WO2020078894A1

WO2020078894A1 - Oncotherapeutic combinations

Info

Publication number: WO2020078894A1
Application number: PCT/EP2019/077764
Authority: WO
Inventors: Adriaan Dirk BINS
Original assignee: Academisch Medisch Centrum
Priority date: 2018-10-15
Filing date: 2019-10-14
Publication date: 2020-04-23
Also published as: TW202019413A

Abstract

An oncotherapeutic product useful for the treatment of cancer. In a preferred embodiment, the oncotherapeutic product is a combination product or kit comprising, the Programmed Death-1 (PD-1) receptor antagonist agent pembrolizumab and the premature stop codon readthrough agent ataluren useful for the treatment of colorectal cancer.

Description

ONCOTHERAPEUTIC COMBINATIONS

FIELD

Presented herein is an oncotherapeutic product useful for the treatment of cancer. In a preferred embodiment, the oncotherapeutic product is a combination product comprising, the Programmed Death 1 (PD-1 ) antagonist agent pembrolizumab and the premature stop codon readthrough agent ataluren useful for the treatment of colorectal cancer (CRC). In another embodiment, the oncotherapeutic product is a kit comprising, the PD-1 antagonist agent pembrolizumab and the premature stop codon readthrough agent ataluren useful for the treatment of CRC.

BACKGROUND

The Programmed Death 1 (PD-1 ) receptor and PD-1 ligands 1 and 2 (PD-L1 and

PD-L2, respectively) play integral roles in immune regulation. Expressed on activated T- cells, PD-1 is activated by PD-L1 and PD-L2 expressed by stromal cells, tumor cells, or both, initiating T-cell death and localized immune suppression, potentially providing an immune-tolerant environment for tumor development and growth. Conversely, inhibition of this interaction can mediate local T-cell responses and enhance antitumor activity in nonclinical animal models.

An immune checkpoint inhibitor (ICI) is a monoclonal antibody (MAb) specifically designed to trigger the immune system’s recognition of proliferating cells in a

tumorogenic environment, binding with either the ligand or tumor cell surface receptor to block these interactions that downregulate the immune response. Certain MAbs such as pembrolizumab bind to the cell PD1 receptor to inhibit binding of the PD-L1 ligand, thus increasing the population of cytotoxic T-cells available to recognize tumor cells for removal.

In certain cancers, a deficient DNA mismatch repair (dMMR) process is known to be a factor in causing uncontrolled cell proliferation. Due to polymerase slippage during DNA replication, the stability of microsatellite Short Tandem Repeats (STR) decreases with increasing length of the STR sequence, resulting in deletions. In healthy cells these deletions are repaired by the MMR process. Contrarily, microsatellite instability (MSI) in dMMR cancers contributes to development of mutations that shift the Open Reading Frame (i.e..creating a’neo-ORF’). These frameshift mutations (FSMs) always result in one or more premature termination codons (PTC) in the neo-ORF, resulting in production of a truncated gene product (i.e., a‘neo-epitope’). In those cancers dependent on MSI, longer microsatellites have a higher rate of FSM.

A clinical study in dMMR metastatic colorectal cancer (mCRC) showed an approximate 60% response (8 out of 13 patients) to ICI treatment with pembrolizumab².

Accordingly, there remains a need to enhance efficacy of immunotherapy treatment for CRC.

SUMMARY

The invention is based on the surprising insight that only 60% of neo-epitopes encoded downstream of insertion-deletion (INDEL) mutations found in coding DNA, as listed in The Cancer Genome Atlas for Colon Adenocarcinoma (TCGA-COAD), were longer than 10 amino acids (AA). Furthermore, only 40% of neo-epitopes were longer than 20AA, as shown in Figure 1 a and Figure 1 b herein. This represents an opportunity to improve the suboptimal response rate of CRC to ICI treatment, as many potentially immunogenic neo-epitopes may have an insufficient length for immune system recognition. Flence, short neo-epitopes cannot be targeted in the anti-tumor immune response. The minimal length required for such neo-epitopes is approximately 10AAs.

Based on the discovery of the large amount of short neo-epitopes in a tumor environment, a combination of cancer immunotherapy with drugs that are capable of elongating short neo-epitopes to longer than 10AAs may potentiate the efficacy of immunotherapy in certain tumor environments.

In colorectal cancer (CRC), the DNA mismatch repair (MMR) process further implicates the production of oncogenic neo-epitopes of various lengths. As a result,

CRC having a deficient MMR (dMMR) process has been found to be moderately responsive to ICI monotherapy while CRC having a proficient MMR (pMMR) process has been found to be refractory to ICI monotherapy. The surprising observation that pMMR CRC is slightly INDEL enriched in coding DNA compared to other cancers, albeit to a lesser extent than dMMR CRC suggests that neo-epitopes of sufficient length in pMMR CRC are not abundant, resulting in a total amount of peptides insufficient for immune recognition. Therefore, the combination product or kit presented herein comprising, the ICI drug pembrolizumab and the read-trough-translatory drug ataluren enhances the therapeutic spectrum of ICI treatment for MMR cancers such as a dMMR CRC moderately responsive to ICI monotherapy or a refractory pMMR CRC.

Accordingly, one embodiment provided herein is a combination product comprising, a combination of the Programmed Death-1 (PD-1 ) receptor antagonist agent

pembrolizumab and the premature stop codon readthrough agent ataluren. In another embodiment, the combination product comprises, an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon readthrough agent ataluren. In another preferred embodiment, the

combination product is for use in the treatment of a mammalian animal. In another preferred embodiment, the combination product is for use in the treatment of a human.

In one preferred embodiment, the combination product is a kit comprising, a combination of the Programmed Death-1 (PD-1 ) receptor antagonist agent

pembrolizumab and the premature stop codon readthrough agent ataluren. In another preferred embodiment, the kit comprises, an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon readthrough agent ataluren. In another preferred embodiment, the kit described herein is prepared for use as a medicament. In another preferred embodiment, the kit is for use in the treatment of a mammalian animal. In another preferred embodiment, the kit is for use in the treatment of a human. In one preferred embodiment, the combination product described herein is for use in the treatment of cancer. In one preferred embodiment, the combination product is for use in the treatment of a colorectal cancer (CRC). In another preferred embodiment, the combination product is for use in the treatment of a CRC having a deficient MMR (dMMR) process (i.e., a dMMR CRC). In another preferred embodiment, the combination product is for use in the treatment of a CRC having a proficient MMR (pMMR) process (i.e., a pMMR CRC). In another preferred embodiment, the combination product is for use in the treatment of an endometrial cancer (EC) having a deficient MMR (dMMR) process (i.e., a dMMR EC). In another preferred embodiment, the combination product is for use in the treatment of a dMMR CRC patient having previously received ICI immunotherapy. In another preferred

embodiment, the combination product is for use in the treatment of a dMMR CRC patient nafve to ICI immunotherapy.

In one preferred embodiment, a method for use of a combination product is described herein for the treatment of cancer in a patient in need thereof comprising, administering to the patient a combination product described herein. In one preferred embodiment, the method for use of a combination product is for the treatment of colorectal cancer (CRC) in a patient in need thereof comprising, administering to the patient a combination product described herein. In another preferred embodiment, the method for use of a combination product is for the treatment of endometrial cancer (EC). In another preferred embodiment, the method for use of a combination product is for the treatment of a CRC having a deficient MMR (dMMR) process (i.e., a dMMR CRC). In another preferred embodiment, the method for use of a combination product is for the treatment of a CRC having a proficient MMR (pMMR) process (i.e., a pMMR CRC). In another preferred embodiment, the method for use of a combination product is for the treatment of an endometrial cancer (EC) having a deficient MMR (dMMR) process (i.e., a dMMR EC). In another preferred embodiment, the method for use of a combination product is for the treatment of a dMMR CRC patient having previously received ICI immunotherapy. In another preferred embodiment, the method for use of a combination product is for the treatment of a dMMR CRC patient nafve to ICI

immunotherapy.

In one preferred embodiment, the method for use of the combination product for the treatment of cancer in a patient in need thereof comprises, administering to a mammalian animal a combination product described herein. In another preferred embodiment, the method for use of the combination product for the treatment of cancer in a patient in need thereof comprises, administering to a human a combination product described herein.

In one preferred embodiment, the kit described herein is for use in the treatment of cancer. In one preferred embodiment, the kit is for use in the treatment of a colorectal cancer (CRC). In another preferred embodiment, the kit is for use in the treatment of a dMMR CRC. In another preferred embodiment, the kit is for use in the treatment of a pMMR CRC. In another preferred embodiment, the kit is for use in the treatment of a dMMR EC. In another preferred embodiment, the kit is for use in the treatment of a dMMR CRC patient having previously received ICI immunotherapy. In another preferred embodiment, the kit is for use in the treatment of a dMMR CRC patient na^'fve to ICI immunotherapy. In one embodiment, a method for use of a kit is described herein for the treatment of cancer in a patient in need thereof comprising, administering to the patient a kit described herein. In one preferred embodiment, the method for use of a kit is for the treatment of colorectal cancer (CRC) in a patient in need thereof comprising, administering to the patient a kit described herein. In another preferred embodiment, the method for use of a kit is for the treatment of colorectal cancer (CRC). In another preferred embodiment, the method for use of a kit is for the treatment of a CRC having a deficient MMR (dMMR) process (i.e., a dMMR CRC). In another preferred

embodiment, the method for use of a kit is for the treatment of a CRC having a proficient MMR (pMMR) process (i.e., a pMMR CRC). In another preferred embodiment, the method for use of a kit is for the treatment of an endometrial cancer (EC) having a deficient MMR (dMMR) process (i.e., a dMMR EC). In another preferred embodiment, the method for use of a kit is for the treatment of a dMMR CRC patient having

previously received ICI immunotherapy. In another preferred embodiment, the method for use of a kit is for the treatment of a dMMR CRC patient na^'fve to ICI immunotherapy.

