disease at baseline, using the same modality as used for baseline imaging until progressive disease per RECIST 1.1 and iRECIST, withdrawal of consent, or initiation of a new anticancer therapy.

[0466] Biopsied and Archived Tumor Samples

[0467]

[0361] Pre-and post-treatment biopsies are required for immunophenotyping, cancer gene expression analysis, and tumor immune contexture assessment by immunohistochemistry. However, in the event that collection of a fresh biopsy with required number of cores are medically infeasible, patient may continue on study and an archival tumor sample will be requested. At baseline, a fresh biopsy (2 cores) is required for immunophenotyping by flow cytometry. Also at baseline, tissue from either a newly obtained (4 cores) or archival biopsy, if fresh biopsy is medically infeasible (tumor block preferred), will be collected for cancer gene expression analysis and tumor immune contexture assessment by immunohistochemistry. Tire baseline sample may be collected any time after enrollment during the 28-day Screening period. On study, a biopsy (6 cores) will be collected between Day 22 and Day 28 of Cycle 1 for immunophenotyping, cancer gene expression analysis and tumor immune contexture assessment by immunohistochemistry. The timing of the second biopsy may shift based on evolving biomarker data.

[0468] Efficacy and Pharmacokinetic Assessments

[0469] Study Endpoints

[0470] Primary Endpoints (Dose Escalation Phase

[0471]

[0362] The primary endpoints of the Dose Escalation Phase of this study are the following:

[0472] • MTD and / or RDE for the IL-2 prodrug alone;

[0473] • MTD and / or RDE for the IL-2 prodrug in combination with pembrolizumab; and

[0474] • Frequency, severity, and relatedness of TEAEs and serious adverse events (SAEs), changes in safety laboratory parameters, and DLTs (if observed) for the IL-2 prodrug alone and in combination with pembrolizumab.

[0475] Primary Endpoints (Dose Expansion Phase)

[0476]

[0363] The primary endpoints of the Dose Expansion Phase of this study are the following:

[0477] • Frequency, severity, and relatedness of TEAEs and SAEs, changes in safety laboratoiy parameters, and DLTs (if observed) for the IL-2 prodrug alone and in combination with pembrolizumab; and

[0478] • ORR (ORR = CR + PR), DOR by RECIST 1.1 and 10RR (iORR = iCR + iPR) by iRECIST.

[0479] Secondary Endpoints (Dose Escalation and Dose Expansion Phases)

[0480]

[0364] The secondary endpoints of the Dose Escalation and Dose Expansion Phases of this study are the following:

[0481] • Plasma concentrations of IL-2 prodrug and free IL-2 and calculated PK parameters;

[0482] • DOR, and PFS by RECIST 1.1 and iDOR, and iPFS by iRECIST;

[0483] • Characterization of changes in peripheral immune cells, including T cell subsets, from baseline in response to IL-2 prodrug alone, and in combination with pembrolizumab;

[0484] Changes in immunological biomarkers in baseline and post-treatment tumor biopsies in response to IL- 2 prodrug alone, and in combination with pembrolizumab, as determined by immunohistochemistry (IHC); and Incidence of immunogenicity of IL-2 prodrug (alone and in combination with pembrolizumab), as indicated by levels of AD As.

[0485] Exploratory Endpoints

[0486]

[0365] Tire exploratory endpoints of the Dose Escalation and Dose Expansion Phases of this study are the following:

[0487] • PD effects of the IL-2 prodrug alone, and in combination with pembrolizumab. as follows: o Modulation of cytokines including, but not limited to, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL- 13, IL-15, soluble CD25, interferon gamma, transforming growth factor beta, tumor necrosis factor alpha o Changes in levels of lymphocytes and eosinophils in peripheral blood; o Characterization of intra-tumoral immune cells, including T-cell subsets; o Changes in gene expression profiles of immune response in tumor: o Changes in intra-tumoral levels of IL-2.

[0488] Safety Assessments: Adverse Events of Special Interest

[0489]

[0366] Tire safety and tolerability profile of IL-2 prodrug, alone and in combination w ith a pembrolizumab, will be assessed by monitoring adverse events (including DLTs, SAEs, and adverse events of special interest [AESIs]), physical examination findings (including ECOG performance status), clinical laboratory evaluations, vital sign measurements, and ECGs. Adverse events will be graded according to the NCI CTCAE version 5.0. For this study, AESIs include tire following:

[0490] • >Grade 2 CRS; and

[0491] • >Grade 2 CLS.

