This is a Phase II, multicenter, single-arm trial designed to evaluate the efficacy and safety of nivolumab (NIVO), ipilimumab (IPI) and temozolomide (TMZ) combination in 27 patients with MSS, MGMT-silenced mCRC with initial clinical benefit following lead-in treatment with single-agent TMZ.

Immune checkpoint inhibitors have been shown to trigger durable antitumor effects in a subset of patients. A high number of tumor mutations (so called 'tumor mutational burden') has recently been found associated with increased immunogenicity (due to a high number of neoantigens) and improved treatment efficacy across several different solid tumors. In mCRCs, only a small fraction of tumors (<5%) display a high mutational load and are usually associated with inactivation of mismatch repair genes such as MLH1, MSH2 and MSH6. Checkpoint inhibitors may have increased activity in dMMR/microsatellite instability-high (MSI-H) tumors, a hypothesis which was tested in various Phase II trials with positive results. On the opposite, mismatch repair proficient colorectal cancer is unresponsive to immune checkpoint inhibitors.

Previous reports indicate that acquired resistance to TMZ may emerge through the induction of a microsatellite-instability-positive phenotype and recent data showed that inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered immune surveillance in mouse models.

On all of the above grounds, the investigators hypothesize that treatment of microsatellite stable MGMT hypermethylated CRCs with alkylating agents could reshape the tumor genetic landscape by increasing the tumor mutational burden, leading to achieve potential sensitization to immunotherapy.

In advanced CRC, the occurrence of chemorefractory disease poses a major therapeutic challenge for presence of an adequate performance status to potentially receive further treatments. Patients who progress after all approved treatments may be generally considered suitable for new investigational drugs or strategies. Thus, in the era of personalized medicine, tumor molecular profiling may lead to the identification of therapeutic targets or predictive biomarkers for pharmacological intervention. The DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is responsible of the elimination of alkyl groups from the O6-position of guanine. If inactive, it may be involved in early steps of colorectal tumor genesis leading to an increase of G-to-A point mutations. Epigenetic silencing of MGMT during colorectal tumor genesis is associated with hypermethylation of the CpG island in its promoter. This transcriptional gene silencing is responsible for diminished DNA-repair of O6-alkylguanine adducts, with the consequence of enhancing chemosensitivity to alkylating agents including dacarbazine and its oral prodrug temozolomide (TMZ). Previous phase II studies showed that TMZ induced an average objective response rate by RECIST criteria in 10% of heavily pre-treated patients with advanced CRC carrying MGMT promoter methylated tumors. Thus, MGMT methylation by methylation-specific PCR (MSP) used for patients screening seemed to be a necessary but not sufficient condition to identify response to TMZ. Digital PCR quantification of MGMT methylation refined patients' selection, with benefit restricted to those with highly hyper-methylated tumors. Further analysis showed that MGMT negative/low expression by immunohistochemistry (IHC) is found in about one third of MSP-methylated samples and is associated with increased response rate. However, even in responding mCRC patients, acquired resistance to single agent TMZ emerges rapidly and almost invariably within 6 months from treatment initiation.

Immune checkpoint inhibitors have been shown to trigger durable antitumor effects in a subset of patients. A high number of tumor mutations (so called 'tumor mutational burden') has recently been found associated with increased immunogenicity (due to a high number of neoantigens) and improved treatment efficacy across several different solid tumors. Early clinical testing indicated that only 1 of 33 CRC patients had a response to anti PD-1 treatment, in contrast to substantial fractions of patients with melanomas, renal-cell cancers, and lung tumors who showed benefit from PD-1 blockade. Similarly, anti CTLA-4 treatment up today brought to unsatisfactory results in unselected mCRC patients. The probability of response has been ascribed to a high mutational burden (that is an elevated number of somatic mutations), which translates in increased number of neo-antigens. In mCRCs, only a small fraction of tumors (<5%) display a high mutational load and are usually associated with inactivation of mismatch repair genes such as MLH1, MSH2 and MSH6. Molecular alterations in these genes occur as an initial step in colon tumor genesis leading to the microsatellite instability (MSI) phenotype. Indeed, mismatch repair-deficient (dMMR) colorectal cancers have 10 to 100 times as many somatic mutations as mismatch repair-proficient colorectal cancers. Moreover, mismatch repair-deficient cancers contain prominent lymphocyte infiltrates, a finding consistent with an immune response. Thus, checkpoint inhibitors may have increased activity in dMMR/microsatellite instability-high (MSI-H) tumors, a hypothesis which was tested in various Phase II trials with positive results. On the opposite, mismatch repair proficient colorectal cancer is unresponsive to immune checkpoint inhibitors.

