Background

Immune checkpoint inhibitors (ICIs) enhance anti-tumor immunity by regulating the interaction between T lymphocytes, antigen presenting cells (APC) and tumor cells, and its mechanism is to reactivate the immune response of T cells to tumor cells and inhibit tumor growth, which has brought long-term and lasting benefits to patients with multiple types of solid tumors.1,2 The dMMR/MSI-H tumors are the main beneficiaries of immunotherapy due to their high immunogenicity, strong lymphocyte infiltration in tumor microenvironment, good prognosis, and insensitivity to conventional radiotherapy and chemotherapy.3–5

The results of KEYNOTE 016, reported by Professor Le’s team at Hopkins Hospital in 2015, showed that dMMR metastatic colorectal cancer (mCRC) could benefit from pembrolizumab monotherapy, This study opened the door of immunotherapy for colorectal cancer and opened the era of immunotherapy for colorectal cancer.6 Subsequently, data from the first phase of the CheckMate142 study, reported in 2016, suggested that recurrent dMMR/MSI-H mCRC could benefit from a combination of nivolumab and ipilimumab.7 Long-term follow-up showed that the combination helped dMMR/MSI-H mCRC patients achieve 77% PFS and 83% OS at 1 year. Based on these data, the US Food and Drug Administration (FDA) has approved the combination of nivolumab and ipilimumab for previously treated dMMR/MSI-H mCRC patients.8 The KEYNOTE-177 study, published in 2020, is the most important clinical trial in first-line therapy to date, which confirmed that pembrolizumab monotherapy can be used as the first-line standard treatment for dMMR/MSI-H colorectal cancer.9,10 KEYNOTE-177 successfully rewrote the NCCN colorectal cancer treatment guidelines.11

Studies on the mechanism have found that somatic mutations in MSI-H colorectal cancer produce a large number of nascent peptides, which can act as specific antigens of tumor cells.11 The combination of these neoantigens with the major histocompatibility complex (MHC) can induce the immune response of CD4+ or CD8+ T cells, while attracting the infiltration of tumor infiltrating lymphocyte (TIL) in the tumor microenvironment, thus making the tumor “hot” and increasing the response of CRC to immunotherapy.12,13

Although dMMR/MSI-H patients belong to the “hot tumor” population that is sensitive to immunotherapy, the proportion of dMMR/MSI-H patients in all mCRC are only 4% to 5%,10 and a larger proportion of pMMR /MSS patients belong to the “cold tumor” population that is insensitive to immunotherapy.14 Studies have found that for pMMR /MSS colorectal cancer, the tumor lacks antigens that induce T cell initiation, and the tumor microenvironment lacks T cell infiltration, so the antitumor effect of ICIs in these “cold tumor” populations that are not sensitive to immunotherapy has certain limitations.15–17 Existing studies have shown that the use of a single ICIs alone is essentially ineffective for pMMR/MSS mCRC.6,18 How to break through this dilemma has always been the challenge. Most of the current treatment strategies use combination therapy to reverse the “cold tumor” into the “hot tumor” sensitive to immunotherapy, so as to improve the efficacy of immunotherapy in pMMR/MSS colorectal cancer, so that immunotherapy can benefit more colorectal cancer patients.16,17,19,20 Although some combination treatment strategies have achieved good short-term efficacy and are promising in the treatment of patients with mCRC, most of them are single-arm small sample exploratory studies, and the research data are not completely stable and consistent (Table 1), and the safety and efficacy need to be verified by more prospective clinical trials with large samples.14,21

Table 1 The Results of Clinical Trials Based on ICIs in Combination with Other Modalities in pMMR/MSS Metastatic Colorectal Cancer

In addition, several studies are looking for other markers with potential to predict immunotherapy efficacy, not only for mismatch repair genes, but also for other molecular markers to enrich the population who can benefit from immunotherapy for pMMR/MSS colorectal cancer. Current studies have found that multiple molecular markers such as tumor mutation burden (TMB), polymerase ε and polymerase δ (POLE/POLD1) mutations, PD-L1 expression, tumor infiltrating lymphocytes, and gut microbiota have the potential and value to predict the efficacy of ICIs for pMMR/MSS colorectal cancer.29,68–76 This article aims to review the research progress of ICIs combination therapy for pMMR/MSS mCRC and related studies on the predictive markers of ICIs efficacy, in order to provide references for finding the best immunotherapy strategy for pMMR/MSS mCRC patients.

ICIs Combined with Multi-Target Tyrosine Kinase Inhibitors

The mechanism of primary resistance to immunotherapy in pMMR/MSS colorectal cancer patients is complex and involves many factors in tumor cells and tumor microenvironment. The tumor microenvironment is composed of blood and lymphovascular networks, extracellular matrix (ECM), various stromal cells (including fibroblasts, mesenchymal stromal cells, and pericytes), and resident and infiltrating immune cells (including T lymphocytes, B lymphocytes, natural killer cells, and tumor-associated macrophages.77,78 Tumor neovascularization plays an important role in tumor microenvironment. Previous studies have shown that overexpression of VEGF and the resulting over-activation of VEGF receptors stimulate endothelial cell proliferation, promote angiogenesis, and improve the availability of oxygen and nutrients in tumors.79 Meanwhile, abnormal activation of receptor tyrosine kinase (RTK), including endothelial growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF), plays a key role in the development and progression of cancer. This makes abnormal RTK a potential therapeutic target for cancer therapy.79,80

Preclinical studies have shown that inhibitors targeting VEGF/VEGF receptor have both effective anti-angiogenesis and immunomodulatory effects, can induce tumor vascular degeneration, improve hypoxia and low pH in the tumor microenvironment, and increase the infiltration of immune T lymphocytes in the tumor microenvironment through tumor revascularization, then an inflammatory tumor microenvironment is generated;In addition, anti-VEGF therapy has a synergistic effect with immunotherapy by up-regulating the expression of PD-L1, reducing immunosuppressive cells [such as tumour-associated macrophages (TAMs) and regulatory T cells (Tregs)], and enhancing the anti-tumor activity of CD8+T cells.81,82 Studies in mouse models have confirmed that the combination of anti-VEGFR2 and anti-PD-1 monoclonal antibodies can increase the production of tumor interferon-gamma, tumor necrosis factor-α and granzyme B, thereby enhancing the immune response of mice.83