In one preferred embodiment, the method for use of the kit for the treatment of cancer in a patient in need thereof comprises, administering to a mammalian animal a kit described herein. In another preferred embodiment, the method for use of the kit for the treatment of cancer in a patient in need thereof comprises, administering to a human a kit described herein.

In one preferred embodiment, the combination product comprises, an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon readthrough agent ataluren for use in the treatment of a cancer selected from the group consisting of a colorectal cancer (CRC) and an endometrial cancer (EC).

In one preferred embodiment, the method for use of a combination product for the treatment of cancer in a patient in need thereof comprises, administering to the patient an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon readthrough agent ataluren for use in the treatment of a cancer selected from the group consisting of a colorectal cancer (CRC) and an endometrial cancer (EC).

In one preferred embodiment, the method for use of a kit for the treatment of cancer in a patient in need thereof comprises, administering to the patient an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon readthrough agent ataluren for use in the treatment of a cancer selected from the group consisting of a colorectal cancer (CRC) and an endometrial cancer (EC).

In one preferred embodiment, the kit comprises, administering an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon readthrough agent ataluren for use in the treatment of a cancer selected from the group consisting of a colorectal cancer (CRC) and an endometrial cancer (EC).

In one preferred embodiment, the effective amount of the PD-1 receptor antagonist agent pembrolizumab to be administered comprises, a single dose of 200 mg pembrolizumab every 3 weeks by injection, preferably by infusion.

In one preferred embodiment, the effective amount of the premature stop codon readthrough agent ataluren comprises, a regimen having a first, second and third daily dose, wherein the second dose is administered about 6 hours after the first dose, the third dose is administered about 6 hours after the second dose, and a subsequent first dose is administered about 12 hours after the third dose of the preceding day,

In another preferred embodiment, the first daily dose is selected from the group consisting of 1.25 mg/kg/day, 2.5 mg/kg/day, 5 mg/kg/day and 10 mg/kg/day.

In another preferred embodiment, the second daily dose is selected from the group consisting of 1.25 mg/kg/day, 2.5 mg/kg/day, 5 mg/kg/day and 10 mg/kg/day.

In another preferred embodiment, the third daily dose is selected from the group consisting of 2.5 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day and 20 mg/kg/day.

In another preferred embodiment, the regimen has a first, second and third daily dose selected from 1.25, 1 ,25 and 2.5 mg/kg/day, respectively.

In another preferred embodiment, the regimen has a first, second and third daily dose selected from 2.5, 2.5 and 5 mg/kg/day, respectively. In another preferred embodiment, the regimen has a first, second and third daily dose selected from 5, 5 and 10 mg/kg/day, respectively.

In another preferred embodiment, the regimen has a first, second and third daily dose selected from 10, 10 and 20 mg/kg/day, respectively.

In another more preferred embodiment, the effective amount of the premature stop codon readthrough agent ataluren comprises, a regimen having a first, second and third daily dose selected from 10, 10 and 20 mg/kg/day, respectively; wherein, the second dose of 10 mg/kg is administered about 6 hours after the first dose of 10 mg/kg, the third dose of 20 mg/kg is administered about 6 hours after the second dose of 10 mg/kg, and a subsequent first dose of 10 mg/kg is administered about 12 hours after the third dose of 20 mg/kg from the preceding day.

In one embodiment, the combination product increases the length of one or more neo-epitopes produced during CRC tumorogenesis to enhance immune checkpoint inhibitor (ICI) therapy.

In another embodiment, the combination product increases the length of one or more neo-epitopes in an amount sufficient to induce complexation with the Major Histocompatibility Complex (MHC).

In another embodiment, the combination product results in a 50% increase in MHC-neo-epitope complexation rate.

In another embodiment, the increased length of one or more neo-epitopes is sufficient to induce an immune response by one or more T-cells.

In another embodiment, the combination product doubles neo-epitope length.

In another embodiment, the combination product increases the immune response to reduce tumor size.

In another embodiment, the combination product increases the immune response to induce CRC remission.

In another embodiment, the combination product increases the immune response to induce remission of immunotherapy refractory CRC.

In another embodiment, the combination product synergistically enhances ICI treatment than with either agent alone.

In one embodiment, the kit increases the length of one or more neo-epitopes produced during CRC tumorogenesis to enhance immune checkpoint inhibitor (ICI) therapy.

In another embodiment, the kit increases the length of one or more neo-epitopes in an amount sufficient to induce complexation with the Major Histocompatibility

Complex (MHC).

In another embodiment, the kit results in a 50% increase in MHC-neo-epitope complexation rate.

In another embodiment, the kit doubles neo-epitope length.

In another embodiment, the kit increases the immune response to reduce tumor size.

In another embodiment, the kit increases the immune response to induce CRC remission.

In another embodiment, the kit increases the immune response to induce remission of immunotherapy refractory CRC.

In another embodiment, the kit synergistically enhances ICI treatment than with either agent alone.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 a shows the frequency distribution of the distance (in number of amino acids [AAs]) between an insertion/deletion (INDEL) FSM and the first PTC on the x-axis, where the left y-axis represents the absolute number of INDEL mutated coding transcripts found in DNA as listed in The Cancer Genome Atlas for Colon

Adenocarcinoma (TCGA-COAD). The right y-axis represents the same measurement as a percentage of the total number of INDEL mutated transcripts.

Figure 1 b shows the frequency distribution of the distance (in number of AAs) between the first PTC and the second PTC on the x-axis, downstream of INDEL mutations in the TCGA-COAD samples, where the left y-axis represents the absolute number of INDEL mutated coding transcripts in the samples and the right y-axis represents the same measurement as a percentage of the total number of INDEL mutated transcripts. Figure 2 is a graph of mRNA levels of frameshift mutated genes identified by analysis of DNA sequencing data from 71 MSI-H colorectal patients from the TCGA- COAD database, ranked by mutation frequency. Each point represents one mutation in one patient.

Figure 3 is a graph of percent of reads of mRNA having frameshift mutations compared to DNA at particular genes of MSI-FI colorectal patients from the TCGA- COAD database identified in Figure 2 has having the highest mRNA levels, ranked by mutation frequency. Each point represents one mutation in one patient.

DETAILED DESCRIPTION

One embodiment provided herein is a combination product comprising, a combination of the Programmed Death-1 (PD-1 ) receptor antagonist agent

pembrolizumab and the premature stop codon readthrough agent ataluren. In another embodiment, the combination product comprises, an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon readthrough agent ataluren.

In one preferred embodiment, the combination product is for use in the treatment of a mammalian animal. In another preferred embodiment, the combination product is for use in the treatment of a human.

More preferably, another embodiment provided herein is a kit comprising, a combination of the Programmed Death-1 (PD-1 ) receptor antagonist agent

pembrolizumab and the premature stop codon readthrough agent ataluren. In another embodiment, the kit comprises, an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon

readthrough agent ataluren. In another embodiment, the kit described herein is prepared for use as a medicament.

In one preferred embodiment, the kit is for use in the treatment of a mammalian animal. In another preferred embodiment, the kit is for use in the treatment of a human.

In one embodiment, the combination product described herein is for use in the treatment of cancer. In one preferred embodiment, the combination product is for use in the treatment of a colorectal cancer (CRC). In another preferred embodiment, the combination product is for use in the treatment of a CRC having a deficient MMR (dMMR) process (i.e., a dMMR CRC). In another preferred embodiment, the combination product is for use in the treatment of a CRC having a proficient MMR (pMMR) process (i.e., a pMMR CRC). In another preferred embodiment, the

combination product is for use in the treatment of an endometrial cancer (EC) having a deficient MMR (dMMR) process (i.e., a dMMR EC). In another preferred embodiment, the combination product is for use in the treatment of a dMMR CRC patient having previously received ICI immunotherapy. In another preferred embodiment, the combination product is for use in the treatment of a dMMR CRC patient nafve to ICI immunotherapy.

In one embodiment, a method for use of a combination product is described herein for the treatment of cancer in a patient in need thereof comprising, administering to the patient a combination product described herein. In one preferred embodiment, the method for use of a combination product is for the treatment of colorectal cancer (CRC) in a patient in need thereof comprising, administering to the patient a combination product described herein. In another preferred embodiment, the method for use of a combination product is for the treatment of endometrial cancer (EC). In another preferred embodiment, the method for use of a combination product is for the treatment of a CRC having a deficient MMR (dMMR) process (i.e., a dMMR CRC). In another preferred embodiment, the method for use of a combination product is for the treatment of a CRC having a proficient MMR (pMMR) process (i.e., a pMMR CRC). In another preferred embodiment, the method for use of a combination product is for the treatment of an endometrial cancer (EC) having a deficient MMR (dMMR) process (i.e., a dMMR EC). In another preferred embodiment, the method for use of a combination product is for the treatment of a dMMR CRC patient having previously received ICI

immunotherapy. In another preferred embodiment, the method for use of a combination product is for the treatment of a dMMR CRC patient nafve to ICI immunotherapy.