[0492]

[0367] For combination amis, AESIs include the following:

[0493] • an overdose of pembrolizumab, as defined in Treatment of Overdose, that is not associated with clinical symptoms or abnormal laboratory results.

[0494] • an elevated AST or ALT lab value that is greater than or equal to 3X the upper limit of normal and an elevated total bilirubin lab value that is greater than or equal to 2X the upper limit of normal and, at the same time, an alkaline phosphatase lab value that is less than 2X the upper limit of normal, as determined by way of protocol-specified laboratory testing or unscheduled laboratory testing.

[0495] Statistical Analysis

[0496]

[0368] Plasma concentrations of IL-2 prodrug and free IL-2 will be determined with a validated bioanalytical assay. The following PK parameters for IL-2 prodrug and free IL-2 will be calculated from plasma concentrations using noncompartmental analyses: maximum observed plasma concentration (Cmax), time to maximum observed plasma concentration (Tmax), area under the concentration versus time curve from time 0 to t (AUCo-t), area under the concentration time versus curve from time 0 to infinity (AUCo-inf), clearance (CL), volume of distribution (Vd), and terminal-elimination half-life (ty). Summary statistics will be generated by dose cohort as appropriate.

[0497]

[0369] Plots of mean IL-2 prodrug and free IL-2 plasma concentrations versus time will be generated by dose group and phase in linear and semi-logarithmic fonn. Individual plasma concentrations versus time graphs will also be provided.

[0498]

[0370] The anti-tumor activity analysis will be conducted on the Safety Analysis Set unless otherwise specified. Tumor response data per RECIST 1.1 and iRECIST criteria will be listed and summarized. ORR will be estimated for each cohort based on the observed proportion of patients whose best overall response is confinned CR or PR. iORR will be estimated for each cohort based on tire observed proportion of patients whose best overall response is confirmed iCR or iPR. Tire DOR, iDOR. PFS, and iPFS will be summarized descriptively using the Kaplan-Meier method.

[0499]

[0371] In general, all safety analyses will be descriptive and will be presented in tabular format with the appropriate summary statistics for the Safety Analysis Set.

[0500]

[0372] Tire safety and tolerability profile of IL-2 prodrug, alone and in combination with pembrolizumab, will be assessed by monitoring adverse events (including DLTs, SAEs, and AESIs), physical examination findings (including ECOG performance status), clinical laboratory evaluations, vital sign measurements, and ECGs. Adverse events will be graded according to the NCI CTCAE version 5.0.

[0501]

[0373] The number and percentage of patients with TEAEs will be summarized by System Organ Class and preferred term. Grade 3 or higher TEAEs, drug -related TEAEs, treatment-emergent SAEs, drug- related treatment-emergent SAEs, TEAEs leading to discontinuation of study drug, drag-related TEAEs leading to discontinuation of study drag, TEAEs leading to dose reduction / interruption of study drag, and drag-related TEAEs leading to dose reduction / interruption of study drag will be summarized in the same manner. DLTs during Cycle I in the Dose Escalation Phase of the study will be summarized by primary System Organ Class and preferred term.

[0502] Analysis of Anti-Tumor Activity

[0503]

[0374] All efficacy will be done using RECIST 1.1 and iRECIST criteria based on local investigator assessment.

[0504]

[0375] Overall Response Rate:

[0505]

[0376] Overall response rate as defined by achieving confirmed CR and / or PR will be presented by percentage rates and 95% confidence intervals (Cis). For changes in solid tumor size, waterfall plots will be presented. For all response assessments, swimmers plots will be presented. All response assessments will be listed.

[0377] Clinical Benefit Rate at Months 3, 6, and 9:

[0506]

[0378] Clinical benefit rate at Months 3, 6, and 9, as defined by achieving CR and / or PR and / or SD, will be presented by percentage rates and 95% Cis. Waterfall plots will be presented.