Regorafenib is the first FDA-approved multi-target TKI in metastatic colorectal cancer that has progressed after treatment with standard treatment, targeting angiogenesis (VEGFR1-3, TIE2), stroma (PDGFR-β) and cancer-causing receptor tyrosine kinases (KIT, RET and RAF).84,85 Regorafenib has been shown to regulate anti-tumor immunity through different mechanisms. In a mouse hepatocellular carcinoma model, regorafenib normalizes tumor blood vessels by targeting VEGFR2/3, promotes the proliferation and activation of CD8+T cells, and improves the anti-tumor immunity of PD-1 blockers.86

In addition, Indoleamine 2,3-dioxygenase 1 (IDO1) overexpression has been shown to induce dendritic cells and regulatory T cells in a variety of cancers, leading to immunosuppressive effects, and therefore IDO1 is likely to be a potential target for anti-tumor immunotherapeutic intervention.87,88 Studies have shown that regorafenib can simultaneously reduce the expression of PD-L1 and IDO1, reduce the TAM infiltration, and produce synergistic effects with anti-PD-1 monoclonal antibodies to significantly improve the anti-tumor effect.89 Doleschel also found that regorafenib significantly reduced the infiltration of immunosuppressive TAM and Treg cells into the tumor microenvironment in his study of CRC mouse models developed after chemotherapy, while anti-PD-1 treatment significantly increased intratumoral IFN-γ levels, thus the two have a synergistic effect in regulating anti-tumor immune response, thus achieving sustained tumor suppression.90 Based on the above theoretical basis, in recent years, the combination of multi-target receptor tyrosine kinase inhibitors, including regorafenib, and immune checkpoint inhibitors has been attempted in pMMR/MSS mCRC.

The REGONIVO study, reported in Japan in 2020, is a Phase I b trial that enrolled 24 patients with pMMR/MSS colorectal cancer after failure of standard treatment to evaluate the safety and efficacy of the combination of regorafenib and nivolumab. The results showed that the objective response rate (ORR) was 33.3% and the median progression free survival (PFS) was 7.9 months. The 1-year PFS rate and overall survival (OS) rate were 41.8% and 68.0%, respectively.22 This study shows that some MSS mCRC patients may benefit from the combination of PD-1 inhibitors with multi-target TKI. However, this result was not replicated in the North American Phase II study, with an ORR of only 7.1%, median PFS of 1.8 months, and median OS (data immature) of 11.9 months. All responders had no liver metastases at baseline.23 It is suggested that the benefit group should be screened in this treatment mode. The researchers subsequently conducted a retrospective exploratory analysis, and the results suggested that there was a certain correlation between some specific biomarkers and treatment response.91

The Phase II REGOMUNE study is evaluating the efficacy and safety of regorafenib in combination with Avelumab in treated MSS colorectal cancer. Of the 40 evaluable patients, the best response was in 23 patients (57.5%) who were stable, with a median PFS and OS of 3.6 and 10.8 months, respectively.24,25 Studies have shown that PFS and OS obtained by this combination therapy are superior to historical data obtained by regorafenib alone.92 The REGOTORI study, led by Professor Xu Ruihua, is a Phase Ib/ II clinical trial evaluating the efficacy of regorafenib combined with the PD-1 inhibitor toripalimab in treated MSS mCRC. The ORR of 33 enrolled patients was 15.2%, and the median PFS and OS were 2.1 and 15.5 months, respectively. Studies have also shown that patients with lung metastasis have a relatively higher effective rate, while patients with only liver metastasis have a relatively poor effect.26

Lenvatinib is another multi-target receptor TKI. In pre-clinical studies, it was found that Lenvatinib combined with pembrolizumab can activate CD8+ T cells by reducing TAM and activating interferon pathway, thus producing synergistic effects with anti-PD-1 antibodies and enhancing anti-tumor effects.93 LEAP-005 (NCT03797326) is a Phase II study of lenvatinib and pembrolizumab in previously treated patients with advanced solid tumors of pMMR/MSS.27 The 32 MSS colorectal cancer patients had an ORR of 22% and a disease control rate (DCR) of 47%; and the median PFS and OS were 2.3 months and 7.5 months, respectively. It showed good antitumor activity and a manageable safety profile. The Phase III LEAP 017 study (NCT04776148) is being recruited worldwide based on the results of pembrolizumab in combination with lenvatinib in treated pMMR/MSS mCRC patients.

Fruquintinib is a potent and highly selective small-molecule TKI against VEGFR1, 2, and 3,94 and its survival benefits have been demonstrated in patients with mCRC in the Phase 3 FRESCO clinical trial.95 Clinical trials of fruquintinib in combination with immunotherapy are also underway. Preliminary results from a Phase 1b/2 study of sintilimab combined with fruquintinib in the treatment of advanced colorectal cancer (NCT03903705) suggest that the combination of sintilimab and fruquintinib is effective and safe to tolerate in patients with mCRC who have failed standard therapy. In this study,44 mCRC patients with disease progression after receiving at least two-line chemotherapy containing fluorouracil, oxaliplatin or irinotecan in the past were included. Patients who received intermittent administration of Fruquintinib combined with sintilimab had ORR of 23.8% and mPFS of 6.9 months, and mOS of 14.8 months respectively.28

It can be seen that despite the exciting results of the REGONIVO Phase Ib study, and ORR in MSS colorectal cancer patients reached 33%,22 thus opening the paradigm of refractory MSS colorectal cancer with regorafenib combined with anti-PD-(L)1 monoclonal antibody. However, subsequent single-arm studies using immune checkpoint inhibitors in combination with different anti-angiogenesis targeting agents (tyrosine kinase inhibitors) as previously treated mCRC therapy have been reported, including the North American REGONIVO study,23 REGOMUNE study (regorafenib combined with Avelumab),25 REGOTORI study (regorafenib combined with toripalimab),26 LEAP-005 colorectal cancer cohort (Lenvatinib combined with pembrolizumab).27 Most studies did not repeat the results consistent with the Japanese REGONIVO study, but in general, the reported ORR of these studies ranged from 7% to 27%, DCR from 39% to 80%, and median OS from 7.5 to 15.5 months. Compared with previous studies on standard third-line monotherapy (regorafenib, fruquintinib or TAS-102), both ORR and OS were numerically improved.92,95,96 However, the sample size of these studies is limited, so more high-level phase III randomized controlled trials are needed for validation, and the preliminary study supports the need to further explore the screening of advantageous populations in the future.