In one embodiment, the kit described herein is for use in the treatment of cancer. In one preferred embodiment, the kit is for use in the treatment of a colorectal cancer (CRC). In another preferred embodiment, the kit is for use in the treatment of a dMMR CRC. In another preferred embodiment, the kit is for use in the treatment of a pMMR CRC. In another preferred embodiment, the kit is for use in the treatment of a dMMR EC. In another preferred embodiment, the kit is for use in the treatment of a dMMR CRC patient having previously received ICI immunotherapy. In another preferred

embodiment, the kit is for use in the treatment of a dMMR CRC patient na^'fve to ICI immunotherapy. In one embodiment, a method for use of a kit is described herein for the treatment of cancer in a patient in need thereof comprising, administering to the patient a kit described herein. In one preferred embodiment, the method for use of a kit is for the treatment of colorectal cancer (CRC) in a patient in need thereof comprising, administering to the patient a kit described herein. In another preferred embodiment, the method for use of a kit is for the treatment of colorectal cancer (CRC). In another preferred embodiment, the method for use of a kit is for the treatment of a CRC having a deficient MMR (dMMR) process (i.e., a dMMR CRC). In another preferred

In another preferred embodiment, the regimen has a first, second and third daily dose selected from 2.5, 2.5 and 5 mg/kg/day, respectively.

In another preferred embodiment, the regimen has a first, second and third daily dose selected from 5, 5 and 10 mg/kg/day, respectively.

In another embodiment, the combination product doubles neo-epitope length.

In another embodiment, the combination product increases the immune response to reduce tumor size. In another embodiment, the combination product increases the immune response to induce CRC remission.

Complex (MHC).

In another embodiment, the kit doubles neo-epitope length.

It is known that out-of-frame mRNA may have one or more PTCs that result in termination of protein translation and the induction of nonsense mediated decay. For the truncated neo-epitope gene product resulting from such translation to be recognized by the immune system, the peptide must be of sufficient length to be bound to Major Histocompatibility Complex (MHC) proteins expressed by antigen presenting cells (APC). However, MHC has a protein binding groove with restrictive sequence specificity, short neo-epitopes having a random sequence produced by an aberrant mRNA in a tumor environment are more unlikely to be complexed with MHC; those having a peptide length of more than about 10AAs are more likely to be complexed with MHC and, thus, may be targeted by T-cells having a receptor specific for the MHC-neo- epitope complex. The longer a neo-epitope, the higher the chance of complexation with MHC.

A 60% response in a dMMR mCRC clinical study suggests that dMMR mCRC is a highly immunogenic tumor². However, the average response rate for ICIs in solid tumors has been shown to be around 20%. In pMMR mCRC this response rate is below 10%. For any tumor to be responsive to ICI monoclonal antibody (MAb) immunotherapy, cytotoxic T-cells must be able to distinguish between normal AA sequences (i.e., ’epitopes’) and aberrant AA sequences (i.e.,’neo-epitopes’) in the tumor proteome. T-cells distinguish between epitopes and neo-epitopes since, in contrast to

neo-epitopes, epitopes do not complex with MHC. The shorter the length of

neo-epitopes, the more unlikely they are to be complexed with MHC and thus

recognized by the T-cells.

The TCGA-COAD data shown in Figure 1 a and Figure 1 b suggest that many potentially immunogenic neo-epitopes may be of insufficient length for complexation with MHC. The minimal length required for any peptide to bind an MHC isomer is approximately 10 AAs. Since MHC has considerable sequence specificity, a random sequence of 10 AAs or less is very unlikely to bind MHC. Further, since MHC binding is proportional to neo-epitope length, longer neo-epitopes may increase the amount of MHC-neo-epitope complexes with the consequence of a greater immune response in the presence of an ICI.

Figure 1 a shows that only 60% of the neo-epitopes encoded downstream of INDEL mutations (in coding DNA) were longer than 10 AAs. Only 40% were longer than 20 AAs. Figure 1 b shows that the potential elongation of neo-epitopes up to the second PTC results in a median increase of just under 20 AA. Hence this should roughly double the size of such neo-epitopes and make them capable of immune recognition.

Compared to dMMR CRC,‘mismatch repair proficient’ (pMMR) CRC is refractory to ICI monotherapy. While pMMR CRC is slightly enriched for INDEL in coding DNA compared to other cancers, but to a lesser extent than dMMR CRC. Without being limited by theory, the unresponsiveness of pMMR CRC to ICI treatment suggests that MHC-neo-epitope complexation is relatively low and insufficient for immune recognition.

However, when an ICI is combined with a readthrough agent, an increase in neo- epitope length is expected to increase MHC-neo-epitope complexation and thereby increase immune recognition, thus enabling treatment of CRC currently refractory to immunotherapy.

Accordingly, the combination of the ICI pembrolizumab with the readthrough agent ataluren may elongate neo-epitopes, increase the amount of MHC-neo-epitope complexes and may engage an increased cytotoxic T-cell population in an anti-cancer response to potentiate the benefit of cancer immunotherapy.

Pembrolizumab (Keytruda^®) is a potent and highly selective humanized

monoclonal antibody (mAb) of the lgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. Keytruda as monotherapy is indicated for the treatment of advanced (unresectable or metastatic) melanoma in adults, as monotherapy for the treatment of locally advanced or metastatic NSCLC in adults whose tumors express PD-L1 with a >1 % TPS and who have received at least one prior chemotherapy regimen.

Ataluren (Translarna™) is an orally administered, non-aminoglycoside, ribosomal readthrough translation enhancing small molecule designed to allow the protein making apparatus (the ribosome) in cells to skip over (i.e., readthrough) a premature stop codon (PTC), allowing translation of the sequence downstream of a PTC in mRNA transcripts. In a tumorogenic environment, having one or more PTCs present in the neo-ORF resulting from FSMs, the presence of a readthrough agent may enable the translation of additional out-of-frame code. Ataluren has received conditional marketing authorization from the European Commission to treat Duchenne muscular dystrophy resulting from a nonsense mutation in the dystrophin gene, in ambulatory patients aged 2 years and older.

Accordingly, the combination product and kit embodiments described herein include:

A combination product comprising, a combination of the Programmed Death-1 (PD-1 ) receptor antagonist agent pembrolizumab and the premature stop codon readthrough agent ataluren.

A method for use of the combination product for the treatment of cancer in a patient in need thereof comprising, administering the combination product to the patient, wherein the cancer is selected from the group consisting of colorectal cancer (CRC) and endometrial cancer (EC).

The method for use, wherein the CRC is selected from the group consisting of a CRC having a deficient MMR (dMMR) process (dMMR CRC), a CRC having a proficient MMR (pMMR) process (pMMR CRC); and, wherein the EC is selected from an EC having a deficient MMR (dMMR) process (dMMR EC).

The method for use, wherein the patient is selected from the group consisting of a mammalian animal and a human.

The method for use, wherein the combination product comprises an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon readthrough agent ataluren for use in the treatment of cancer in a patient in need thereof comprising, administering the combination product to the patient.

The method for use, wherein the effective amount of the PD-1 receptor antagonist agent pembrolizumab to be administered comprises, a single dose of 200 mg pembrolizumab every 3 weeks by infusion.

The method for use, wherein the effective amount of the premature stop codon readthrough agent ataluren comprises, a regimen having a first, second and third daily dose, wherein the second dose is administered about 6 hours after the first dose, the third dose is administered about 6 hours after the second dose, and a subsequent first dose is administered about 12 hours after the third dose of the preceding day,

The method for use, wherein the regimen has a first, second and third daily dose selected from 1.25, 1 ,25 and 2.5 mg/kg/day, respectively.

The method for use, wherein the regimen has a first, second and third daily dose selected from 2.5, 2.5 and 5 mg/kg/day, respectively.

The method for use, wherein the regimen has a first, second and third daily dose selected from 5, 5 and 10 mg/kg/day, respectively. The method for use, wherein the regimen has a first, second and third daily dose selected from 10, 10 and 20 mg/kg/day, respectively.

The method for use, wherein the effective amount of the premature stop codon readthrough agent ataluren comprises, a regimen having a first, second and third daily dose selected from 10, 10 and 20 mg/kg/day, respectively; wherein, the second dose of 10 mg/kg is administered about 6 hours after the first dose of 10 mg/kg, the third dose of 20 mg/kg is administered about 6 hours after the second dose of 10 mg/kg, and a subsequent first dose of 10 mg/kg is administered about 12 hours after the third dose of 20 mg/kg from the preceding day.

A kit comprising, a combination of the Programmed Death-1 (PD-1 ) receptor antagonist agent pembrolizumab and the premature stop codon readthrough agent ataluren.

A use of the kit, wherein the kit is prepared for use as a medicament.

A use of the kit, for the treatment of cancer in a patient in need thereof

comprising, administering the kit to the patient, wherein the cancer is selected from the group consisting of colorectal cancer (CRC) and endometrial cancer (EC).

The use of the kit, wherein the CRC is selected from the group consisting of a CRC having a deficient MMR (dMMR) process (dMMR CRC), a CRC having a proficient MMR (pMMR) process (pMMR CRC); and, wherein the EC is selected from an EC having a deficient MMR (dMMR) process (dMMR EC).

The use of the kit, wherein the patient is selected from the group consisting of a mammalian animal and a human.

The use of the kit, wherein the kit comprises an effective amount of the PD-1 receptor antagonist agent pembrolizumab and an effective amount of the premature stop codon readthrough agent ataluren for the treatment of cancer in a patient in need thereof comprising, administering the kit to the patient.

The use of the kit, wherein the effective amount of the PD-1 receptor antagonist agent pembrolizumab to be administered comprises, a single dose of 200 mg

pembrolizumab every 3 weeks by infusion.