[0507]

[0379] Duration of Response :

[0508]

[0380] The duration of response defined as time from first assessment of PR or CR to follow-on first assessment of PD will be summarized by descriptive statistics including median duration of response and respective 95% Cis. Duration of response will also be listed.

[0509]

[0381] Progression Free Survival (PFS) and Overall Survival (OS):

[0510]

[0382] Time from first treatment received until PD / OS will be summarized by Kaplan-Meier estimates, median PFS / OS and respective 95% Cis. Patients with no event will be censored at the last available tumor assessment for PFS and at the last timepoint known alive for OS.

[0511]

[0383] iORR will be estimated for each cohort based on the observed proportion of patients whose best overall response is confirmed iCR or iPR. The DOR, iDOR, PFS, and iPFS will be summarized descriptively using the Kaplan-Meier method.

[0512] Analysis of Pharmacokinetics

[0513]

[0384] The PK Analysis Set will be used for summaries of all PK data. No fomial statistical analysis beyond descriptive statistics is planned. For each PK parameter, individual and mean data and summary statistics (including number of patients, arithmetic mean, geometric mean (for time to maximum plasma concentration (tmax) and time to last measurable plasma concentration (tiast) no geometric mean will be calculated), standard deviation (StD), confidence value (CV), median, Min and Max) will be presented.

[0514] Analysis of Pharmacodynamics

[0515]

[0385] The result, change, percent change, and maximum percent change in immunologic changes to serum cytokines and immune cell subsets in the blood and tumor microenvironment will be summarized descriptively.

[0516] Sample Size Determination

[0517]

[0386] The overall sample size for this study is approximately 150 patients between the Dose Escalation Phase and Dose Expansion Phase and will depend on the observed DET profiles of IL-2 prodrug monotherapy and IL-2 prodrug in combination with pembrolizumab.

[0518] Example 2: Initial Results of the IL-2 Prodrug Monotherapy Dose Escalation and Combination Therapy Dose Escalation with Pembrolizumab

[0387] This example provides initial results of the first in human study of IL-2 prodrug as monotherapy or in combination with pembrolizumab. The subjects enrolled in the dose escalation study had solid tumors for which immunotherapy is indicated and had progressed on all available standard of care therapies. IL-2 prodrug was administered as a flat dose intravenously (IV) every two weeks (Q2W) alone or with pembrolizumab 400 mg IV every six weeks (Q6W) in 28-day cycles for monotherapy and 42-day cycles for combination therapy. The dose limiting toxicity observation (DLT) period was 28 days for both monotherapy and combination therapy. Expansion arms are planned as follows: monotherapy for renal cell carcinoma (N=20), monotherapy for melanoma (N=20), monotherapy for cutaneous squamous cell carcinoma (n=10), combination therapy for renal cell carcinoma (N=20) and combination therapy for melanoma (n=20). Forty-seven subjects have been treated with at least one does of IL-2 prodrug monotherapy or combination therapy. Tire IL-2 prodrug used in Example 1 can be Compound 1. The IL- 2 prodrug used in this Example 1 can be Compound 2. The IL-2 prodrug used in this Example 1 can be Compound 3. The IL-2 prodrug used in this Example 1 can be Compound 4.

[0519] Table 7. Subject Enrollment for Monotherapy and Pembrolizumab Combination Therapy

[0520]

[0388] Subject study population are shown in Table 8. Tire subject study population with melanoma included eleven subjects with cutaneous melanoma, three subjects with uveal melanoma, and 4 subjects with mucosal melanoma under the monotherapy arm, and six subjects with cutaneous melanoma under the combination therapy arm. All subjects were considered resistant or refractory to immune checkpoint inhibitor (ICI) therapy and may have previously received anywhere from 1 to more than 4 lines of systemic therapy or immunotherapy. Lines of immunotherapy included immune-oncology (IO) agents used alone or in combination with each other or standard of care therapy (e.g. chemotherapy, targeted therapy).