Previous reports indicate that acquired resistance to TMZ may emerge through the induction of a microsatellite-instability-positive phenotype. On the other hand, TMZ by itself has been shown to induce an increase of mutational load in other MGMT deficient solid tumors such as melanoma or glioblastoma. In parallel, other studies have demonstrated that alkylating agents' side effects can influence the immune cell compartment by selectively depleting the immuno-suppressive T regulator lymphocytes (Tregs), and activating the immuno-active T cytotoxic lymphocytes (Tc) and natural killers (NK). The investigators recently showed that inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered immune surveillance in mouse models.

On all of the above grounds, the investigators hypothesize that treatment of microsatellite stable MGMT hypermethylated CRCs with alkylating agents could reshape the tumor genetic landscape by increasing the tumor mutational burden either directly (by inducing G>A mutations) or/and indirectly (by inactivating DNA repair genes such as MLH1, MSH2 or MSH6, which in turn could lead to hypermutated phenotype) therefore enhancing formation of cancer neoantigens and immunogenicity. TMZ treatment can also modulate the repertoire of immune cells (Tregs, Tc, NK) favoring T cell activation. To achieve potential sensitization to immunotherapy by means of TMZ-induced MSI-like status, treatment with TMZ should be active (i.e. inducing a SD/PR/CR).

Inclusion Criteria:

1. Have provided written informed consent prior to any study specific procedures
2. Willing and able to comply with the protocol
3. ≥18 years of age
4. ECOG status 0 - 1
5. At least 12 weeks of life expectancy at time of entry into the study
6. Histologically confirmed metastatic or inoperable adenocarcinoma of the colon and/or rectum, with centrally confirmed mismatch repair proficiency (microsatellite stable [MSS]) by multiplex polymerase chain reaction (PCR), MGMT promoter methylation by methylation-specific PCR (MSP) and MGMT low expression by IHC
7. Patients with progressive disease or that are not candidate for oxaliplatin irinotecan fluoropirimidin based chemotherapy and anti EGFR mAbs (in RAS/BRAF wild type tumors) in the metastatic setting
8. Patients with documented disease relapsed within 6 months from the completion of adjuvant oxaliplatin-based chemotherapy are considered eligible
9. Measureable, unresectable disease according to RECIST 1.1. Subjects with lesions in a previously irradiated field as the sole site of measurable disease will be permitted to enroll provided the lesion(s) have demonstrated clear progression and can be measured accurately.
10. Is willing and able to provide an adequate archival tumor sample (FFPE) available for tissue screening for central tissue screening. If the tumour block is not available, a minimum of twenty 3-micron unstained sections on charged slides of tumor will be required.

Exclusion Criteria:

1. Requirement for treatment with any medicinal product that contraindicates the use of any of the study medications, may interfere with the planned treatment, affects patient compliance or puts the patient at high risk for treatment-related complications

2. Inability to swallow pills

3. Refractory nausea and vomiting, malabsorption, external biliary shunt or significant bowel resection that would preclude adequate absorption

4. Inadequate haematological function indicated by all of the following:

   • White Blood Cell (WBC) count < 2 x 109/L
   • Absolute neutrophil count (ANC) < 1.5 x 109/L
   • Platelet count < 100 x 109/L
   • Haemoglobin < 9 g/dL (patients may have transfusions and/or growth factors to attain adequate Hb)