The use of the kit, wherein the effective amount of the premature stop codon readthrough agent ataluren comprises, a regimen having a first, second and third daily dose, wherein the second dose is administered about 6 hours after the first dose, the third dose is administered about 6 hours after the second dose, and a subsequent first dose is administered about 12 hours after the third dose of the preceding day,

The use of the kit, wherein the regimen has a first, second and third daily dose selected from 1.25, 1 ,25 and 2.5 mg/kg/day, respectively

The use of the kit, wherein the regimen has a first, second and third daily dose selected from 2.5, 2.5 and 5 mg/kg/day, respectively.

The use of the kit, wherein the regimen has a first, second and third daily dose selected from 5, 5 and 10 mg/kg/day, respectively.

The use of the kit, wherein the regimen has a first, second and third daily dose selected from 10, 10 and 20 mg/kg/day, respectively.

The use of the kit, wherein the effective amount of the premature stop codon readthrough agent ataluren comprises, a regimen having a first, second and third daily dose selected from 10, 10 and 20 mg/kg/day, respectively; wherein, the second dose of 10 mg/kg is administered about 6 hours after the first dose of 10 mg/kg, the third dose of 20 mg/kg is administered about 6 hours after the second dose of 10 mg/kg, and a subsequent first dose of 10 mg/kg is administered about 12 hours after the third dose of 20 mg/kg from the preceding day.

Definitions

As used herein, the term“about” means a range around a given value wherein the resulting value is substantially the same as the expressly recited value. In one embodiment,“about” means within 25% of a given value or range. For example, the phrase“about 70% by weight” comprises at least all values from 52% to 88% by weight. In another embodiment, the term“about” means within 10% of a given value or range. For example, the phrase“about 70% by weight” comprises at least all values from 63% to 77% by weight. In another embodiment, the term“about” means within 7% of a given value or range. For example, the phrase“about 70% by weight” comprises at least all values from 65% to 75% by weight.

Concentrations, amounts, cell counts, percentages and other numerical values may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range was explicitly recited.

As used herein, the terms“therapies” and“therapy” can refer to any protocol(s), method(s), compositions, formulations, and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a condition or disorder or one or more symptoms thereof {e.g., one or more symptoms or one or more conditions associated therewith).

In certain embodiments, the terms“therapies” and“therapy” refer to drug therapy such as chemotherapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, antiviral therapy and/or other therapies useful in treatment, management, prevention, or amelioration of a condition or disorder or one or more symptoms thereof {e.g., one or more symptoms or one or more conditions associated therewith). In certain embodiments, the term“therapy” refers to a therapy other than the combination product or kit described herein. In specific embodiments, an

“additional therapy” and“additional therapies” refer to a therapy other than a treatment using the combination product or kit described herein. In a specific embodiment, a therapy includes the use of the combination product or kit described herein as an adjuvant therapy. For example, using the combination product or kit described herein in conjunction with a drug therapy such as chemotherapy, biological therapy, surgery, supportive therapy, antiviral therapy and/or other therapies useful in treatment, management, prevention, or amelioration of a condition or disorder or one or more symptoms thereof {e.g., one or more symptoms or one or more conditions associated therewith).

As used herein, the term“human infant” refers to a newborn to 1 year old year human.

As used herein, the term“human toddler” refers to a human that is 1 year to 3 years old.

As used herein, the term“human child” refers to a human that is 1 year to 18 years old.

As used herein, the term“human adult” refers to a human that is 18 years or older.

As used herein, the term“middle-aged human” refers to a human between the ages of 30 and 64.

As used herein, the term“elderly human” refers to a human 65 years or older.

As used herein, the term“subject” may be used interchangeably with the term “patient” where both terms refer to an individual being administered a therapy as described herein. In a specific embodiment, the individual is a human.

As used herein, the term“effective amount” in the context of administering the combination product or kit described herein to a subject having a dMMR mCRC, dMMR mEC, or pMMR mCRC refers to that amount of the combination product or kit described herein

that results in a beneficial or therapeutic effect. In specific embodiments, an “effective amount” of the combination product or kit described herein refers to an amount of the combination product or kit which is sufficient to achieve at least one, two, three, four or more of the beneficial or therapeutic effects described herein.

As used herein, the term“premature stop codon readthrough agent” refers to the small molecule compound ataluren having demonstrated translational readthrough of PTCs, enabling the synthesis and expression of full-length functional proteins at sufficient levels to achieve therapeutic benefit. W02004/091502 discloses ataluren and its use for translational readthrough of PTCs.

As used herein, the term“PD-1 receptor antagonist” refers to a compound capable of blocking or reducing the interaction of PD-L1 on tumor cells while enhancing T-cell interaction with the PD-1 receptor

Combination Product or Kit for use in the Treatment of Cancer

The importance of intact immune surveillance in controlling outgrowth of neoplastic transformation has been known for decades. Accumulating evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in various malignancies. In particular, the presence of CD8+ T-cells and a high ratio of CD8+ effector T-cells to FoxP3+ regulatory T-cells seems to correlate with improved prognosis and long-term survival in many solid tumors.

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 Pdcdl ) is an Ig superfamily member related to CD28 and CTLA-4 which has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2).

Binding of PD-L1 to PD-1 inhibits T-cell activation triggered through the T-cell receptor. PD-L1 is expressed at low levels on various non-hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is only detectably expressed on antigen-presenting cells found in lymphoid tissue or chronic inflammatory

environments. PD-L2 is thought to control immune T-cell activation in lymphoid organs, whereas PD-L1 serves to dampen unwarranted T-cell function in peripheral tissues. Although healthy organs express little (if any) PD-L1 , a variety of cancers were demonstrated to express abundant levels of this T-cell inhibitor. PD-1 has been suggested to regulate tumor-specific T-cell expansion in subjects with melanoma (Mel). This suggests that the PD-1/PD-L1 pathway plays a critical role in tumor immune evasion and should be considered as an attractive target for therapeutic intervention.

An open label, Phase 1 -2 pilot study will evaluate the toxicity and clinical activity of the combination product or kit pembrolizumab and ataluren to treat unstable microsatellite mismatch repair deficient (dMMR) metastatic colorectal adenocarcinoma and metastatic endometrial carcinoma with an additional exploratory cohort to evaluate the effect of the combination product or kit to treat stable microsatellite mismatch repair proficient (pMMR) metastatic colorectal adenocarcinoma.

The study described herein is designed to test the combination of

immunotherapy with pembrolizumab combined with ataluren in dMMR colorectal adenocarcinomas and endometrial carcinomas with an additional exploratory cohort in microsatellite stable (pMMR) colorectal adenocarcinomas. Microsatellite instability (dMMR) results from the accumulation of INDEL DNA mutations in microsatellites. This occurs in tumors with a deficiency in the DNA mismatch repair (dMMR) process.

dMMR CRC patients can be included whether they are treatment na^'fve or have had one or more prior chemotherapy regimens. For medical reasons, certain pMMR CRC patients can be included if they have received two or more prior chemotherapy regimens. For medical reasons, dMMR EC patients can be included if they have received one prior chemotherapy regimen.

In Phase-1 of the study, after a one-week run-in period, the effective ataluren dose is determined in 2-4 dose escalation cohorts of 3 patients each, who will then receive pembrolizumab at a fixed dose of 200 mg i.v. every 21 days in combination with 3 days oral ataluren continuously. In all cohorts the ataluren dose will be based on body weight according to a standard regimen. In the first cohort, ataluren will be dosed at 25% of body weight (2.5 mg/kg morning dose; 2.5 mg/kg midday dose; 5 mg/kg evening dose); the second cohort will be dosed at 50% of body weight (5 mg/kg morning dose; 5 mg/kg midday dose; 10 mg/kg evening dose) and the third cohort will be dosed at 100% body weight (10 mg/kg morning dose; 10 mg/kg midday dose; 20 mg/kg evening dose).

In Phase-2 of the study, two cohorts of patients will receive pembrolizumab combined with ataluren dosed at the Maximum Tolerated Dose (MTD) of ataluren.

Pembrolizumab will be administered at a fixed dose of 200 mg i.v. every 21 days.

Cohort A may include up to 20 dMMR CRC or EC patients and Cohort B may include up to 15 pMMR CRC patients; each Cohort will be treated with the combination product or kit (pembrolizumab combined with ataluren) in an open label Phase 2 pilot design.

Patients that have received prior treatment with either anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) or anti-PD1 monoclonal antibodies will be excluded.

The study is designed to evaluate the toxicity and the treatment benefit of pembrolizumab combined with ataluren. The added benefit of ataluren will be assessed by case-matched comparison of the results of this trial with those of the pembrolizumab metastatic melanoma (mMel) trial (ClinicalTrials.gov Identifier NCT01876511 ). In the NCT01876511 study, patients with comparable characteristics were treated with pembrolizumab only. The study protocol described herein is analogous to the

NCT01876511 study, with the exception herein that dMMR patients can also be included after one prior treatment regimen with pembrolizumab only for metastatic CRC. The primary endpoints for Cohorts A and B are the immune-related progression- free survival (irPFS) rate at 21 weeks and the immune-related objective response rate (irORR) assessed using immune-related response criteria (irRC); the irPFS is defined as the proportion of subjects alive and free of disease progression at 21 weeks as determined by irRC; the irORR is defined as the proportion of subjects whose best overall response is a Complete Response (CR) or Partial Response (PR). The results within each cohort will be assessed separately.

Archived tumor samples or newly obtained biopsies will be used for determining MMR status. dMMR tumors will be defined using standard clinical criteria and require at least two loci to be evaluable¹.

Patient immune and clinical responses will be evaluated through the following methods at baseline and during treatment using: i). tumor biopsies; ii). peripheral blood lymphocytes; iii). serum and plasma; and. iv). CT scan.

