Researchers at the Icahn School of Medicine at Mount Sinai in New York, with contributions from Memorial Sloan Kettering Cancer Center, report that a four-drug immune checkpoint combination targeting PD-1, LAG-3, CTLA-4, and TREM2 achieved complete tumor clearance in preclinical models of both mismatch repair-deficient and mismatch repair-proficient colorectal cancer, according to a study published May 6, 2026 in Cell Reports Medicine. The findings address one of the most persistent problems in colorectal cancer immunotherapy: the near-complete failure of anti-PD-1 therapy in the majority of patients whose tumors carry intact mismatch repair machinery.
Approved checkpoint inhibitors, including Keytruda (pembrolizumab) and Opdivo (nivolumab), have demonstrated activity in colorectal cancer only within the mismatch repair-deficient subgroup, which accounts for roughly 5% of metastatic cases. The mismatch repair-proficient, microsatellite-stable population — the vast majority of patients — has remained without an effective immunotherapy option. The Mount Sinai study, led by Nina Bhardwaj, set out to define why immune resistance colorectal cancer is so pervasive and whether it could be dismantled through rational combination design.
The team's central finding is that resistance is not a single-pathway problem. Using single-cell RNA sequencing, spatial transcriptomics, TCR clonal diversity profiling, and spectral flow cytometry across multiple murine tumor models and patient-derived tissue, the researchers mapped a convergence of T cell exhaustion, myeloid spatial exclusion, and tumor-intrinsic evasion mechanisms operating simultaneously within the tumor microenvironment.
Mapping the myeloid-T cell axis
A core mechanistic finding concerns TREM2-expressing macrophages. Spatial transcriptomics showed that TREM2-positive macrophages accumulate preferentially in tumor core regions, where they co-localize with exhausted T cells and physically exclude effector lymphocytes from sites of active tumor growth. These macrophages expressed high levels of immunosuppressive mediators including IL-10, TGF-β1, and VEGF-A, and their abundance correlated negatively with T cell infiltration and cytotoxic function across both murine and human datasets.
In the T cell compartment, LAG-3 emerged as the dominant co-inhibitory receptor on tumor-infiltrating CD8-positive T cells in mismatch repair-deficient tumors. Anti-PD-1 therapy alone expanded T cell receptor clonotype diversity and activated CD8-positive cells but triggered compensatory upregulation of LAG-3, TIM-3, and TIGIT, creating redundant suppressive signals that monotherapy could not overcome. Non-responders in reanalyzed human single-cell RNA sequencing datasets from mismatch repair-deficient colorectal cancer patients showed persistent co-expression of TOX, PDCD1, and HAVCR2 — a signature of deep T cell exhaustion — while responders demonstrated expansion of clonally diverse effector populations with upregulated cytotoxic gene signatures.
The spatial data further showed that in animals achieving tumor clearance, the immune architecture reorganized: effector T cells redistributed toward the tumor core while TREM2-positive macrophage density at the tumor-immune interface declined. This architectural shift was absent in non-responders.
The quadruple combination and its results
The study tested more than ten checkpoint blockade combinations per model across CT26, 4T1, B16F10, and MC38 cell lines in both orthotopic and subcutaneous settings, as well as in three-dimensional spheroid cultures. The lead combination — anti-PD-1 plus anti-LAG-3 plus anti-CTLA-4 plus anti-TREM2 — produced complete responses in approximately 100 percent of mismatch repair-deficient CT26 tumors, compared with roughly 40 to 50 percent for anti-PD-1 monotherapy. In mismatch repair-proficient models, where anti-PD-1 alone produced near-zero complete responses, the quadruple combination achieved approximately 73 percent complete response rates.
The combination immunotherapy CRC results were replicated in patient-derived mismatch repair-deficient colorectal cancer spheroids, where the quadruple regimen produced superior tumor killing relative to anti-PD-1 monotherapy, with increased secretion of IFN-γ, TNF-α, and IL-2 confirmed by Luminex cytokine profiling. Depletion of TREM2-positive macrophages in co-culture systems directly enhanced T cell cytotoxicity, providing a mechanistic link between the myeloid targeting component and the observed anti-tumor activity.