Combination Therapy with Two ICIs Anti-PD-(L)1 Combined with Anti-CTLA-4 Therapy

Cytotoxic T lymphocyte antigen-4(CTLA-4) mainly inhibits T cell activation in the initial phase, thereby inhibiting T cell activity.97 Anti-CTLA-4 monoclonal antibody can block the inhibitory signal transduction of CTLA-4, thereby promoting T cell activation and immune response to tumor cells; However, anti-PD-(L)1 monoclonal antibody removes the inhibition of the combination of PD-1 and PD-L1 on activated T cells and promotes the killing effect of T cells on tumor cells.1,98 Therefore, the combination of the two can synergistically promote the anti-tumor immune response through the blocking complementary mechanism, thereby the acquired resistance to ICIs monotherapy is overcome1,99 and has a good safety profile.100 It has been observed that anti-PD (L)-1 antibody combined with anti-CTLA-4 antibody treatment has shown relatively excellent anti-tumor effects in a variety of malignant tumors (including melanoma, lung cancer, etc),101,102 and has begun to be explored in colorectal cancer, although the exact mechanism of action is still unclear.103

The CCTG CO.26 randomized Phase II trial evaluated whether the combination of durvalumab and tremelimumab improved survival in patients with advanced refractory colorectal cancer compared to best supportive care (BSC). A total of 180 patients were included in the study, excluding patients with dMMR, and the results showed that the study met the primary endpoint, with the median OS increased from 4.1 months in the BSC group to 6.6 months in the dual immunotherapy group, while the incidence of grade 3 or 4 adverse events also increased significantly.29 Although this study was the first to report positive results for PD-L1 monoclonal antibody combined with CTLA-4 monoclonal antibody in MSS mCRC, it was not widely accepted in the clinic, mainly because no patients in the study achieved complete response (CR). Only one patient with MSS type achieved partial response (PR). This study suggests the potential role of dual immunotherapy in MSS mCRC.29

In a clinical trial (NCT02754856), a combination of durvalumab and tremelimumab was administered to 21 pMMR patients with only liver metastasis before surgery, and evidence of extended relapse-free survival (RFS) and T cell activation was initially observed.104 In addition, the Fakih team conducted a phase I study on nivolumab combined with ipilimumab and regorafenib in MSS mCRC patients who had not been treated with regorafenib and anti-PD (L) −1 monoclonal antibody,30 and also achieved surprising results. ORR reached 31%, and the median PFS and OS were 4 months and 19.6 months, respectively, which were significantly improved compared with traditional third-line treatment for mCRC.92,95,96

Botensilimab (AGEN1181) is a new generation FC-enhanced CTLA-4 inhibitor that blocks the inhibitory function of CTLA-4 interactions with its ligands CD80 and CD86. A Phase 1A/B study by Professor Bullock et al explored the efficacy and safety of botensilimab in combination with balstilimab (AGEN2034, a PD-1 inhibitor) in treated MSS patients with mCRC. 34% of enrolled patients had previously received immunotherapy, and the median number of prior treatment lines was 4. Results showed that dual immunotherapy had an ORR of 24% and a disease control rate (DCR) of 73% in 41 evaluable patients. At the same time, the safety status was good, and most treatment-related adverse events were grade 1–3.31 A randomized Phase II trial (NCT05608044) is about to start.

Anti- PD-(L)1 Combined with New Generation of Immune Checkpoint Inhibitors

To enable more patients to benefit from immunotherapy, there is growing interest in other immune checkpoints and new potential targets have been identified, such as LAG-3, TIGIT, TIM-3 or VISTA. These receptors, like PD-1 or CTLA-4, are physiologically classified as inhibitory immune checkpoint receptors that are induced after T cell activation to prevent excessive stimulation.105–108 LAG-3,109 TIGIT,110 TIM-3,105,111 and VISTA112 are frequently co-expressed with PD-1 on tumor infiltrating lymphocytes (TILs) in a variety of cancers, including renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), ovarian cancer, and melanoma.

Lymphocyte activation Gene-3(LAG-3) is an inhibitory receptor on the surface of T cells and is often overexpressed in colorectal cancer.113 LAG-3 molecule can negatively regulate T lymphocytes, and the combination of LAG-3 inhibitor and PD-(L)1 inhibitor can enhance anti-tumor activity.114 As one of the new targets, LAG-3 has great potential in tumor immunotherapy. Targeting LAG-3 and its corresponding inhibitors may have a synergistic effect with anti-PD-(L)1 drugs to improve the immune response to cancer cells.113

The LAG-3 antibody favezelimab combined with pembrolizumab is currently in Phase I studies,32 with an ORR of 6.3% in 89 patients with pMMR/MSS mCRC who progressed after prior ≥2line therapy. The median PFS and OS were 2.1 and 8.3 months, respectively. Meanwhile, the median OS (12.7 months) in Combined Positive Score (CPS) ≥1 group was better than that in CPS<1 group (only 6.7 months), suggesting that further screening according to the level of PD-L1 expression in pMMR/MSS patients can identify the advantaged patients receiving LAG-3 antibody combined with anti-PD -(L)1 therapy.

T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory domains (TIGIT) is an inhibitory receptor shared by T cells and natural killer (NK) cells. On the one hand, TIGIT can inhibit the function of NK cells, prevent their killing of tumor cells and release of tumor cell antigens. On the other hand, it can directly inhibit the function of CD8+T cells, thereby inhibiting the killing effect of CD8+T cells on tumors; Meanwhile, Tregs with high expression of TIGIT can inhibit a variety of immune cells in the tumor immune cycle.115–118 Therefore, antibodies targeting TIGIT can restore the tumor killing ability of NK cells and CD8+T cells, and restore the anti-tumor immune response to normal.118,119 Based on the inhibitory effect of TIGIT in anti-tumor immune response, it has become a new target of tumor immunotherapy with potential.