Patients who have a confirmed CR by two scans > 6 weeks apart and who have been on combined pembrolizumab/ataluren treatment for at least 6 months may discontinue the treatment after completing at least two cycles beyond the initial determination of CR. The pembrolizumab/ataluren treatment may be resumed upon disease recurrence in these patients.

Patients with tumor progression on two consecutive scans by Response

Evaluation Criteria In Solid Tumors (RECIST) imaging or as determined by laboratory testing parameters prior to a 30 week/7 month evaluation but without rapid clinical deterioration or significant change in performance status who do not require additional immediate therapy, may continue to be treated with pembrolizumab combined with ataluren and clinically observed following the assigned imaging schedule to allow detection of a subsequent tumor response. Tumor assessments will be made using RECIST 1.1 and immune-related response criteria (irRC). Subjects that meet the above criteria and continue on study therapy must discontinue pembrolizumab/ataluren upon confirmed documentation of PD at 30 weeks using irRC unless there are signs of delayed stabilization: i.e., evidence of disease progression at week 21 with stabilization at week 28 (by comparison to week 21 ). Primary Objective

The study will determine immune-related progression free survival (irPFS) rate at 21 weeks and objective response rate (irORR) at 30 weeks in patients with dMMR mCRC, pMMR mCRC and dMMR mEC treated with pembrolizumab combined with ataluren using immune related Response Criteria (irRC). The use of ataluren is intended to synergistically augment the efficacy of pembrolizumab without additional toxicity in dMMR mCRC, pMMR mCRC and dMMR mEC, wherein use of ataluren may enhance the therapeutic spectrum of pembrolizumab to include pMMR mCRC.

Secondary Objectives

The study will also be used to: i). Determine the overall survival of patients with dMMR mCRC, pMMR mCRC and dMMR mEC treated with pembrolizumab combined with ataluren; ii). Estimate irPFS and PFS in patients with dMMR mCRC, pMMR mCRC and dMMR mEC treated with pembrolizumab combined with ataluren at 30 weeks using irRC and RECIST 1.1 iii). Estimate best overall response rate and disease control rate in patients with dMMR mCRC, pMMR mCRC and dMMR mEC treated with

pembrolizumab combined with ataluren; iv). Assess safety and characterize toxicities of pembrolizumab combined with ataluren in patients with dMMR mCRC, pMMR mCRC and dMMR mEC; v). Compare the outcomes of the present primary and secondary study objectives with a series of case-matched controls treated within the

NCT01876511 study.

Exploratory Objectives

The study will also be used to: i). Compare mRNA levels in the tumor before and after start of ataluren; ii). Assess tumor tissue for disease stability over the course of treatment using next generation sequencing technology; iii). Compare archived tumor and polyp tissue (when available) obtained at baseline and after treatment failure;

iv). Assess tumor burden dynamics using both standard protein biomarkers such as CEA, CA19-9, C-reactive protein, and other exploratory circulating biomarkers in serial collections of sera and plasma at baseline and throughout treatment; v). Assess molecular characteristics in tumor tissue of the subjects enrolled and correlate these with treatment response and toxicity; vi). Collect peripheral blood lymphocytes to explore the association of lymphocyte activation markers and T cell reactivity’s against neo-epitopes with clinical responses.

Rationale for Pembrolizumab Dose Selection/Reqimen/Modifications

The dose escalation portion of a first-in-human (FIH) open-label Phase 1 trial for the safety and clinical activity of pembrolizumab as a single agent therapy

(NCT01295827) evaluated three dose levels, 1 mg/kg, 3 mg/kg, and 10 mg/kg, administered every 2 weeks (Q2W) in subjects with advanced solid tumors. All three dose levels were well tolerated and no dose-limiting toxicities were observed.

NCT01295827 showed evidence of pembrolizumab target engagement and objective evidence of tumor size reduction at all Q2W dose levels (1 mg/kg, 3 mg/kg and 10 mg/kg). No MTD has been identified to date. The highest NCT01295827 dose tested 10.0 mg/kg will be the dose and schedule utilized in Cohorts A, B, C and D of the protocol herein to test for initial tumor activity. Recent data from other pembrolizumab clinical studies have shown that a lower dose and a less frequent schedule may be sufficient for target engagement and clinical activity.

NCT01295827 PK data analysis of pembrolizumab administered Q2W and Q3W showed a slower systemic clearance, limited volume of distribution, and a long half-life (refer to pembrolizumab IB). Pharmacodynamic data (IL-2 release assay) suggested that peripheral target engagement was durable (>21 days). This early PK and pharmacodynamic data provides scientific rationale for testing a Q2W and Q3W dosing schedule.

A population PK analysis has been performed using serum concentration time data from 476 patients. Within the resulting population PK model, clearance and volume parameters of pembrolizumab were found to be dependent on body weight. The relationship between clearance and body weight, with an allometric exponent of 0.59, is within the range observed for other antibodies and would support both body weight normalized dosing or a fixed dose across all body weights. Pembrolizumab has been found to have a wide therapeutic range based on NCT01295827. The differences in exposure for a 200 mg fixed dose regimen relative to a 2 mg/kg Q3W body weight based regimen are anticipated to remain well within the established exposure margins of 0.5 - 5.0 for pembrolizumab in the melanoma indication. The exposure margins are based on the notion of similar efficacy and safety in melanoma at 10 mg/kg Q3W vs. the proposed dose regimen of 2 mg/kg Q3W (i.e. 5-fold higher dose and exposure). The population PK evaluation revealed that there was no significant impact of tumor burden on exposure. In addition, exposure was similar between the NSCLC and melanoma indications. Therefore, there are no anticipated changes in exposure between different indication settings.

The rationale for further exploration of 2 mg/kg and comparable doses of pembrolizumab in solid tumors is based on: i). similar efficacy and safety of

pembrolizumab when dosed at either 2 mg/kg or 10 mg/kg Q3W in melanoma patients; ii). the flat exposure-response relationships of pembrolizumab for both efficacy and safety in the dose ranges of 2 mg/kg Q3W to 10 mg/kg Q3W; iii). the lack of effect of tumor burden or indication on distribution behavior of pembrolizumab (as assessed by the population PK model); and, iv). the assumption that the dynamic of pembrolizumab target engagement will not vary meaningfully with tumor type.

The choice of the 200 mg Q3W as an appropriate dose for the switch to fixed dosing is based on simulations performed using the population PK model of

pembrolizumab showing that the fixed dose of 200 mg every 3 weeks will provide exposures that i). are optimally consistent with those obtained with the 2 mg/kg dose every 3 weeks; ii). will maintain individual patient exposures in the exposure range established in melanoma as associated with maximal efficacy response; and, iii). will maintain individual patient’s exposure in a range established in NCT01295827 that are well tolerated and safe.

A fixed dose regimen will simplify the dosing regimen to be more convenient for physicians and to reduce potential for dosing errors. A fixed dosing scheme will also reduce complexity in the logistical chain at treatment facilities and reduce wastage.

Rationale for Dose Selection/Reqimen/Modifications of Ataluren

The safety and efficacy of ataluren were assessed in a randomized, double-blind, placebo-controlled, multicenter nonsense mutation Duchenne muscular dystrophy (nmDMD) study of 174 male patients ages 5 to 20 years. nmDMD is caused by a congenital nonsense mutation in the dystrophin gene, resulting in production of a non- functional truncated dystrophin protein. Patients in the trial were required to have documented confirmation of the presence of a nonsense mutation in the dystrophin gene as determined by gene sequencing. The majority of patients in all treatment groups were Caucasian (90%). Patients were randomized in a 1 :1 :1 ratio and received ataluren or placebo 3 times per day (morning, midday, and evening) for 48 weeks, with 57 receiving placebo, 57 receiving ataluren 10-, 10-, 20-mg/kg, and 60 receiving ataluren 20-, 20-, 40 mg/kg; 173 patients completed the study. The primary efficacy endpoint evaluated the effect of ataluren on ambulation as assessed by the change in distance (6MWD) walked during a 6MWT. The post hoc analysis showed that from baseline to Week 48, patients receiving ataluren 10-, 10-, 20-mg/kg had a 12.9 meter mean decline in 6MWD, and patients receiving placebo had a 44.1 -meter mean decline in 6MWD. Thus, the mean change in observed 6MWD from baseline to Week 48 was 31.3 meters better in the ataluren 10-, 10-, 20-mg/kg arm than in the placebo arm (p=0.056). In a statistical based model, the estimated mean difference was 31.7 meters (adjusted p=0.0367). These results indicate that ataluren 10-, 10-, 20-mg/kg slows the loss of walking ability in nmDMD patients. Likely, this effect is attributable to an increase in the translation of full length dystrophin from the PTC containing dystrophin

transcripts.

