Neoadjuvant immune checkpoint inhibition and novel IO combinations in early-stage colon cancer. The NICHE trial.

Currently, it is not standard of care to give pre-operative treatment is to
patients with colon cancer. However, based on the FOXTROT data national
guidelines for the treatment of colon cancer will advise consideration of
neoadjuvant chemotherapy when a T4 tumor is suspected. There are no specific
advices with regards to MSI or MSS tumors yet, awaiting the next update of
these guidelines and incorporation of the FOXTROT data for MSI tumors after
publication (considering the lack of pathological response in most patients
with MSI tumors). Post-operative adjuvant chemotherapy is commonly administered
to patients with stage 3 tumors and in high risk stage 2 tumors (only MSS),
and has been shown to marginally increase PFS and OS, the latter by
approximately 5%.

Patients within the NICHE trial are exposed to immunotherapeutic drugs, which
may lead to immune related adverse events. We expect the limited exposure to
these drugs within this study to carry a low risk of adverse events and no
significant delays in surgical resection of the primary tumor. The first
results of the NICHE trial have recently been published, showing that this
treatment is safe and feasible, without any delays in surgery and without any
unexpected toxicities. As anticipated, immune-related adverse events were
limited and comparable to data from studies with monotherapy PD-1 blockade,
namely 13% (n=5) grade 3-4 adverse events, of which n=3 were asymptomatic
increases in lipase and amylase, which resolved without intervention.
Furthermore, surgical interventions in patients on immunotherapeutic regimens
does not seem to increase the risk of complications, albeit with wide
confidence intervals. The benefit of this treatment in MSI tumors in terms of
pathological response was recently published by Chalabi M. et al, Nature
Medicine, 2020, with 100% pathological response rate. Furthermore, a 27%
response rate was seen in MSS tumors.

Previous reports of clinical studies using combinations of ipilimumab and
nivolumab show grade 3-4 adverse events in 44-58% of patients, with 37% of
treatments-related events leading to discontinuation of treatment. More recent
data from the Checkmate-142 trial using the combination of nivo 3mg/kg and low
dose ipilimumab (1mg/kg) every three weeks (Morse, Overman et al, The
Oncologist 2019), showed 56% grade 1-2 and 24% grade 3-4 immune-related adverse
events. In total, 25%, 23%, 19%, 5%, 5% and 29% experienced an endocrine,
gastrointestinal, hepatic, pulmonary, renal, or skin irAE, respectively.
These findings show a favorable toxicity profile for the alternatively dosed
combination of nivolumab and ipilimumab, with a low frequency of grade 3 and 4
treatment related events. Also, considering the fact that patients in this
study will only receive one single low dose of ipilimumab and two cycles of
nivolumab, the incidence of treatment related events seems lower than above
mentioned in the first 40 patients included in the NICHE trial.

Rationale for nivolumab plus anti-IL8
The next cohort within this adaptive design will include treatment of patients
with nivolumab plus anti-interleukin 8 (IL-8). In cancer, serum IL-8, released
by tumor cells, has been shown to promote tumor immune evasion by recruitment
of immunosuppressive neutrophils and myeloid-derived suppressor cells to the
TME, which in turn could contribute to immune resistance by dampening the
cytotoxic ability of T cells.
Additionally, IL-8 may promote angiogenesis and epithelial to mesenchymal
transition, increasing metastatic potential and resistance to therapy. Serum
IL-8 together with tumor neutrophil infiltration are associated with worse
prognosis in cancer and may also have predictive value in patients treated with
immunotherapy. Increases in IL-8 serum levels can be attributed to various
sources, including the surrounding stromal tissue, immune cells in the TE and
tumor cells.
A recent retrospective study in over 1300 patients showed that an elevated
serum IL-8 is associated with poor outcome in patients treated with
immunotherapy. Elevated serum IL-8 was also found to be negatively associated
with tumor IFNy and T-cell infiltration signatures in the transcriptome.
Additionally, serum-IL8 can easily be measured in conventional blood specimens
in clinical settings, making it a potentially useful, but also broadly
implementable biomarker. In colorectal cancer specifically, several studies
have demonstrated that IL-8 and its receptor CXCR2 are the most significantly
upregulated chemokines.
Based on these data, the combination of PD-1 blockade with IL-8 blockade could
have synergistic antitumor activity, increasing the potential of response to
anti-PD1 and decreasing the risk of resistance. Also, the combination of
nivolumab plus anti-IL8 in 120 patients was very well-tolerated with limited
toxicity and only 5% grade 3-4 treatment-related adverse events.
As soon as 30 patients have been included in the original MSS cohort (nivolumab
plus ipilimumab), the next cohort will be opened. Depending on efficacy in that
cohort, and the safety of the combination, expansion to triplet therapy and/or
other combinations may be considered.