[0521] Table 8. Subject Study Population

[0522] | therapy | Combination | Total

[0523] * Abbreviations: SD: standard deviation; ECOG: Eastern Cooperative Oncology Group; PS: performance status; NSCLC: non-small cell lung cancer; SCC: squamous cell carcinoma

[0524] 2.1 Safety Profile

[0525]

[0389] Treatment-Emergent Adverse Events (TEAEs) that were at least possibly related to IL-2 prodrug were assessed in the monotherapy study population and were primarily mild to moderate, including at clinically active doses (>12 mg IV Q2W). Hie most frequent TEAEs, which occurred in five or more subjects, included arthralgia, fatigue, pruritus, eosinophilia, myalgia, maculo-papular rash, rash, chills, nausea and headache. The majority of TEAEs that were at least possibly related to IL-2 prodrug and occurred in more than one subject (n=35) were mild to moderate (grade 1, grade 2) and no severe TEAEs (grade 4, grade 5) occurred. Moderate TEAEs (grade 3) occurred at IL-2 prodrug doses of >18 mg IV Q2W and included arthralgia, eosinophilia, maculo-papular rash, stomatitis, and non-cardiac chest pain. All grade 3 TEAEs were manageable and reversible. There was no evidence of vascular leakage syndrome (VLS), grade 2 or higher cytokine release syndrome (CRS) or infusion-related reactions in any subjects. No reoccurrence of immune-related adverse events (irAEs) that previously occurred on immune checkpoint inhibitors were observed.

[0390] Similar to the monotherapy dose escalation arm, the majority of related TEAEs for combination therapy ranged from mild to moderate (grade 1, grade 2) with no severe TEAEs being observed (grade 4, grade 5). There was no increase in frequency and / or severity of related TEAEs in subjects treated with combination therapy in comparison to IL-2 prodrug monotherapy. (Eleven subjects received IL-2 prodrug monotherapy at a dose of 12 mg while four subjects received the 12 mg dose of IL-2 prodrug in combination with pembrolizumab.) A single subject in tire combination arm experienced arthralgia at grade 3.

[0526] 2.2 Clinical Response

[0527]

[0391] Antitumor activity was demonstrated in patients who received IL-2 prodrug monotherapy at doses >12 mg IV Q2W. Three patients were observed to have objective responses to IL-2 prodrug monotherapy dosed at 12 mg IV Q2W (n=2. one with cutaneous squamous cell cancer and one with melanoma) or 18 mg IV Q2W (n=l, one patient with gastroesophageal junction adenocarcinoma), and eight patients demonstrated stable disease at points during the study (FIG. 2). The three subjects with objective responses (characterized as a complete response (CR) or partial response (PR)) showed complete, durable regression of target lesions. (FIG. 3). Notably, the antitumor responses in all the three subjects with durable regression of target lesions were documented within the first two cycles of therapy (i.e.. on initial restaging CT scan at approximately 8 weeks).

[0528] 2.3 Subject Case Reports

[0529] A. CSCC Complete Response

[0530] A complete response to 12 mg IL-2 prodrug IV Q2W was observed in a 72-year old man with ICI- refractory cutaneous squamous cell carcinoma (CSCC). Hie subject had progressed on RT / cetuximab and had no response to four doses (12 weeks) of cemiplimab. Baseline CT scan showed a 2.6 cm premaxillary target lesion and anon-target lesion extending into the ptery gopalatine fossa. The subject, after three weeks on IL-2 prodrug treatment, showed no tumor on collected on-treatment biopsy of target legion.

[0531] After eight weeks, the subject showed a PR with a 62% decrease in the target lesion and no increase in the non-target legion. After twelve weeks, it was observed on PET-CT scan a complete metabolic response to both target and non-target lesions consistent with a CR. TEAEs included a grade 2 rash and grade 2 arthralgias, thus, subject tolerated therapy well. Monotherapy was stopped at 21 weeks and subject has been observed to have durable complete remission ongoing for more than eight months and is being followed off drug.

[0532] B. Cutaneous Melanoma Partial Response A partial response to 12 mg IL-2 prodrug IV Q2W was observed in a 78-year old man with cutaneous melanoma observed to have secondary ICI resistance. The subject previously discontinued adjuvant nivolumab due to toxicity and then progressed on nicolumab / relatlimab as first line therapy for metastatic disease. At five weeks a target liver metastasis lesion (T) biopsied at baseline could not be reidentified. At eight weeks, the subjects CT scan showed a complete response at the target lesion with no increase of a non-target lesion. At sixteen weeks, the target lesion remained absent, but non-target lesion had increased. Further, new lesions were identified in the sternum and periportal lymph nodes.