Some studies have shown that although blocking TIGIT or PD-1 alone can enhance the function of CD8+ T cells, blocking both receptors at the same time is more effective in enhancing anti-tumor immunity of CD8+ T cells.120–122 Thibaudin et al found in vitro that Atezolizumab alone could only reactivate T cells from MSI-H tumors. The combination of Atezolizumab and anti-TIGIT monoclonal antibody tiragolumab reactivated T cells in 46% of MSS CRC samples, suggesting that the combination could initiate clinical trials in colorectal cancer patients with MSS status.123

GO30103 is the first human study to study the treatment of advanced solid tumors with anti-TIGIT monoclonal antibody tiragolumab.124 In this non-randomized controlled trial, tiragolumab was well tolerated with or without atezolizumab and showed initial antitumor activity of the combination therapy. Later, some other anti- TIGIT monoclonal antibodies such as vibostolimab and etigilimab combined with anti-PD-1 monoclonal antibodies in the treatment of advanced solid tumors were also reported, and the results were basically consistent.125,126 CITYSCAPE study is a phase II clinical study of TIGIT inhibitor tiragolumab combined with PD-L1 inhibitor Atezolizumab in the treatment of patients with PD-L1 positive metastatic NSCLC.127 The combination of tiragolumab and Atezolizumab resulted in a significant improvement in PFS compared to Atezolizumab monotherapy, with no increased safety risk. However, unfortunately, the PFS of the subsequent Phase III SKYSCRAPER 01 clinical study on NSCLC patients with high PD-L1 expression did not reach positive results, and the OS data was not mature.

T cell immunoglobulin and mucin domain 3 (TIM-3) are activation-induced inhibitory molecules involved in tumor immune tolerance and T cell depletion, while blocking TIM-3 pathway can promote the production of interferon-γ by T cells and enhance the immune function of tumor cells.128 Studies have found that in colorectal cancer, tumor-infiltrating CD8+ T cells that express both PD-1 and TIM3 have significantly lower levels of IFN-γ production, indicating that PD-1+ TIM-3+ CD8+ T cells are more dysfunctional than PD-1+ TIM-3-CD8 +T cells. Blocking TIM-3 may restore T cell response.129 Therefore, targeting TIM-3 is a promising approach to further improve the effectiveness of current immunotherapy. Data from several I/II clinical trials indicate that the anti-TIM-3 antibodies LY3321367 and MBG453 are well tolerated as monotherapy and in combination with anti-PD-L1 LY3300054 or anti-PD-1 PDR001 monoclonal antibodies, respectively; and showed preliminary antitumor activity.130,131 Encouragingly, partial responses were observed in 2 of the 6 patients with colorectal cancer who received the combination therapy.130

The immune regulation of colorectal cancer is a multi-link, multi-step and multi-mechanism process. These findings suggest that the use of single-agent anti-PD-1 monoclonal antibodies is not always sufficient to effectively restore T cell function, especially when these T cells co-express a variety of other immunosuppressive receptors. The results of the above clinical studies initially confirmed the effectiveness and safety of the combined treatment strategy of PD-L1 inhibitor and CTLA-4 inhibitor in MSS colorectal cancer, but most of the current studies are in the early stage, and more randomized controlled studies are still needed to continue to expand the sample for further verification. In addition, although the efficacy and safety of the combined treatment strategy of PD-L1 inhibitor and novel immune checkpoint inhibitors (LAG-3, TIGIT, TIM-3, VISTA, etc.) in MSS colorectal cancer has not yet been studied in clinical studies, according to the results of current clinical studies, They are expected to block multiple inhibitory signal pathways through combination therapy, thereby enhancing the anti-tumor immune response of the body to tumor cells, which is a promising anti-tumor immunotherapy strategy. In conclusion, combined immunotherapy strategies based on different mechanisms of action should be one of the models to continue to be explored in MSS colorectal cancer in the future.

ICIs Combined with Chemotherapy ICIs Combined with Chemotherapy Alone

Chemotherapy-induced tumor cell death can promote the release and presentation of tumor antigens, increase tumor immunogenicity, and activate and improve the activity of CD8+ T lymphocytes, so immune checkpoint inhibitors combined with cytotoxic chemotherapy can theoretically improve the efficacy.19,132–134 At present, basic chemotherapy drugs for colorectal cancer include platinum (such as oxaliplatin), topoisomerase inhibitors (such as irinotecan) and antimetabolic drugs (such as 5-fluorouracil).135 It was found that both 5-fluorouracil and oxaliplatin were immunogenic. 5-fluorouracil (5FU) has selective cytotoxicity to myeloid-derived suppressor cells (MDSCs). In vivo, 5FU treatment of tumor-bearing mice resulted in a significant decrease in the number of MDSCs in the mice’s spleen and tumor bed, but had no significant effect on T lymphocytes, natural killer cells, dendritic cells, or B lymphocytes.136 Oxaliplatin can also induce immunogenic cell death and increase the immunogenicity of MSS tumors, thereby improving the efficacy of immunotherapy when combined with PD-(L)1 inhibitors.137,138

Studies of Dosset et al showed that 5-FU combined with oxaliplatin can improve anti-tumor immune response in MSS CRC mouse model, and then FOLFOX regimen can induce PD-L1 expression and high CD8+T lymphocyte infiltration in tumor microenvironment in tumor samples of colorectal cancer patients. Thus, the FOLFOX regimen has a synergistic effect with anti-PD-(L)1 monoclonal antibody.139 These preclinical data suggest that the combination of a PD-(L)1 inhibitor with an inducer of immunogenic cell death can improve immunotherapy efficacy and support a therapeutic strategy of combining ICIs with chemotherapy such as FOLFOX regimens. Based on these data, a series of clinical studies were gradually carried out.

The METIMMOX study evaluated the efficacy of short-course repeat sequential oxaliplatin-based chemotherapy (FLOX) (oxaliplatin + fluorouracil) combined with nivolumab in first-line treatment of MSS mCRC. A total of 54 patients were enrolled, all of whom were RAS/BRAF mutation patients. ORR rates in the combined immunotherapy group and chemotherapy group were 48% and 23%, CR rates were 16% and 0%, respectively, indicating that immunotherapy combined chemotherapy could improve the chances of immunotherapy response for patients. However, the difference in median PFS between the two groups was not significant, 6.6 months and 5.6 months, respectively.33 KEYNOTE-651 evaluated the efficacy and safety of Pembrolizumab in combination with mFOLFOX7 or FOLFIRI in mCRC.34 The latest results showed that Pembrolizumab combined with mFOLFOX7 had an ORR of 61% for first-line therapy and Pembrolizumab combined with FOLFIRI had an ORR of 25% for second-line therapy. Both combination regimens showed antitumor activity in pMMR/MSS mCRC patients.