Rationale for nivolumab/relatlimab
Lymphocyte-activation gene 3 (LAG-3) is an inhibitory checkpoint molecule that
is often expressed together with PD-1 on tumor infiltrating lymphocytes (TILs),
contributing to T-cell exhaustion. Relatlimab has shown promising PFS
improvement in melanoma patients, compared to nivolumab alone (Tawbi et al).
Very recently the combination of nivolumab and relatlimab was approved by the
FDA for the first-line treatment of patients with metastatic melanoma. In
addition, the response rates to neoadjuvant nivolumab/relatlimab in melanoma
patients is similar to the neoadjuvant treatment with nivolumab/ipilimumab.
Based on these data, it is expected that this combination will be effective in
a substantial proportion of patients with dMMR colon cancer. The favorable
toxicity profile compared to nivolumab/ipilimumab makes it an attractive
option, especially in the neoadjuvant setting. High response rates with a
better toxicity profile compared to nivolumab/ipilimumab would be an incentive
to further develop registrational studies for nivolumab/relatlimab in dMMR CC
patients, in addition to organ-sparing approaches for also lower risk dMMR CC.

This study has been transitioned to CTIS with ID 2024-513314-35-00 check the CTIS register for the current data.

Primary Objective: To determine the safety and feasibility of pre-operative
immunotherapy in CRC.

Additional Primary Objective for the expanded the MSI cohort: To assess
efficacy of neoadjuvant ipilimumab plus nivolumab in terms of disease-free
survival.

Secondary Objectives:
• To assess the major pathological response rate (MPR, <10% viable tumor rest)
and complete response rate;
• To assess post-surgical outcome and infectious complications following
neoadjuvant immunotherapy;
• To find biomarkers and evaluation strategies able to accurately assess
complete and near-complete responses in order to pursue organ-sparing treatment
(omission of surgery) in this patient population:
o Post-treatment CT-scans: can we use radiomics to accurately assess complete
and near-complete response?
o Post-treatment MRI scans (pilot, possibly additional pilots with novel
PET-tracers): is assessment of complete response rate more accurate with MRI
(and/or PET) than CT-scans?
o ctDNA analysis: can we use ctDNA to assess complete response and is there a
difference between complete and near-complete response in terms of minimal
residual disease on ctDNA and risk of relapse?
• To expand current translational analyses, in terms of:
o RNA sequencing and inflammatory signatures to validate current findings and
identify predictors of response in pMMR tumors.
o Analysis of immune cell infiltration and the difference with inflamed pMMR
tumors pre- and post-treatment: why is there no pathological response in pMMR
tumors showing immune activation?
o ctDNA analysis: can we predict complete and near-complete responses and if
so; can we move to omitting surgery in this patient population?
• Analysis of immune cell infiltration and the difference with inflamed pMMR
tumors pre- and post-treatment: why is there no pathological response in pMMR
tumors showing immune activation? And the differences between complete and
near-complete responders?
• To assess the immunogenic mutational load by DNA WES and correlation with
putative markers of response;
• To assess relapse free survival for the MSS cohort;
• To explore the immunogenicity and in vitro T-cell sensitivity of tumor
organoid cultures and explore efficacy of novel treatment combinations for a
selection of patients;