[0533] C. Gastroesophageal Junction Adenocarcinoma Partial Response

[0534]

[0392] A partial response to 18 mg IL-2 prodrug IV Q2W was observed in a 63 -year old man with gastroesophageal junction adenocarcinoma. Tire subject had previously progressed on FOLFOX / nivolumab and nivolumab / BMS986253, with the best overall response for each prior line being stable disease. The baseline CT showed a mesenteric lymph node target lesion and four lymph node non- target lesions. At three weeks, the L axillary lymph node biopsied at baseline could not be reidentified. At eight weeks, a restaging CT showed a 56% reduction of the target legion (with compete normalization to <10mm) and no increase in non-target lesion, consistent with PR. At sixteen weeks, the subject response at the target lesion continued but progression was observed at one of the four non-target lesions. Subject discontinued therapy and has not progressed nor needed additional therapy for three months.

[0535] 2.4 NanoString Analysis of T Cell Activation Genes in Patient Tumor Biopsies After Treatment with

[0536] IL-2 Prodrug

[0537]

[0393] RNA was purified from FFPE sample derived from pre- and on-treatment patient biopsies. Analysis of gene expression was performed using a PanCaner IO panel. Normalized mRNA counts were used to generate a list of the top forty T Cell activation genes (NanoString curated) that were upregulated or downregulated with increase in monotherapy dose level of on treatment biopsies compared to baseline. Gene expression changes were plotted as a heat map of Log2 fold change in gene expression of on treatment / baseline (FIG. 4A-4B). Notably, gene expression signatures of subjects receiving 3 mg or 6 mg of IL-2 prodrug in combination with pembrolizumab were observed to have an increase in log2 fold change of T Cell activation genes compared to subjects receiving 3 mg or 6 mg of IL-2 prodrug alone. This demonstrates that the effects seen at doses of IL-2 prodrug > I2mg are due to the activity of the IL-2 prodrug and free IL-2 released from the prodrug. The data show that there is no carry over effect of prior discontinued ICI therapy, as the combination therapy arm showed that when ICI was present and IL-2 prodrug was dosed at 3 or 6 mg there were significant increases in expression of some genes that were not increased at the same IL-2 prodrug doses in the monotherapy arm. See, FIG. 4A.

[0394] The data also show that monotherapy with IL-2 prodrug induced expression of IL2RA but not FOXP3. FIG. 4B. Tire ratio of IL2RA / FOXP3 also increased with dose level demonstrating that IL-2 prodrug activates effector T cells in the tumor microenvironment without simultaneously expanding Tregs.

[0538] 2.5 Multiplex IF Analysis Identify Unique Baseline Signature of Patient that Respond to IL- 2 Prodrug

[0539]

[0395] Multi-plex immunofluorescence (IF) analysis was performed on patient tumor biopsies at baseline and on treatment with IL-2 prodrug. FFPE slides were strained for CD8 and GRZB expression and imaged. The densities of CD8+ T Cells and activated CD8+ T Cells (CD8+ GrB+ T Cells) were analyzed using HALO software. Quantification of images shown in FIG. 4D. The analysis showed substantial infiltration of activated effector T cells at baseline prior to IL-2 prodrug therapy in patients with ICI resistance who had CR / PR or SD in comparison to those patients who experienced progressive disease.

[0540]

[0396] Baseline transcription signature analysis was performed using DeSeq2 analysis to identify genes differentially expressed between responders (CR / PR) and subjects with stable / progressive disease, among subjects treated at active dose levels > 6 mg WTX-124 Q2W. The differential expression analysis was plotted as a volcano plot between -log 1 Op value and log2 fold change between responder and nonresponders, with cutoffs at p<0.01 (Wald test) and log2FC > 0.5 (FIGs. 6A and 6B). Data were also plotted in a heat map based of Z scores, which reflect the deviation from the mean baseline mRNA count from all patients (FIGs. 4C). The heat map shows that responders (with CR / PR) have a unique transcriptional signature at base line and that this signature can be used to identify candidates who are likely to respond (CR or PR) to IL-2 prodrug therapy.