5. Inadequate liver function indicated by all of the following:

   • Total bilirubin ≥ 1.5 x upper limit of normal (ULN)
   • Aspartate transaminase (AST) and alanine aminotransferase (ALT) ≥ 3 x ULN (≥ 5 x ULN in patients with known liver metastases)
   • Alkaline phosphatase (ALP) ≥ 2 x ULN (≥ 5 x ULN in patients with known liver metastases)

6. Inadequate renal function indicated by all of the following:

   - Serum creatinine > 1.5 x ULN or calculated creatinine clearance < 40 ml/min

7. INR > 1.5 and aPTT > 1.5 x ULN within 7 days prior to the start of study treatment for patients not receiving anti-coagulation

   a. NOTE: The use of full-dose oral or parenteral anticoagulants is permitted as long as the INR or aPTT is within therapeutic limits (according to the medical standard of the enrolling institution) and the patient has been on a stable dose of anticoagulants for at least two weeks prior to the start of study treatment

8. Active infection requiring intravenous antibiotics at the start of study treatment

9. Previous or concurrent malignancy, except for adequately treated basal or squamous cell skin cancer, superficial bladder cancer, or carcinoma in situ of the prostate, cervix, or breast, or other cancer for which the patient has been disease-free for three years prior to study entry

10. Evidence of any other disease, neurologic or metabolic dysfunction, physical examination finding or laboratory finding giving reasonable suspicion of a disease or condition that contraindicates the use of any of the study medications, puts the patient at higher risk for treatment-related complications or may affect the interpretation of study results

11. Clinically significant (i.e. active) cardiovascular disease, for example cerebrovascular accidents ≤ 6 months prior to start of study treatment, myocardial infarction ≤ 6 months prior to study enrolment, unstable angina, New York Heart Association (NYHA) Functional Classification Grade II or greater congestive heart failure, or serious cardiac arrhythmia uncontrolled by medication or potentially interfering with protocol treatment

12. History or evidence upon physical or neurological examination of central nervous system (CNS) disease (e.g. seizures) unrelated to cancer unless adequately treated with standard medical therapy

13. Active brain metastases or leptomeningeal metastases. Subjects with brain metastases are eligible if these have been treated and there is no magnetic resonance imaging (MRI except where contraindicated in which CT scan is acceptable) evidence of progression for at least 8 weeks after treatment is complete and within 28 days prior to first dose of study drug administration. Cases should be discussed with the medical monitor. There must also be no requirement for immunosuppressive doses of systemic corticosteroids (>10mg/day prednisone equivalents) for at least 2 weeks prior to study drug administration.

14. Surgical procedure (including open biopsy, surgical resection, wound revision, or any other major surgery involving entry into a body cavity) or significant traumatic injury within 28 days prior to start of study treatment, or anticipation of need for major surgical procedure during the course of the study.

15. Treatment with any chemotherapy, curative intent radiation therapy, biologics for cancer, or investigational therapy within 28 days of first administration of study treatment (subjects with prior cytotoxic or investigational products < 4 weeks prior to treatment might be eligible after discussion between investigator and sponsor, if toxicities from the prior treatment have been resolved to Grade 1 (NCI CTCAE version 4). Prior focal palliative radiotherapy must have been completed at least 2 weeks before study drug administration.

16. All toxicities attributed to prior anti-cancer therapy other than alopecia and fatigue must have resolved to Grade 1 (NCI CTCAE version 4) or baseline before administration of study drug. Subjects with toxicities attributed to prior anti-cancer therapy which are not expected to resolve and result in long lasting sequelae, such as neuropathy after platinum based therapy, are permitted to enroll.

17. Known hypersensitivity to any of the study medications or Known hypersensitivity or allergy to Chinese hamster ovary cell products or any component of the NIVO formulation

18. History of severe allergic, anaphylactic, or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins

19. History of autoimmune disease including but not limited to myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, vascular thrombosis associated with antiphospholipid syndrome, Wegener's granulomatosis, Sjögren's syndrome, Guillain-Barré syndrome, multiple sclerosis, vasculitis, or glomerulonephritis (see Appendix IV for a more comprehensive list of autoimmune diseases)

    a. Note: history of autoimmune-related hypothyroidism on a stable dose of thyroid replacement hormone may be eligible. Subjects with controlled type I diabetes mellitus on a stable insulin regimen, vitiligo or psoriasis not requiring systemic treatment may be eligible.