In nonclinical neurological, respiratory, and cardiovascular safety pharmacology studies, ataluren has shown no adverse effects. Secondary pharmacodynamic (PD) assessments performed in vitro and in vivo have documented selective readthrough of premature stop codons without readthrough of normal stop codons. Toxicology studies in mice through 1 month, in rats through 26 weeks, and in dogs through 52 weeks have identified safety signals to be monitored in the clinic. Nephrosis was observed in mice as soon as within 1 or 2 days of dosing; a no observed adverse effect level (NOAEL) was not identified. At the lowest observed adverse effect level (LOAEL), mean steady state exposure in mice was 0.3-fold exposure in patients. The nephrosis primarily involved the distal nephron and was characterized by degenerative changes and epithelial cell proliferation in association with renal tubular dilatation and proteinaceous material in the tubules. The renal finding was reversible and no drug-related effects on the kidney were observed in rats dosed for 24 months or in dogs dosed for 52 weeks despite achievement of exposures in rats and dogs that were comparable to or greater than those observed in mice. In dogs dosed for > 20 weeks, lymphohistiocytic infiltrates with adjacent foci of parenchymal degeneration in sites of corticosteroid production were observed; sites of mineralocorticoid production were unaffected. The histologic findings in the adrenal gland were not reversible up to 8 weeks following chronic administration. Functional findings included an increase in serum basal

adrenocorticotropic hormone (ACTH) and a reduction in mean cortisol responses to ACTH stimulation. Serum aldosterone was unaffected and no evidence of

hyperkalemia consistent with a mineralocorticoid deficiency was seen. A NOAEL was not identified for the histologic finding, but exposure at the LOAEL for functional effects on the adrenal gland was 0.8-fold exposure in patients.

Ataluren had no effect on male and female fertility in rats. Ataluren was not teratogenic in rats and rabbits; fetal toxicity and postnatal development effects were observed only at doses which resulted in maternal toxicity. Maternal administration of ataluren in rats had no effect on reproduction of offspring and on embryo/fetal development in the next generation. Ataluren is not genotoxic. No ataluren-related tumors were observed in a 26-week carcinogenicity study conducted in a transgenic mouse model (Tg.rasH2 mice). Tumors observed in rats occurred at exposures that exceeded clinical exposure and were not considered relevant to humans. The completed toxicology program supports dosing in patients >2 years old. A juvenile toxicology program in dogs is ongoing, and when completed, will bridge to the completed toxicology program and will support dosing in neonates and older patients. Findings (vacuolation of the ganglion cell, optic fiber and inner nuclear layers of the retina) not present in the completed toxicology program which supports dosing in patients > 2 years of age were observed in the 28-day dose range finding study in neonatal dogs. There was no necrosis or degeneration and the appearance of the retinal vacuolation and the lack of associated tissue reaction was suggestive of edema.

Rationale for Efficacy Endpoints

The endpoints are similar to the endpoints in the pembrolizumab NCT01876511 trial, with minor adaptations to accommodate the new Q3W treatment regimen.

For medical reasons, dMMR EC patients can be included after one metastatic treatment (instead of two metastatic or non-metastatic chemotherapy treatments required in the NCT01876511 trial).

Given the caveats of cross-trial comparisons, the close analogy to the

NCT01876511 study facilitates case-matched cross-trial comparisons of the results of both studies.

Entry Criteria

Phase-1 / Dose Escalation: 6-12 patients with histologically proven metastatic or locally advanced deficient mismatch repair (dMMR) colorectal adenocarcinoma (CRC), dMMR endometrial carcinoma (EC) or mismatch repair proficient (pMMR) CRC.

Cohort A: 20 patients with histologically proven metastatic or locally advanced deficient mismatch repair (dMMR) colorectal adenocarcinoma (CRC) or dMMR

endometrial carcinoma (EC)

Cohort B: 15 patients with histologically proven metastatic or locally advanced pMMR CRC.

Subject Inclusion Criteria: A subject must have at least one lesion with measurable disease as defined by 10 mm in longest diameter for a soft tissue lesion or 15mm in short axis for a lymph node by RECIST 1.1 and irRC criteria for response assessment.

For dMMR mEC Inclusion: A subject must have received at least 1 prior cancer therapy regimen for metastatic EC.

For pMMR mCRC Inclusion: A subject must have: i). Received at least 2 prior cancer therapy regimens for metastatic or non-metastatic CRC; ii). A life expectancy of greater than 3 months; and, ii). Normal organ and marrow function.

The treatment regimen to be used in the instant trial is outlined in Table 1 , where

QD OD: once daily oral dose; Q3W: three doses/week; PBZ: pembrolizumab.

Table 1 Treatment Regimen

In Phase 1 , the dose of ataluren will be escalated according to the dose scheme outlined in Table 2, wherein the subgroup minimally requires three patients, each dosed based on body weight at the indicated percentage of the approved 10,10,20 regimen.

Table 2 Ataluren Dose Escalation Scheme

In Phase 1 , patients will start treatment in a staged approach. For this purpose, the start dates of the study treatment for the included patients will be spaced minimally 3 weeks apart. Dose limiting toxicity (DLT) will be evaluated after 28 days (Cycle 0 and Cycle 1 ).

A minimum of three patients will be entered on each dose level. Subsequent enrollment of new dose levels will be based on the toxicity assessment at Cycle 0 and Cycle 1.

Timing of Dose Administration

Pembrolizumab treatment (200 mg) should be administered on Day 1 of each cycle (except Cycle 0) as a 30 minute IV infusion every 3 weeks.

Discontinuation of Study Therapy After CR

Discontinuation of study treatment may be considered for subjects who have attained a confirmed CR after having been treated for at least 24 weeks with the ataluren/pembrolizumab combination product or kit and had at least two treatment cycles beyond the date when the initial CR was declared. Subjects who then experience radiographic disease progression may be eligible for up to one year of additional treatment with the combination product or kit if: i). no cancer treatment was

administered since the last dose of pembrolizumab; and, ii). the subject meets the safety parameters listed in the Inclusion/Exclusion criteria. Subjects will resume therapy at the same dose of ataluren at the time of initial discontinuation. Continuation of Study Therapy After Disease Progression At 7 Months/30 Weeks

Treatment with the combination product or kit is expected to trigger immune- mediated responses, which require activation of the immune system prior to the observation of clinical responses. Such immune activation may take weeks to months to be evident. Some patients may have objective volume increase of tumor lesions or other disease parameters within weeks following the start of the treatment. Such patients may not have had sufficient time to develop the required immune activation or, in some patients, tumor volume or other disease parameter increases may represent infiltration of lymphocytes into the original tumor. In conventional studies, such tumor volume or relevant laboratory parameter increases during the first 2-4 months of the study would constitute PD and lead to discontinuation of imaging to detect response, thus disregarding the potential for subsequent immune-mediated clinical response.

Therefore, patients with tumor progression by RECIST imaging or laboratory parameters prior to their 7 month (30 week) evaluation but without rapid clinical deterioration or change in PR who do not require additional immediate therapy, may continue to be treated with the combination product or kit and be clinically observed following the assigned imaging schedule to allow detection of a subsequent tumor response. Tumor assessments will be made using RECIST 1.1 and immune-related RECIST criteria (irRC). Subjects that meet the above criteria and continue on study therapy must discontinue the treatment if there are no signs of disease stabilization by 7 months using irRC.

Solid Tumor Imaging and Assessment of Antitumor Effect

For the purposes of this study, patients should be evaluated for response at the 12 week timepoint and then every 9 weeks thereafter. Response and progression will be evaluated in this study using the new international criteria proposed by the Revised RECIST Guideline (version 1.1 ) and the irRC. Changes in the largest diameter

(unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST criteria. STATISTICAL ANALYSIS PLAN

Sample Size/Accrual Rate

Cohort A will enroll 20 patients; Cohort B will enroll 15 patients. For Cohort A and Cohort B, there will be 1 final analysis for irPFS and 1 final analysis for irORR.

Stratification Factors

There is no randomization nor stratification in this study. Two Cohorts of patients will be enrolled based on presence of metastatic colorectal adenocarcinoma (mCRC) or metastatic endometrial carcinoma (mEC) and dMMR status: Patients with dMMR mCRC or mEC (Cohort A); and, patients with pMMR mCRC (Cohort B);

Analysis of Primary Endpoints

For Cohort A (n=20) and Cohort B (n=15), immune-related Progression-Free

Survival (irPFS) rate at 21 weeks and immune-related Objective Response Rate

(irORR) assessed using immune-related Response Criteria (irRC) are co-primary endpoints. The irPFS rate at 21 weeks will be estimated as the proportion of patients alive and free of disease progression at 21 weeks per irRC, along with its 95%

confidence interval (Cl); irORR will be estimated as the proportion of subjects whose best overall response is either a CR or PR with corresponding 95% Cl. A non-responder to therapy are those patients who drop out of the study due to toxicity and do not have a follow-up scan. The formal statistical testing for irORR will take place only if 21 -week irPFS is determined to be statistically significant. Therefore, an adjusted alpha for two co-primary endpoints will not be used.

The added benefit of ataluren will be assessed by case-matched comparison of the irORR of the pembrolizumab/ataluren combination with the irORR found in the NCT01876511 trial². In the NCT01876511 study similar patients were treated with pembrolizumab only. For this purpose, the study protocol is analogous to that of the NCT01876511 study.

The case-matching will be performed on the following variables at the time of last infusion (or first day of last cycle for oral treatment) of the previous study: i). Toxicity; ii). Progression as reason for discontinuation; iii). Number of previous treatment groups administered; iv). Age; ii). ECOG performance status; v). RAS status for colon patients (mutant / wildtype); vi). LDH levels (normal / above upper limit of normal); and, vii). Synchronous/metachronous (>6 months) metastatic disease.

Data will be compared between groups for primary and secondary outcomes using chi-squared tests for categorical variables.

For Cohort B in the instant study, the irORR results will be case-matched and compared with the irORR results from the NCT01876511 study. A pilot study has determined that a 50% power is sufficient to detect a 20% irORR rate in the pMMR patients at an alpha of 5%.

For Cohort A in the instant study, the irPFS results will be case-matched and compared with the irPFS results from the NCT01876511 study. A pilot study has determined that a 50% power is sufficient to detect a 25% % increase in the irPFS rate at 21 weeks at an alpha of 5%.