[0541] 2.6 Pharmacokinetic (PK Profile

[0542]

[0397] Plasma PK was collected from patients dosed with IL-2 prodrag monotherapy and / or IL-2 prodrag and pembrolizumab combination therapy. Samples collected at the end of infusion, 4, 8, 24-, 48-, 168- and 336-hours post infusion were analyzed using validated Mesoscale Discovery (MSD) based PK assays that specifically detect total IL-2 prodrag or free IL-2. Mean and standard error of the PK data for each cohort was plotted using R and ggplot2 package. Sample below LLOQ were replaced with zero. IL-2 prodrug anti-drag antibodies (ADA) were measured from pre-dose plasma samples and analyzed using MSD based ASA assay. High dose IL-2 Cmax was estimated using a 2-compartment IV PK model published in Konrad ct al., 1990. It was observed that IL-2 prodrug dosed at 18 mg IV Q2W has an approximately 1.5 -fold higher Cmax than HD IL-2, while peak free IL-2 exposure after 18 mg IV Q2W is approximately 136-fold lower than HD-IL-2. Further, at all dose levels, free IL-2 levels were below 1.6% of prodrug exposure. It was found that repeat dosing did not cause accumulation of IL-2 prodrug or free IL-2 and IL-2 prodrug anti -drug antibodies (ADA) are transient with primarily low titer, and also had no impact on repeat dose exposure. Pembrolizumab was observed to have no effect on IL-2 prodrug PK. PK profiles are shown in FIG. 5.

[0543] 2. 7 Summary and Conclusions

[0544]

[0398] When treating subjects with IL-2 prodrug as a monotherapy, objective clinical responses were achieved in patients who previously experienced relapse or refractory to all standard of care therapies, including ICIs. One subject achieved a durable, confirmed complete response, while two other subjects had observable partial responses. These responding subjects had rapid, complete, and durable regression of target lesions within two cycles of therapy.

[0545]

[0399] TEAEs that were at least possibly related to IL-2 prodrug monotherapy and combination therapy with IL-2 prodrug and Pembrolizumab, were primarily mild to moderate in severity, and deemed manageable and reversible. Further, IL-2 prodrug when combined with pembrolizumab exhibited similar TEAEs, with no new safety signals identified.

[0546]

[0400] Through quantification of gene expression in tumor biopsies of IL2RA and FOXP3, an increase in IL2RA but not FOXP3 demonstrated activation and expansion of effector T cells without expanding Tregs. Gene expression analysis of T cell activation genes demonstrated the potential for improved efficacy by combining IL-2 prodrug with pembrolizumab. Additionally, comparison of T cell activation gene expression of the monotherapy ami with the combination therapy arm, supports that there is no carryover effect of prior discontinued ICI therapy. Finally, the PK profile showed approximate doseproportionality and low peripheral free IL-2.

[0547]

[0401] In conclusion. IL-2 prodrug is clinically active and well tolerated in patients ineligible for high- dose IL-2 treatment. Further, based on the clinical activity and acceptable safety- in outpatient settings, 18 mg IV Q2W of IL-2 prodrug was selected as the monotherapy RDE.

[0548] Table 9. Sequences

Claims

CLAIMS1. A method for treating a solid tumor, comprising: administering to a subject in need thereof an effective amount of an IL-2 prodrug, wherein prior to administration of tire IL-2 prodrug the solid tumor has a gene expression signature indicative of activated intratumoral effector CD8+ T cells.

2. A method for treating cancer, comprising: a) identifying or selecting a subject with a solid tumor that comprises a gene expression signature indicative of activated intratumoral effector CD8+ T cells prior to administration of the IL-2 prodrug; and b) administering to the subject an effective amount of an IL-2 prodrug.

3. The method of claim 1 or 2. wherein the gene expression signature comprises an increase in the expression of at least one gene selected from the group consisting of SLAMF1, IDOL S100A8, CXCL9, GZMB, IL1B, GZMK, GZMA, PRF1, GZMH, CD3D, CCL11, SLAMF7, CCL5, CD5, CD3E, CXCR6, SH2D1A, CCR5, CD3G, CXCR3, CTLA4, CD27, and LAG3.