In addition, some studies have found that chemotherapy drugs related to DNA repair enzymes can produce better synergies with ICIs. O 6-methylguanine-DNA methyltransferase (MGMT) is designed to repair DNA damage caused by alkylating agents.140 If the tumor has MGMT gene silence (promoter methylation and protein expression loss), it will enhance the sensitivity of tumor cells to alkylate agents such as temozolomide (TMZ),141 and more secondary gene mutations may occur in the tumor after receiving TMZ, resulting in secondary high tumor mutation burden (TMB). The special synergistic mechanism makes TMZ a sensitizer for immunotherapy. Studies have reported that nearly 40% of mCRC patients have MGMT silencing.142 The MAYA study is based on this theory to explore a TMZ induction chemotherapy followed by combined immunotherapy strategy in MSS mCRC. This Phase II study included MSS mCRC patients who failed to respond to multi-line therapy and had silent MGMT expression, and received 2 cycles of TMZ induction chemotherapy followed by nivolumab combined with low-dose ipilimumab. The results showed that the median PFS and OS were 7.1 months and 18.5 months, respectively, and the ORR was as high as 39%; the incidence of grade 3 and above adverse reactions is low.35,36 This appears to be better than the results of a previous Phase II study of TMZ monotherapy in MGMT- silenced mCRC, which enrolled 41 patients with an ORR rate of only 10% and a median PFS and OS of only 1.9 and 5.1 months.143

ICIs Combined with Chemotherapy and Bevacizumab

The anti-angiogenic drug bevacizumab has been an important component of targeted therapy for mCRC and is approved for mCRC when used in combination with chemotherapy. By inhibiting the VEGF/VEGFR pathway, bevacizumab normalizes blood vessels, increases the infiltration of T lymphocytes, and activates immune effector cells by stimulating the maturation of dendritic cells and reducing the expansion of Tregs and MDSCs.144–146 Therefore, ICIs combined with chemotherapy and anti-angiogenesis targeted therapy have been the focus of research.

A Phase II placebo-controlled BACCI study evaluated the clinical activity of capecitabine, bevacizumab combined with atezolizumab in advanced refractory MSS mCRC, showing that the addition of atezolizumab failed to significantly improve PFS and OS.37 Another Phase II study evaluated the safety and tolerability of capecitabine, bevacizumab and pembrolizumab in the treatment of MSS mCRC. The ORR in the three-drug combination treatment group was 5%, which did not meet the predetermined target, but the median PFS and OS reached 4.3 and 9.6 months, respectively, and were tolerable.38 Meanwhile, after FOLFOX combined with bevacizumab induction therapy in MSS mCRC patients in MODUL Cohort 2 studies, atezolizumab is added to Fluoropyrimidine (FP) + bevacizumab as first-line maintenance therapy. The results showed that although no new safety signals were found, there was no improvement in efficacy.39,40

ICIs in combination with standard chemotherapy and bevacizumab is also being explored as a first-line treatment. The researchers first evaluated the efficacy of atezolizumab combined with bevacizumab and FOLFOX in the first-line treatment of MSS mCRC patients, with an ORR of 52% and a median PFS of 14.1 months, showing no significant activity compared with standard treatment.147 CHECKMATE-9X8 evaluated nivolumab combined with mFOLFOX6/ bevacizumab (NIVO+SOC) versus mFOLFOX6/ bevacizumab (SOC) for first-line treatment of mCRC. Most of the patients were MSS type. The ORR was 60% in the NIVO+SOC group and 46% in the SOC group, but the median PFS in both groups was 11.9 months, which did not meet the primary endpoint of PFS. However, after 12 months, the PFS rate in the NIVO + SOC group was higher than that in the SOC group.41

Two other clinical studies conducted in China have given some positive signals in terms of survival benefits. The Phase II BBCAPX study explored the efficacy of sintilimab combined with CAPEOX/bevacizumab in the first-line treatment of RAS mutation and MSS mCRC.42 The results showed that ORR and DCR were 84.0% and 100.0%, respectively. This efficacy data shows that the addition of immunotherapy improves efficacy by about 20% compared to the existing standard regimen. The results of follow-up were published in 2023, suggesting that the ORR and DCR of the combination treatment regimen remained stable, with a median PFS of 18.2 months, and were generally well tolerated.42 The study offers new hope for the up to 95% of MSS mCRC patients. However, the sample size of this study was small, only 25 people were included, and further large-scale studies are needed for verification. At present, the Phase III study of BBCAPX is in progress (NCT04194359). Another randomized, double-blind, phase 2 part of a phase 2/3 trial in China assessed the preliminary anti-tumor activity and safety of serplulimab plus HLX04 and XELOX as a treatment option for patients with MSS mCRC.148 The results showed that the ORR reached 65.5% and the median PFS reached 17.2 months. Although the median overall survival (OS) was not reached for either group, a trend of an OS benefit was observed for the serplulimab group (HR, 0.77; 95% CI, 0.41–1.45).

In addition, ICIs combined with three-drug chemotherapy and bevacizumab have also been explored for first-line treatment, equivalent to the use of the strongest induction chemotherapy, in order to kill more tumor cells and produce more neoantigens, so as to enhance the effect of immunotherapy and improve the survival outcome of patients. Recently, two related clinical studies (NIVACOR study and AtezoTRIBE study43,44 were published. Unfortunately, there was only some improvement in efficiency, but the survival benefit was not obviously. The single-arm Phase II NIVACOR study evaluated the efficacy of nivolumab combined with FOLFOXIRI/bevacizumab in first-line treatment of RAS/BRAF mutated MSS mCRC patients. The results showed that ORR and DCR were 78.9% and 96.2%, but mPFS were only 9.8 months.43 The AtezoTRIBE study is a multicenter, open-label, randomized, controlled phase 2 clinical trial that included unresectable mCRC patients regardless of MMR status. The results show that adding atezolizumab to first-line FOLFOXIRI/bevacizumab is safe. The median PFS of 13.1 months in the altazomab group was better than that of 11.5 months in the control group.44 At present, the OS data of AtezoTRIBE is still immature, and people are interested in further results of this study.