Analysis of Secondary Endpoints

Overall survival (OS) is the time from the first day of study treatment with the pembrolizumab/ataluren combination to the date of death due to any cause. A subject who has not survived will be censored as of the last known survival date. Kaplan-Meier curves will be used to summarize OS. The analyses below will be conducted on the basis of responses as assessed by both RECIST v1.1 and irRC. Summary statistics will be provided for each Cohort.

Progression Free Survival (PFS) is defined as the time from the first day of study treatment to the date of the first documented tumor progression or failure to survive due to any cause, whichever occurs first. Subjects who did not progress or survive will be censored on the date of their last evaluable tumor assessment. Kaplan-Meier curves will be used to summarize PFS.

Objective Response Rate (ORR) is defined as the proportion of subjects whose Best Overall Response (BOR) from baseline is either a CR or PR. The BOR is determined by the best response designation recorded between the date of the first study treatment and the date of objectively documented progression or the date of subsequent anti-cancer therapy, whichever occurs first. For subjects without

documented progression or subsequent anticancer therapy, all available response designations will contribute to the BOR determination. For subjects who continue use of the combination product or kit beyond progression, the BOR should be determined based on response designations recorded at the time of the initial progression. DCR is the proportion of patients with CR, PR, or SD.

Among patients with an objective response, the Date of Objective Response Rate (DOOR) is defined as the time between the date of initial complete or partial response to the date of the first documented tumor progression or death due to any cause. Subjects who neither progress nor die will be censored on the date of their last assessment. The Time to Objective Response (TTOR) is defined as the time from the first day of study treatment to the date of the first documented CR or PR. DOOR and TTOR will be evaluated for responsive patients (CR or PR) only.

Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 Criteria for Evaluating Response in Solid Tumors

RECIST version 1.1 (As published in the European Journal of Cancer³) will be used for assessment of tumor response in this study. While either CT or MRI may be utilized, as per RECIST 1.1 , CT is the preferred imaging technique in this study. In addition, volumetric analysis will be explored by central review for response

assessment.

Immune Related Response Criteria (irRC) Criteria for Evaluating Response in Solid Tumors⁴

Measurable disease:

Neoplastic masses that can be precisely measured in 2 in-plane perpendicular diameters. Both its longest diameter and its longest perpendicular must be greater than or equal to 10 mm. Lymph nodes must have a short-axis line-length of > 15 mm.

Malignant lymph nodes must be measurable in 2 perpendicular diameters. Both its longest diameter and its longest perpendicular must be greater than or equal to 15 mm. The quantitative endpoint will be defined as the product of the longest diameter with its longest perpendicular.

Non-measurable disease:

Non-measurable lesions are those that are not suitable for quantitative

assessment over time. These include: i). Neoplastic masses that are too small to measure, having a longest uninterrupted diameter or longest perpendicular less than 10 mm); ii). Neoplastic masses whose boundaries cannot be distinguished, including masses which cannot be demarcated from surrounding tissue because of inadequate contrast, masses with overly complex morphology, or those with highly heterogeneous tissue composition; iii). Other types of lesions that are confidently felt to represent neoplastic tissue, but difficult to quantify in a reproducible manner, including bone metastases, leptomeningeal metastases, malignant ascites, pleural/pericardial effusions, inflammatory breast disease, lymphangitis cutis/pulmonis, cystic lesions, ill-defined abdominal masses, etc.

For irRC, only target lesions selected at baseline and measurable new lesions are taken into account. At the baseline tumor assessment, the sum of the products of the two largest perpendicular diameters (SPD) of all index lesions (five lesions per organ, up to 10 visceral lesions and five cutaneous index lesions) is calculated.

At each subsequent tumor assessment, the SPD of the index lesions and of new, measurable lesions (> 5 X 5 mm; up to 5 new lesions per organ: 5 new cutaneous lesions and 10 visceral lesions) are added together to provide the total time-point tumor burden.

Response Definitions Using irRC:

Complete Response (irCR): Complete disappearance of all tumor lesions (whether

measureable or not, and no new lesions). CR must be confirmed by repeated, consecutive assessments made no less than 4 weeks from the date first documented.

Partial Response (irPR): Decrease in SPD of 50% or greater by a consecutive

assessment at least 4 weeks after first documentation.

Stable Disease (irSD): Failure to meet criteria for irCR or irPR, in absence of irPD.

Progressive Disease (irPD): At least 25% increase in SPD relative to nadir (minimum recorded tumor burden); Confirmation by a repeat, consecutive assessment no less than 4 weeks from the data first documented.

In a specific embodiment, use of the combination product or kit described herein for preventing, treating or ameliorating a dMMR mCRC, dMMR mEC, or pMMR mCRC inhibits proliferation or reduces an in vitro or in vivo proliferating cell or cell line population by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95 %, or 100% relative to proliferation or in vitro or in vivo proliferating cell or cell line population prior to use of the combination product or kit described herein, as assessed by methods well known in the art.

In a specific embodiment, use of the combination product or kit described herein for preventing, treating or ameliorating a dMMR mCRC, dMMR mEC, or pMMR mCRC inhibits proliferation or reduces an in vitro or in vivo proliferating cell or cell line population in a range of from about 5% to about 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, or from about 40% to about 100%, or any range in between, relative to proliferation or in vitro or in vivo proliferating cell or cell line population prior to use of the combination product or kit described herein, as assessed by methods well known in the art.

Kits

In one embodiment provided herein the combination product is a kit comprising, a combination of a Programmed Death-1 (PD-1 ) receptor antagonist agent

readthrough agent ataluren. In another embodiment, the kit described herein is prepared for use as a medicament. In another preferred embodiment, the kit is for use in the treatment of a mammalian animal. In another preferred embodiment, the kit is for use in the treatment of a human.

Complex (MHC).

In another embodiment, the kit doubles neo-epitope length. In another embodiment, the kit increases the immune response to reduce tumor size.

In one embodiment, the kit may contain each agent in one or more containers. Additionally, one or more other therapies useful for the treatment of a dMMR mCRC, dMMR mEC, or pMMR mCRC, or other relevant agents can also be included in the kit. In another embodiment, the kit may contain instructions printed with information to aid the patient in taking the combination product doses including, but not limited to, the product name for each agent and approved uses, the relative order of use for each agent, instructions for when and how to administer the agents and the dose required for therapeutic benefit. In another embodiment, the kit may contain a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency for manufacture, use or sale for human administration.

Example 1

For a specific frameshift mutation (FSM), measure the distance between the

FSM and the first encountered premature termination codon (PTC) and the distance between the first encountered PTC and the next following PTC in each corresponding transcript.

Samples

A dataset of mutations is required for the analysis, having a sample id, a mutated gene id, the location of the mutation on the chromosome (start, stop) and the mutation subtype.

An lllumina DNA mutation dataset from 461 colorectal adenocarcinoma patients obtained from The Cancer Genome Atlas (TCGA-COAD) was used. The dataset was derived from mutational data downloaded from the legacy archive of the TCGA

(https://p0rtal.gdc.cancer.g0v/legacy-archive/search/f) filtered for frameshift mutations.

Data Processing Software

A Perl based Ensembl API (https://www.perl.org/ and

https://www.ensembl.org/info/docs/api/index.html; GRch37) was used to calculate FSM to PTC distances. The proprietary Perl Script (pPS) used for identification of FSM and PTC locations and distances was run on a Linux Ubuntu 14.04 platform, using a module Spreadsheet: :XLSX (http://www.cpan.org/modules/index.html).

Data Processing Method

The pPS defines the exact position of the mutation within the cDNA of the corresponding transcript, the position of the mutation in the normal reading frame of the transcript and the strand where the mutation is located, calculating the shift in reading frame resulting from the mutational length and subtype, which is either an insertion or a deletion.

The Script checks every codon in the adjusted reading frame one by one, starting from the mutation locus, to determine the presence of a PTC. Upon finding a first PTC (PTC1 ), the Script continues to run until the presence of a second PTC (PTC2) is localized. The data obtained was formatted as a tab delimited file.

Analysis

The raw data was imported into a Microsoft Excel file used to calculate absolute and percent counts of transcripts containing specific PTCs, distances from PTC1 to PTC2 and their corresponding means, as shown in Figures 1 a and 1 b, the cumulative number of mutated transcripts are referenced on the x-axis with distances between FSMs and PTCs (Figure 1 a) and distances between PTC1 -PTC2 greater than a specific number of AAs shown on the y-axis (Figure 1 b).

Example 2

The first premature termination codon in a set of frequently mutated

microsatellites in MSI colorectal carcinoma was identified as follows. This was done in order to identify loci where ataluren is likely to affect frame shifting in such populations. Frameshifted mutated loci were identified in coding DNA in DNA sequencing data from 71 MSI-H colorectal cancer patients from the TCGA-COAD database. The frameshifted loci that were found to appear the most frequently in that database were in the following genes: BRAF, AC004687-2-RNF 43, ACVR2A, DOCK3, BMPR2, ZBTB20, LARP4B, QKI, USP35, MVK, RAB28, SETD18, SVIL, AL031847-2-RPL22, CCR5, PLEKHA6, HNRNPL, MECOM, and NFASC.

RNA sequencing data from the frameshifted loci identified above, that appeared the most frequently in that cohort, was plotted for each of those loci, as illustrated in Figure 2. Loci with the highest RNA levels identified in Figure 2, with levels above the dotted line shown in that figure (specifically, AC004687-2-RNF-43, ACVR2A, BMR2, LAP4B, QKI, USP35, MVK, RAB28, SETD1 B, SVIL, AL031847-2-RPL22, CCR5, PLEKHA6, HNRNPL, and MECOM), were studied further. The percent of reads mutated in RNA compared to DNA was plotted for each such loci, as shown in Figure 3.