Translational Objectives:
* To analyse expression and changes in expression of immune cell subsets,
including but not limited to CD4+, CD8+, FOXP3+ regulatory T cells, MDSCs);
* To assess changes in immune suppressive pathway and inflammation signatures,
IFNy induced gene expression and the added effects of COX2-i when combined with
nivolumab and ipilimumab;
* To assess serum-IL8 and its predictive value in response to nivolumab plus
ipilimumab, and to anti-IL8 + nivolumab
* To assess safety and feasibility of neoadjuvant immunotherapy and the effects
hereof on post-surgical outcome and complications;
* To analyse gain/change in presence of other biomarkers and immune
checkpoints, (depending on material availability for comparison); To analyse
clonality and changes in the TCR repertoire pre- and post-therapy in the subset
of patients showing significant changes in the TME;
* To identify subtypes of MSS CRC with signs of response and find predictive
biomarkers based on IHC or gene expression analyses as mentioned above;
* To assess immunogenic mutational load by DNA WES;
* To explore the association between microbiota composition and treatment
outcomes and the effect of neoadjuvant nivolumab plus ipilimumab on the gut
microbiota composition;
* To identify and explore the predictive value of checkpoint expression,
cytokines and chemokines, CRP/ESR/LDH/CEA;
* To evaluate and explore post-immunotherapy effects on draining lymph nodes
using resection material;
* To explore the immunogenicity of autologous tumor organoids and their T cell
reactivity in vitro, using normal tissue organoids as controls;
* To explore the phenomenon of exhausted T cells (Tex) by assessing IHC and
gene expression signatures and combinations thereof;
* To assess ctDNA before and during therapy and after resection of the primary
tumor and during follow-up;
* To explore changes in inflammation signatures, immune checkpoints, immune
suppressive pathways and IFN* induced gene expression by using RNA sequencing.

DMMR cohort 3: In this multi-center, open-label phase II study, we will enroll
70 patients in the dMMR cohort after accrual of the first 30 patients in the
original dMMR cohort, making a total of 100 patients with dMMR tumors to be
analyzed together for disease-free survival. The study was amended to include
these additional patients, including a formal sample size calculation and
primary endpoint of 3-year disease-free survival (DFS) for this group.

DMMR Cohort 6: nivolumab + relatlimab: will enroll 19 patients in stage I, and
with more than 14 responders accrual can continue into stage 2 for an
additional 40 patients, making 59 total. Power calculation was performed using
a one-sided alpha of 0.05 and power of 80%. The study will be considered a
success when more than 46 pathologic responses are observed in the total cohort
of 59 patients.

PMMR cohorts:
For the pMMR subgroup accrual will be continued until 30 patients have been
treated with nivolumab plus ipilimumab after which additional amendments can be
done to include other treatment combinations.
New cohorts (4 and 5)

For the pMMR/MSS cohort, after accrual of 30 evaluable patients has been
complete:
- cohort 4: in which patients will receive nivolumab plus anti-IL8 (BMS-986253)
- cohort 5: in which patients will receive nivolumab plus relatlimab (anti-LAG3)
- additional cohorts will open depending on the possibility of new treatment
combinations

Patients will be treated with short-term immunotherapy + COX2-inhibitors. This
treatment will be given during the window period until surgical resection of
the tumor. The duration of treatment will be approximately 4 weeks.
After completion of accrual, analysis of the primary endpoint will be
performed. Based on these findings and available compounds at that time, the
study will be amended to include new drug combinations with one or more of the
study drugs used in the first group of patients.

For the MSI subgroup, analysis of disease-free survival will be performed 6
months after inclusion of the last patient.
Analysis of pathological response rate may be presented or published prior to
DFS data.