So far, immunotherapy combined with chemotherapy (including three drugs) and bevacizumab in the first-line treatment of MSS mCRC has shown very limited efficacy, and whether it can bring survival benefits to patients is still controversial. Further consideration should be given to the combination mode and related value of this treatment mode in the future. We look forward to the Phase III clinical study to obtain more data.

Dual ICIs Regimen Combined with Chemotherapy

The single-arm Phase 1b/2 MEDITREME trial evaluated whether the addition of durvalumab and tremelimumab to standard chemotherapy mFOLFOX6 improved therapeutic efficacy.45,46 Preliminary results showed that ORR and CR were 62.5% and 25%, respectively, and 1-year PFS were 50%, indicating that the above regimen was initially effective in treating MSS mCRC patients.47 In 2023, the results of the MEDETREME study were republished after long-term follow-up. Among them, 48 patients were MSS type, with a median PFS of 8.2 months, showing good safety. The study met its primary endpoint and achieved survival outcomes similar to those previously observed in bevacizumab combined chemotherapy regimens, which resulted in approximately 8 months of PFS.149 This study showed that the dual immunotherapy combined with standard chemotherapy was significantly better than chemotherapy alone or immunotherapy alone for MSS mCRC on two key efficacy indicators of ORR and PFS. Researchers have also explored predictive biomarkers, and the relevant information is expected to guide more accurate CRC combined immunotherapy strategies in the future.46

To sum up, the overall efficacy of ICIs combined with chemotherapy in MSS mCRC patients has been limited. Compared with previous phase III clinical studies of first-line or second-line chemotherapy combined with targeted therapy, there seems to be no obvious advantage in ORR and survival. In the context of targeted therapy being the front-line standard treatment, In the future, more exploration directions will focus on the triple regimen such as immunotherapy combined with chemotherapy and anti-vascular targeted therapy, as well as the “AtezoTRIBE” model with three-drug chemotherapy, and double immunotherapy combined with chemotherapy. The optimal combined model is worthy of further exploration.

ICIs Combined with Radiotherapy

The combination of radiotherapy and immunotherapy may have synergistic effects based on several mechanisms. Radiotherapy can induce tumor cell death, release tumor antigens and activate immune signals, thus promoting systemic immune response.150,151 Radiotherapy changed the tumor immune microenvironment and inhibited the immune escape of tumor cells, thus enhancing the killing ability of immune cells to tumor cells.152–154 Radiotherapy can also promote the diversification of tumor antigen pool, promote the antigen presentation of dendritic cells and stimulate the body’s specific immune response.152 Radiotherapy can induce the aggregation of TILs and the up-regulation of PD-L1 expression, thereby increasing the sensitivity of immunotherapy.155

Preclinical data suggest that the combination of ICIs with an inducer of immunogenic cell death, such as radiotherapy, improves the efficacy of immunotherapy.152,156 The synergistic effect of radiation therapy and ICIs has also been demonstrated in several studies of preclinical CRC tumor models.157,158 These mechanisms provide a basis for the clinical study of radiotherapy combined with immunotherapy for mCRC patients.

The ANAVA study enrolled 101 patients with locally advanced rectal cancer (LARC), and 96 patients who were pathologically evaluable underwent preoperative chemoradiotherapy (capecitabine +conventional fractionated long-term radiotherapy + avelumab). The results showed that 22 cases (23%) achieved pathological complete response (pCR),59 cases (61.5%) achieved obvious pathological remission, the rates of non-immune and immune-related adverse reactions above grade 3 were only 8% and 4%. There were 39 cases of MSS rectal cancer in the study, suggesting that radiotherapy combined with immunotherapy may be a potentially effective program for patients with MSS rectal cancer.48 The VOLTAGE-A study showed that 39 MSS LARC patients treated with nivolumab after the end of long-term concurrent chemoradiotherapy achieved 38% major pathological response (MPR) and 30% pathological complete response (pCR).49

A single-arm, non-randomized Phase II clinical trial (NCT03104439) enrolled 40 patients with MSS mCRC and administered nivolumab + ipilimumab + hypofractionated short-course radiotherapy. The results showed that the ORR and DCR were 10% and 25% respectively, and the mOS was 7.1 months, which indicated that radiotherapy combined with immunotherapy could improve the ICIs response rate and prolong the overall survival of MSS mCRC patients, providing a new treatment idea for MSS mCRC patients.50 However, in a Phase II study, 24 patients with chemotherapy-refractory MSS mCRC who received durvalumab combined with tremelimumab and radiotherapy achieved an 8.3% response rate, which did not meet the preset ORR of at least 25%, despite a median OS of 11.4 months.51 Another study explored the feasibility of Y90 liver radioembolization combined with durvalumab and tremelimumab for MSS mCRC and found that although the combination regimen was well tolerated by patients, there was no significant benefit.159 Similarly, Monjazeb et al found that the addition of hypofractionated radiotherapy to PD-1 and CTLA-4 inhibitors had no significant clinical benefit for MSS mCRC patients resistant to first-line chemotherapy.52 Therefore, the role of radiation therapy in overcoming therapeutic resistance to MSS mCRC remains to be further validated.

In summary, the preclinical model showed that combined radiotherapy on the basis of ICIs induced and enhanced systemic anti-tumor immune response. Relevant clinical studies have also confirmed that it can improve the pathological remission of MSS LARC patients, showing a relatively optimistic short-term efficacy. However, the safety and efficacy of this combination therapy model in MSS mCRC are not very consistent, and more large-scale prospective clinical trials are needed to further confirm it. At the same time, the choice of radiotherapy mode (including dose, segmentation, treatment order, etc.) is still inconclusive, and more studies are expected to further explore.

ICIs Combined with Targeted Drug ICIs Combined with Anti-EGFR Monoclonal Antibody

Anti-EGFR therapy is the standard treatment for RAS wild-type mCRC. The Fc segment of cetuximab can bind to CD16 receptors on NK cells and dendritic cells, and direct killing of target cells by effector cells is mediated through antibody-dependent cell-mediated cytotoxicity (ADCC). Meanwhile, cetuximab can effectively improve the immune microenvironment and promote the infiltration of immune cells within the tumor.160,161 In addition, cetuximab alone or in combination with chemotherapy can induce immunogenic cell death (ICD) of colorectal cancer cells, thereby activating an effective anti-tumor response.162,163 Therefore, anti-EGFR monotherapy or combination chemotherapy and ICIs have a good synergistic mechanism basis.