One could expect ataluren to affect T-cell reactivity, measured by interferon gamma production on an enzyme-linked immunospot (ELISpot) assay against frameshifted peptides behind (post) the first premature termination codon in such frequently mutated microsatellites in MSI colorectal carcinoma. Thus, such an ELISpot assay or similar assay could be used to detect the effectiveness of ataluren at such sites in such a population.

Example 3

A phase 1 dose escalation study is conducted as follows to determine the MTD of ataluren. 6 to 12 subjects with histologically proven metastatic or locally advanced deficient mismatch repair (dMMR) colorectal adenocarcinoma (CRC), dMMR

endometrial carcinoma (EC) or mismatch repair proficient (pMMR) CRC are selected for the study. The subjects selected for the study all meet the following criteria:

· Each has at least one lesion with measurable disease as defined by 10 mm in longest diameter for a soft tissue lesion or 15mm in short axis for a lymph node by RECIST 1.1 and irRC criteria for response assessment.

• For dMMR mEC Inclusion: Each subject has received at least 1 prior cancer therapy regimen for metastatic EC. • For pMMR mCRC Inclusion: A subject has: i). Received at least 2 prior cancer therapy regimens for metastatic or non-metastatic CRC; ii). A life expectancy of greater than 3 months; and, ii). Normal organ and marrow function.

After a one-week run-in period, during which the subjects are administered three daily doses of 1.25 mg/kg, 1.25 mg/kg, and 2.5 mg/kg, respectively, the effective ataluren dose is determined in 2-4 dose escalation cohorts of at least 3 subjects each, who then receive pembrolizumab at a fixed dose of 200 mg i.v. every 21 days in combination with 3 days oral ataluren continuously. In all cohorts the ataluren dose is based on body weight according to a standard regimen. In the first cohort, ataluren is dosed at 25% of body weight (2.5 mg/kg morning dose; 2.5 mg/kg midday dose; 5 mg/kg evening dose); the second cohort is dosed at 50% of body weight (5 mg/kg morning dose; 5 mg/kg midday dose; 10 mg/kg evening dose) and the third cohort is dosed at 100% body weight (10 mg/kg morning dose; 10 mg/kg midday dose; 20 mg/kg evening dose).

The subjects start treatment in a staged approach, with the start dates of the study treatment spaced minimally 3 weeks apart. Dose limiting toxicity (DLT) is evaluated after 28 days (Cycle 0 and Cycle 1 ). A minimum of three patients are entered on each dose level. Subsequent enrollment of new dose levels are based on the toxicity assessment at Cycle 0 and Cycle 1. Pembrolizumab treatment (200 mg) is administered on Day 1 of each cycle (except Cycle 0) as a 30 minute IV infusion every 3 weeks.

Example 4

A phase 2 part two study is conducted on two cohorts of patients in which each patient receives pembrolizumab combined with ataluren dosed at the MTD of ataluren determined from the study described in Example 2. Pembrolizumab is administered at a fixed dose of 200 mg i.v. every 21 days. The two cohorts of patients are as follows:

• Cohort A: 20 patients with histologically proven metastatic or locally advanced deficient mismatch repair (dMMR) colorectal adenocarcinoma (CRC) or dMMR endometrial carcinoma (EC)

• Cohort B: 15 patients with histologically proven metastatic or locally advanced pMMR CRC.

Patients that have received prior treatment with either anti-CTLA4 or anti-PD1 monoclonal antibodies are excluded. The added benefit of ataluren is assessed by case-matched comparison of the results of this trial with those of a trial in which subjects with comparable characteristics are administered pembrolizumab alone. The primary endpoints for cohorts A and B are the immune-related progression-free survival

(irPFS) rate at 21 weeks and the objective response rate (irORR) assessed using immune related response criteria. An added benefit from administration of ataluren with pembrolizumab is found in at least some of the patients.

References

(1 ) Dietmaier, W., Wallinger, S., Bocker, T., Kullmann, F., Fishel, R., and

ROschoff, J. (1997) Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression. Cancer Res. 57, 4749-4756.

(2) Le, D. T., Uram, J. N., Wang, H., Bartlett, B. R., Kemberling, H., Eyring, A. D., Skora, A. D., Luber, B. S., Azad, N. S., Laheru, D., Biedrzycki, B., Donehower, R. C., Zaheer, A., Fisher, G. A., Crocenzi, T. S., Lee, J. J., Duffy, S. M., Goldberg, R. M., de la Chapelle, A., Koshiji, M., Bhaijee, F., Fluebner, T., Hruban, R. H., Wood, L. D., Cuka,

N., Pardoll, D. M., Papadopoulos, N., Kinzler, K. W., Zhou, S., Cornish, T. C., Taube, J. M., Anders, R. A., Eshleman, J. R., Vogelstein, B., and Diaz, L. A. (2015) PD-1

Blockade in Tumors with Mismatch-Repair Deficiency. N. Engl. J. Med. 372, 2509- 2520.

(3) Eisenhauer, E. A., Therasse, P., Bogaerts, J., Schwartz, L. H., Sargent, D., Ford, R., Dancey, J., Arbuck, S., Gwyther, S., Mooney, M., Rubinstein, L., Shankar, L., Dodd, L., Kaplan, R., Lacombe, D., and Verweij, J. (2009) New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1 ). Eur. J. Cancer Oxf.

Engl. 1990 45, 228-247.

(4) Wolchok, J. D., Hoos, A., O’Day, S., Weber, J. S., Hamid, O., Lebbe, C.,

Maio, M., Binder, M., Bohnsack, O., Nichol, G., Humphrey, R., and Hodi, F. S. (2009) Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 15, 7412-7420. Without regard to whether a document cited herein was specifically and individually indicated as being incorporated by reference, all documents referred to herein are incorporated by reference into the present application for any and all purposes to the same extent as if each individual reference was fully set forth herein.

Having now fully described the subject matter of the claims, it will be understood by those having ordinary skill in the art that the same can be performed within a wide range of equivalents without affecting the scope of the subject matter or embodiments described herein. It is intended that the appended claims be interpreted to include all such equivalents.

Claims

Claims:

1. A kit comprising a combination of the Programmed Death-1 (PD-1 ) receptor

antagonist agent pembrolizumab and the premature stop codon readthrough agent ataluren.

2. The kit according to claim 1 , wherein the kit is for use as a medicament.

3. The kit according to claims 1 or 2, for use in the treatment of cancer in a patient in need thereof, wherein the cancer is selected from the group consisting of colorectal cancer (CRC) and endometrial cancer (EC).

4. The kit for use according to claim 3, wherein the CRC is selected from the group consisting of a CRC having a deficient MMR (dMMR) process (dMMR CRC), a CRC having a proficient MMR (pMMR) process (pMMR CRC); and, wherein the

EC is selected from an EC having a deficient MMR (dMMR) process (dMMR EC).

5. The kit for use according to claim 3 or 4, wherein the patient is a mammalian

animal, preferably a human.

6. The kit for use according to any one of claims 3-5, wherein the treatment

comprises administering an effective amount of pembrolizumab and an effective amount of ataluren.

7. The kit for use according to claim 6, wherein said treatment comprises

administering pembrolizumab in a single dose of 200 mg every 3 weeks by infusion.

8. The kit for use according to claim 6 or 7, wherein said treatment comprises

administering ataluren in a regimen having a first, second and third daily dose, wherein the second dose is administered about 6 hours after the first dose, and the third dose is administered about 6 hours after the second dose, and a subsequent first dose is administered about 12 hours after the third dose of the preceding day.

9. The kit for use according to claim 8, wherein said regimen has a first, second and third daily dose selected from 1.25, 1 ,25 and 2.5 mg/kg/day, respectively.

10. The kit for use according to claim 8, wherein the regimen has a first, second and third daily dose selected from 2.5, 2.5 and 5 mg/kg/day, respectively.

11. The kit for use according to claim 8, wherein the regimen has a first, second and third daily dose selected from 5, 5 and 10 mg/kg/day, respectively.

12. The kit for use according to claim 8, wherein the regimen has a first, second and third daily dose selected from 10, 10 and 20 mg/kg/day, respectively.

13. A method of treatment of a cancer selected from the group consisting of

colorectal cancer (CRC) and endometrial cancer (EC), comprising administering to a human in need thereof an effective amount of ataluren and pembrolizumab.

14. The method of claim 13, wherein when the cancer is CRC the CRC has a

deficient mismatch repair (dMMR) process (dMMR CRC) or a proficient MMR

(pMMR) process (pMMR CRC); and wherein the cancer is EC the EC has a deficient MMR process (dMMR EC).

15. The method of claim 13, wherein the pembrolizumab is administered in a single dose of 200 mg by infusion every three weeks.

16. The method of claim 13, wherein the ataluren is administered orally in a regimen having a first, second and third daily dose, wherein the second dose is

administered about 6 hours after the first dose, and the third dose is administered about 6 hours after the second dose, and a subsequent first dose is administered about 12 hours after the third dose of the preceding day.

17. The method of claim 16, wherein the regimen has a first, second and third daily dose of 2.5, 2.5 and 5 mg/kg/day of ataluren respectively.

18. The method of claim 16, wherein the regimen has a first, second and third daily dose of 5, 5 and 10 mg/kg/day of ataluren, respectively.

19. The method of claim 16, wherein the regimen has a first, second and third daily dose selected from 10, 10 and 20 mg/kg/day of ataluren, respectively.