20. Prior allogeneic bone marrow transplantation or prior solid organ transplantation

21. History of idiopathic pulmonary fibrosis (including pneumonitis), drug-induced pneumonitis, organizing pneumonia (i.e., bronchiolitis obliterans, cryptogenic organizing pneumonia), or evidence of active pneumonitis on Screening chest CT scan

22. Treatment with systemic immunostimulatory agents (including but not limited to interferons or interleukin-2) within 4 weeks or five half-lives of the drug, whichever is shorter, prior to start of study treatment

23. Treatment with systemic corticosteroids (>10 mg daily prednisone equivalents) or other systemic immunosuppressive medications (including but not limited to prednisone, dexamethasone, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-tumour necrosis factor [TNF] agents) within 2 weeks prior to start of study treatment, or requirement for systemic immunosuppressive medications during the trial. The use of inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone) is allowed.

    a. Note: Patients who have received acute, low-dose, systemic immunosuppressant medications (e.g., a one-time dose of dexamethasone for nausea) may be enrolled in the study after discussion with and approval by the Sponsor.

24. Positive test for human immunodeficiency virus (HIV)

25. Active hepatitis B (defined as having a positive hepatitis B surface antigen [HBsAg] test prior to randomization) or hepatitis C

    1. Note: Patients with past hepatitis B virus (HBV) infection or resolved HBV infection (defined as having a negative HBsAg test and a positive antibody to hepatitis B core antigen antibody test) are eligible. Patients with detectable HBV-DNA are not eligible.
    2. Note: Patients positive for hepatitis C virus (HCV) antibody are eligible only if polymerase chain reaction testing is negative for HCV ribonucleic acid (RNA).

26. Active tuberculosis

27. Administration of a live, attenuated vaccine within 4 weeks prior to start of study treatment or anticipation that such a live attenuated vaccine will be required during the study

28. Prior treatment with CD137 agonists, anti-CTLA4, anti-PD-1, or anti-PD-L1 therapeutic antibody or drug specifically targeting T-cell co-stimulation or immune checkpoint pathways, including prior therapy with anti-tumor vaccines.

29. Pregnancy or lactation. A serum pregnancy test is required within 7 days prior to start of study treatment, or within 14 days with a confirmatory urine pregnancy test within 7 days prior start of study treatment

30. For women who are not post-menopausal (< 12 months of non-therapy-induced amenorrhea) or surgically sterile (absence of ovaries and/or uterus): refusal to use a highly effective contraceptive method (i.e. with a failure rate of < 1% per year such as sexual abstinence, hormonal implants, combined oral contraceptives, vasectomised partner), during the study drug administration and for at least 6 months after the last dose of study medication. Periodic abstinence [e.g., calendar, ovulation, symptothermal, postovulation methods] and withdrawal are not acceptable methods of contraception. A combination of male condom with cap, diaphragm or sponge with spermicide (double barrier methods) is not considered highly effective, birth control methods. Acceptable methods of contraception may include total abstinence in cases where the lifestyle of the patient ensures compliance. A Vasectomised partner is a highly effective birth control method provided that partner is the sole sexual partner of the trial participant and that the vasectomised partner has received medical assessment of the surgical success.

31. For men: refusal to use a highly effective contraceptive method (i.e. with a failure rate of < 1 % per year such as vasectomy, sexual abstinence or female partner use of hormonal implants or combined oral contraceptives) during the study drug administration and for a period of at least 6 months after the last dose of study medication. Periodic abstinence [e.g., calendar, ovulation, symptothermal, post ovulation methods] and withdrawal are not acceptable methods of contraception. A combination of male condom with either, cap, diaphragm or sponge with spermicide (double barrier methods) is not considered highly effective, birth control methods. Acceptable methods of contraception may include total abstinence in cases where the lifestyle of the patient ensures compliance. A vasectomised trial participant is a highly effective birth control method provided that the trial participant has received medical assessment of the surgical success.