Several studies have confirmed the initial efficacy of ICIs combined with anti-EGFR mab. The Phase II CAVE study included RAS wild-type mCRC patients treated with cetuximab, and 92% of enrolled patients had MSS status. When cetuximab and Avelumab were combined for third-line treatment, the median PFS and OS of the population were 3.6 months and 11.6 months, respectively, but the overall objective response rate was not high, only about 7%.53 An updated long-term follow-up of the study found that the median OS was 18.6 months. At the same time, the significant effectiveness of cetuximab rechallenge combined with Avelumab was further verified in patients with RAS wild-type mCRC based on ctDNA cues.54

Another AVETUXIRI study was A Phase II study of Avelumab combined with cetuximab combined with irinotecan in the treatment of refractory MSS mCRC, in which cohort A was RAS wild type and cohort B was RAS mutant. The interim analysis results showed that the ORR of cohort A and cohort B were 30% and 0%, respectively, which seemed to be quite different. However, 6-month PFS rates were 40% and 38.5%, and 1-year OS rates were 50% and 46.2%, respectively. Finally, the RAS wild-type cohort reached the primary efficacy endpoint, and the RAS mutant cohort also saw some survival benefits. These results also suggest that regardless of RAS status, ICIs combined with anti-EGFR mab can improve the outcome of immunotherapy to some extent.55 The study is ongoing. A similar study evaluated the efficacy and safety of tislelizumab in combination with cetuximab and irinotecan in previously treated RAS wild-type mCRC patients in a single-arm Phase II TEC trial.56 The results showed an ORR of 33%, exceeding the study’s preset value of 30%, and a DCR of 79%, and the median PFS and OS were 7.3 and 17.4 months respectively.

Immunotherapy combined with anti-EGFR mab in first-line therapy is also being explored. A single-arm, multicenter Phase II AVETUX trial evaluated avelumab in combination with cetuximab and mFOLFOX6 in previously untreated RAS/BRAF wild-type, MSI-independent mCRC patients.57 Among the 41 MSS mCRC patients, the 12-month PFS rate was 40% and the median PFS was 11.1 months. The overall ORR and DCR were 81% and 89%, respectively. This study preliminarily suggests that immunotherapy combined with anti-EGFR monoclonal antibody and chemotherapy as first-line treatment may further improve efficacy.

In addition, the Phase II AVETRIC study of cetuximab combined with Avelumab and mFOLFOXIRI three-drug chemotherapy regimen for first-line treatment of MSS RAS wild-type mCRC patients is ongoing and is expected to bring more tips on immunotherapy for advanced bowel tumors. There is also a phase II study that is changing ICIs from single to double immunotherapy, with a stronger combination therapy. In RAS wild-type and MSS type mCRC patients who had not been treated with anti-EGFR mab, the combination of panitumumab, ipilimumab, and nivolumab resulted in an ORR of 35%. This study showed that wild-type patients with RAS could have more choices and could try to adopt a combination regimen of anti-EGFR monoclonal antibody plus dual immunotherapy.58

In conclusion, the combination mode of anti-EGFR monoclonal antibody plus immunotherapy has a theoretical basis, and it also shows us better disease control. In particular, the effectiveness is more evident in the posterior treatment and in the RAS wild-type population, which is worthy of further exploration in the future.

ICIs Combined with Anti-EGFR Monoclonal Antibody and BRAF Inhibitor

Preclinical studies suggest possible synergies between BRAF inhibitors and PD-1/PD-L1 inhibitors. It has been reported that BRAF inhibitors combined with anti-EGFR mab can induce transient MSI-H phenotype in MSS colorectal cancer, suggesting that this combination model may bring better survival benefits for BRAF mutated MSS patients.164

A Phase I/II study (NCT04017650) evaluated the efficacy and safety of encorafenib (BRAF inhibitor) in combination with cetuximab and nivolumab in the treatment of BRAF V600E mutant MSS mCRC. A total of 26 patients were enrolled, and the ORR and DCR of 24 patients that could be evaluated reached 50% and 96%, respectively, and the median PFS and OS were 7.4 months and 15.1 months, respectively.59 This is the best outcome for second-line and above treatment in patients with BRAF mutations, and it is well tolerated, and the randomized Phase II SWOG 2107 study is also ongoing.

ICIs Combined with MAPK Signal Transduction Pathway Inhibitors

Overexpression and activation of mitogen-activated protein kinase (MAPK) pathways are often detected in mCRC, and dysregulation of these signal transduction pathways can be used as potential targets for cancer therapy.165 RAS, RAF, MEK and ERK proteins are key factors in this pathway. Some studies have shown that RAS-MAPK pathway can directly promote tumor cell proliferation,166 while BRAF mutations often lead to continuous activation of RAS/RAF/MEK/ERK pathway. Combined inhibition of upstream and downstream nodes of RAS/MAPK pathway is an important strategy to overcome drug resistance. Studies have found that the ORR of BRAF inhibitor monotherapy is only 0–5%,167,168 and the co-inhibition of BRAF and MEK contributes to the inhibition of MAPK pathway. Therefore, the combination of anti-EGFR drugs with BRAF inhibitor and MEK inhibitor is one of the treatment options for BRAF V600E mutant mCRC. However, the therapeutic effect is still limited.169–171

Based on the available clinical evidence, the US Food and Drug Administration (FDA) approved BRAF inhibitor encorafenib in combination with cetuximab for BRAFV600E mCRC. However, with an ORR of only 20% and a median PFS of only 4.3 months, the clinical benefit was not long-lasting.170,171

In the era of immunotherapy, more and more studies have found that the activation of RAS-MAPK pathway is also related to the decrease of T lymphocyte infiltration in tumors, while inhibition of MEK can enhance T lymphocyte infiltration in tumors and synergistic effect with PD-1 inhibitors to enhance anti-tumor activity.164,172,173 Therefore, on this basis, the exploration of clinical research has been carried out.