4. The method of any one of claims 1-3, wherein the IL-2 prodrug comprises Compound 1 (SEQ ID NO: 1 / SEQ ID NO:5), Compound 2 (SEQ ID NO:2 / SEQ ID NO:5), Compound 3 (SEQ ID NO:3 / SEQ ID NO:5), Compound 4 (SEQ ID NO:4 / SEQ ID NO:5) or an amino acid sequence variant of any of the foregoing.

5. The method of any one of claims 1-4, wherein the effective amount of the IL-2 prodrug is administered intravenously or by intravenous infusion.

6. The method of any one of claims 1-5, wherein about 6 mg to about 28 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

7. The method of claim 6, wherein about 12 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about even’ two weeks.

8. The method of claim 6, wherein about 18 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

9. The method of claim 6, wherein about 23 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

10. Tire method of claim 6, wherein about 28 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

11. The method of any one of claims 1-10, further comprising administering to the subject an effective amount of another therapeutic agent.

12. The method of claim 11, wherein the another therapeutic agent is the standard of care therapy for the solid tumor.

13. The method of claim 11, wherein the another therapeutic agent comprises an immune checkpoint inhibitor.

14. The method of claim 13, wherein the immune checkpoint inhibitor binds to and inhibits a protein selected from the group consisting of PD-1, PD-L1 (B7-H1. CD274) and PD-L2 (B7-DC, CD273), CTLA-4 (CD152), B7-1 (CD80), B7-2 (CD86). LAG 3 (CD223); TIM3 (HAVCR2); TIGIT (VSTM3, WUCAM); BTLA (CD272) which binds HVEM (TNFRSF14), B7-H3 (CD276), B7-H4 (VTCN1), VISTA (B7-H5), KIR, CD44 (2B4), CD160 (BY55), CD134 (TNRFSR4, 0X40) and CD252 (OX-40L).

15. The method of claim 14, wherein the immune checkpoint inhibitor is an anti-PD-Ll antibody.

16. The method of claim 15, wherein the anti-PD-Ll antibody comprises avelumab, durvalumab. or atezolizumab.

17. Tire method of claim 15, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

18. The method of claim 17, wherein the anti-CTLA-4 antibody comprises ipilimumab.

19. The method of claim 15, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or antigen binding fragment thereof.

20. The method of claim 19, wherein the anti-PD-1 antibody comprises pembrolizumab, dostarlimab. cemiplimab-rwlc, nivolumab, camrelizumab, tislelizumab, toripalimab, or sintilimab.

21. Tire method of claim 20, wherein the anti-PD-1 antibody or antigen binding fragment thereof comprises: (a) light chain CDRs SEQ ID NOs: 6, 7, 8 and (b) heavy chain CDRs SEQ ID NOs: 11, 12, 13.

22. The method of claim 20, wherein the anti-PD-1 antibody comprises: (a) a heavy chain variable region comprising SEQ ID NO: 14 or a variant hereof and (b) a light chain variable region comprising SEQ ID NO: 9 or a variant thereof.

23. The method of claim 20, wherein the anti-PD-1 antibody comprises a heavy chain and a light chain, and wherein the heavy chain and light chain comprise the amino acid sequences in SEQ ID NO: 10 and SEQ ID NO: 15, respectively.

24. Tire method of claim 20, wherein the anti-PD-1 antibody is pembrolizumab or a pembrolizumab variant.

25. The method of claim 20, wherein the anti-PD-1 antibody is pembrolizumab (or a biosimilar).

26. The method of any one of claims 19-25, wherein about 100 mg to about 600 mg of the anti-PD-1 antibody or antigen binding fragment thereof, is administered about every three to six weeks.

27. The method of claim 26, wherein 200 mg of the anti-PD-1 antibody, or antigen binding fragment thereof, is administered about every three weeks.

28. The method of claim 26, wherein 600 mg of the anti-PD-1 antibody, or antigen binding fragment thereof, is administered about every six weeks.

29. The method of any one of claims 1-28, wherein the subject has failed to achieve a complete response to a prior treatment or to an ongoing treatment prior to administration of the IL-2 prodrug.

30. Tire method of claim 29, wherein the prior treatment or ongoing treatment comprises treatment with an immune checkpoint inhibitor.