Patients with MSI tumors will be treated with a single dose of ipilimumab
1mg/kg on day 1 and two cycles of nivolumab 3mg/kg on day 1 and 15,
respectively.
Patients with MSS intumors in group 1 will be treated with a single dose of
ipilimumab 1mg/kg on day 1 and two cycles of nivolumab 3mg/kg on day 1 and 15,
respectively. Additionally, patients with MSS tumors will be randomized to
receive celecoxib 200mg once daily until the day before surgery (group 2),
until accrual of 30 patients within the MS subgroup has been completed, after
which the study may be amended to include new treatment combinations for the
MSS group.The next cohort for the pMMR/MSS group of patients will consist of
nivolumab 3mg/kg plus BMS-986253 (anti-IL8) 2400mg on day 1 and 15.

A minimum of 12 tumor biopsies is required at baseline, acquired through
colonoscopy. Tumor and normal tissue will be collected at resection. One CT
scans will be required at baseline and post-treatment, prior to surgery. Blood
draws (incl. PBMC, serum and plasma collection) will be required at baseline,
before every cycle of therapy, peri-operatively and.during follow-up.

Extra blood samples taken:
- all new inclusions
- 20ml per sample extra for ctDNA en PBMCs
- Time points: 3, 6, 12, 24, 36, 48, 60 months.

In cohorts 5 and 6, patients will be treated with a combination of nivolumab
and relatlimab.
Cohort 5: patients with MSS tumors will be coadministered nivolumab and
relatlimab at a flat dose of 240/240mg
Cohort 6: patients with MSI tumors will be coadministered nivolumab and
relatlimab at a flat dose of 480/480mg

Currently, no it is not standard of care to give pre-operative treatment is
given to patients with colon cancer. However, based on the FOXTROT data
national guidelines for the treatment of colon cancer will advise consideration
of neoadjuvant chemotherapy when atu T4 tumor is suspected. There are no
specific advices with regards to MSI or MSS tumors yet, awaiting the next
update of these guidelines and incorporation of the FOXTROT data for MSI tumors
after publication (considering the lack of pathological response in most
patients with MSI tumors). Post-operative adjuvant chemotherapy is commonly
administered to patients with stage 3 tumors and in some high risk cases in
stage 2 tumors (only MSS), and has been shown to marginally increase PFS and
OS, the latter by approximately 5%.

Patients within this the NICHE trial are exposed to immunotherapeutic drugs,
which may lead to immune related adverse events. We expect the limited exposure
to these drugs within this study to carry a low risk of adverse events and no
significant delays in surgical resection of the primary tumor. The first
results of the NICHE trial have recentlyl been published, showing that this
treatment is safe and feasible, without any delays in surgery and without any
unexpected toxicities. As anticipated, immune-related adverse events were
limited and comparable to data from studies with monotherapy PD-1 blockade,
namely 13% (n=5) grade 3-4 adverse events, of which n=3 were asymptomatic
increases in lipase and amylase, which resolved without intervention.
Furthermore, surgical interventions in patients on immunotherapeutic regimens
does not seem to increase the risk of complications, albeit with wide
confidence intervals. . The benefit of this treatment in MSI tumors in terms of
pathological response was recently published by Chalabi M. et al, Nature
Medicine, 2020, with 100% pathological response rate. Furthermore, a 27%
response rate was seen in MSS tumors.

Previous reports of clinical studies using combinations of ipilimumab and
nivolumab show grade 3-4 adverse events in 44-58% of patients, with 37% of
treatments-related events leading to discontinuation of treatment. More recent
data from the Checkmate-142 trial using the combination of nivo 3mg/kg and low
dose ipilimumab (1mg/kg) every three weeks (Morse, Overman et al, The
Oncologist 2019), showed 56% grade 1-2 and 24% grade 3-4 immune-related adverse
events. In total, 25%, 23%, 19%, 5%, 5% and 29% experienced an endocrine,
gastrointestinal, hepatic, pulmonary, renal, or skin irAE, respectively.