First, in a Phase IB study, Atezolizumab in combination with cobimetinib (a MEK inhibitor) achieved manageable safety and clinical activity in mCRC patients after standard treatment failure, with ORR and DCR of 17% and 39%, respectively. The 12-month PFS and OS rates were 11% and 43%, respectively.60,61 Unfortunately, the IMblaze370 Phase III study of atezolizumab in combination with cobimetinib, published in the Lancet, yielded negative results. Patients who had previously failed multiple lines of therapy were enrolled, and regorafenib was used as the standard control. Results showed that the efficacy of the combination regimen and regorafenib standard third-line therapy was similar, and atezolizumab alone or combined with cobimetinib achieved OS of 7.1 and 8.9 months, respectively, which was not significantly better than the standard treatment regorafenib monotherapy (OS of 8.5 months).62

In addition, durvalumab plus the MEK inhibitor trametinib had a similar effect in MSS mCRC patients, showing acceptable tolerability, but no improvement in OS was seen.63 A trial of another MEK inhibitor binimetinib combined with pembrolizumab combined with beizumab in mCRC patients who had failed multiline therapy showed that a total of 21 patients with pMMR/MSS mCRC were enrolled, with ORR and DCR of 12% and 94%, respectively. The median PFS was 6.4 months and the adverse reactions were tolerable.64 Studies have shown initial clinical efficacy, but Phase III clinical trials are needed to confirm this. Therefore, whether MEK inhibitor combined with immunotherapy has a synergistic effect remains to be confirmed by further studies.

The co-inhibition of BRAF and MEK contributes to the inhibition of MAPK pathway, so the combination of BRAF inhibitor and MEK inhibitor is also one of the treatment options for BRAF V600E mutant mCRC, although the efficacy is limited.169 Studies have shown that combining drugs targeting the RAS/MAPK pathway with immunotherapy may lead to an enhanced immune response, which may improve patient outcomes.65 A single-arm phase 2 clinical trial of combined PD-1, BRAF and MEK inhibition with sparatlizumab (PDR001), dabrafenib and trametinib in 37 patients with BRAFV600E CRC, met its primary end point. In 32 patients with microsatellite stabilization, ORR and DCR reached 25% and 75%, respectively, and durability that were favorable relative to historical controls of BRAF-targeted combinations alone, and the combination regimen was well tolerated.65

Based on the regulatory effect of RAS/MAPK pathway targeting drugs on tumor immune microenvironment, continuing to explore new combined strategies of RAS/MAPK pathway targeting drugs and immunotherapy will likely bring lasting survival benefits for tumor patients with RAS/MAPK pathway mutations.

ICIs Combined with KRAS-G12C Inhibitors

Kirsten rat sarcoma virus oncogene homology (KRAS) mutation is an important genetic driver of colorectal cancer (CRC).174,175 KRAS mutation can lead to activation of downstream signaling pathways, promote tumorigenesis and invasion, and thus affect the prognosis of colorectal cancer.176,177

Drug targeting of KRAS mutations is challenging. Although KRAS G12C inhibitors show significant clinical response after their emergence, the resistance mechanisms of KRAS G12C inhibitors are more complex and diverse. The main driving factors include secondary mutations of KRAS itself, reactivation of multiple nodes upstream and downstream of KRAS on the MAPK pathway, and immune deficiency.178 Therefore, combination therapy may be an effective means to overcome drug resistance.

Preclinical trials of KRAS G12C inhibitors in combination with PD-1/PD-L1 inhibitors are underway. In 2017, British scholars found that KRAS signal could inhibit T lymphocytes’ immune surveillance of tumor cells by up-regulating the protein expression of PD-L1 in tumor cells.179 It has been demonstrated in preclinical models that the KRAS-G12C inhibitor sotorasib can promote anti-tumor immunity by inhibiting PD-L1 signaling in tumors, by increasing and activating T cell infiltration and making tumor microenvironment (TME) a highly sensitive to immune checkpoint inhibitors. Therefore, the combination of KRAS-G12C inhibitor and immune checkpoint inhibitor can produce durable activity.180

Another KRAS G12C inhibitor MRTX849, had similar results when used in combination with anti-PD-1 antibodies. MRTX849 reversed the immunosuppressed tumor microenvironment, thereby sensitizing the tumor to ICIs therapy. The results showed that in a genetically engineered mouse (GEM) model, the combination of MRTX849 and anti-PD-1 antibodies improved progression-free survival compared with either drug alone.181

In summary, preclinical studies have found that the combination of immune checkpoint inhibitors with anti-EGFR mab, BRAF inhibitors, MAPK signaling pathway inhibitors, etc., has a good mechanism basis. At the same time, some clinical studies have shown that immunotherapy combined with these targeted therapies has a certain effect in the treatment of MSS colorectal cancer, showing a certain therapeutic prospect. Unfortunately, most of these existing studies are single-arm small sample exploratory studies, and the data direction is not completely stable and consistent, and more research results and long-term follow-up are still needed to prove the efficacy of immunotherapy combined with targeted therapy in MSS colorectal cancer.

ICIs Combined with Novel Development Drugs ICIs Combined with Cancer Vaccines

Cancer vaccines are considered an option for immunotherapy in most solid tumors, including colorectal cancer. Several cancer vaccines have been studied, including autologous vaccines, dendritic cell vaccines, viral vector vaccines, and peptide-based vaccines, which stimulate anti-tumor immunity by containing tumor antigens, nucleic acids, whole cells, and peptides.182,183 These vaccines work by stimulating tumor antigen-specific cytotoxic T lymphocytes, which in turn recognize and potentially eliminate cancer cells in an antigen-specific manner.183,184 These results provide a theoretical basis for the combination of cancer vaccine and ICIs. Therapies involving cancer vaccines in combination with conventional treatments or different immunotherapies have been shown to be beneficial in combating tumor resistance and improving clinical outcomes, but clinical trials are still in the early stages and more research is needed to demonstrate their therapeutic potential.184–189

Oncolytic viruses (OVs) are cancer vaccines that use viruses as carriers to selectively infect tumor cells with natural or genetically modified viruses with replication ability, then induce tumor cell lysis and death, release cytokines and cell antigens, and make the tumor change from “cold” to “hot”. Thus, the anti-tumor effect of ICIs can be improved.190–194 Therefore, combining ICIs with OVs can enhance the anti-tumor ability of ICIs. OVs combined with PD-1/PD-L1 inhibitors has achieved good therapeutic ef