31. The method of claim 30, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

32. Tire method of any one of claims 1-31, wherein the solid tumor is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer. Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, squamous cell carcinoma, adenocarcinoma, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary' syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

33. The method of any one of claims 1-32, wherein the cancer is colon cancer, lung cancer, renal cell carcinoma, breast cancer, melanoma, squamous cell carcinoma, or adenocarcinoma.

34. The method of claim 33, wherein the melanoma comprises cutaneous melanoma.

35. The method of claim 33, wherein the squamous cell carcinoma comprises cutaneous squamous cell carcinoma.

36. The method of claim 33, wherein the adenocarcinoma comprises gastroesophageal junction adenocarcinoma.

37. A method for treating a solid tumor, comprising: administering to a subject in need thereof an effective amount of an IL-2 prodrug as a monotherapy, wherein prior to administration of the IL-2 prodrug the solid tumor has a gene expression signature comprising an increase in the expression of at least one gene selected from the group consisting of SLAMFL IDO1, S100A8, IL-1B, GZMB, CCL11, CXCL9, GZMK, GZMH, IL-8, GZMA, SLAMF7, CD3G, CD3D, PRF1, CTLA4, CD5, SH2D1A, CD22, CD3E, TNFRSF17, CCR5, CXCL11, ITK, and CD247; and wherein the gene expression signature is indicative of responsiveness to the IL-2 prodrug.

38. A method for treating cancer, comprising : a) identifying or selecting a subject with a solid tumor that comprises a gene expression signature indicative of activated intratumoral effector CD8+ T cells prior to administration of the IL-2 prodrug, wherein the gene expression signature comprises an increase in the expression of at least one gene selected from the group consisting of SLAMF1, IDO1, S100A8, IL-1B, GZMB. CCL11, CXCL9, GZMK, GZMH. IL-8. GZMA, SLAMF7, CD3G, CD3D, PRF1. CTLA4, CD5, SH2D1A, CD22, CD3E, TNFRSF17. CCR5, CXCL11. ITK, and CD247: and b) administering to the subject an effective amount of an IL-2 prodmg as a monotherapy, wherein the gene expression signature is indicative of responsiveness to the IL-2 prodmg.

39. The method of any one of claims 37 or 38, wherein the IL-2 prodmg comprises Compound 1 (SEQ ID NO: 1 / SEQ ID NO:5), Compound 2 (SEQ ID NO:2 / SEQ ID NO:5), Compound 3 (SEQ ID NO:3 / SEQ ID NO:5). Compound 4 (SEQ ID NO:4 / SEQ ID NO:5) or an amino acid sequence variant of any of the foregoing.

40. Tire method of any one of claims 37-39, wherein the effective amount of the IL-2 prodmg is administered intravenously.

41. The method of claim 40, wherein the effective amount of the IL-2 prodmg is administered by intravenous infusion.

42. The method of any one of claims 37-41. wherein about 6 mg to about 28 mg of Compound 1, Compound 2, Compound 3, Compound 4. or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

43. Tire method of claim 42, wherein about 12 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

44. The method of claim 42, wherein about 18 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

45. The method of claim 42, wherein about 23 mg of Compound 1, Compound 2, Compound 3. Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

46. Tire method of claim 42, wherein about 28 mg of Compound 1, Compound 2, Compound 3, Compound 4, or an amino acid sequence variant of any of the foregoing is administered about every two weeks.

47. The method of any one of claims 37-46, wherein the solid tumor is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, lary ngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignantfibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, squamous cell carcinoma, adenocarcinoma, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezar ' syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

48. The method of any one of claims 37-47, wherein the cancer is colon cancer, lung cancer, renal cell carcinoma, breast cancer, melanoma, squamous cell carcinoma, or adenocarcinoma.

49. The method of claim 48, wherein the melanoma comprises cutaneous melanoma.

50. The method of claim 48, wherein the squamous cell carcinoma comprises cutaneous squamous cell carcinoma.

51. The method of claim 48, wherein the adenocarcinoma comprises gastroesophageal junction adenocarcinoma.

52. The method of any one of claims 37-51 , wherein the subject does not receive any other cancer treatment within 30 days prior to or following administration.

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