Immunological Assessment of Recent Immunotherapy for Colorectal Cancer

ABSTRACT Colorectal cancer (CRC) is the third most prevalent malignancy with increased incidence and mortality rates worldwide. Traditional treatment approaches have attempted to efficiently target CRC; however, they have failed in most cases, owing to the cytotoxicity and non-specificity of these therapies. Therefore, it is essential to develop an effective alternative therapy to improve the clinical outcomes in heterogeneous CRC cases. Immunotherapy has transformed cancer treatment with remarkable efficacy and overcomes the limitations of traditional treatments. With an understanding of the cancer-immunity cycle and tumor microenvironment evolution, current immunotherapy approaches have elicited enhanced antitumor immune responses. In this comprehensive review, we outline the latest advances in immunotherapy targeting CRC and provide insights into antitumor immune responses reported in landmark clinical studies. We focused on highlighting the combination approaches that synergistically induce immune responses and eliminate immunosuppression. This review aimed to understand the limitations and potential of recent immunotherapy clinical studies conducted in the last five years (2019–2023) and to transform this knowledge into a rational design of clinical trials intended for effective antitumor immune responses in CRC.


Introduction
Globally, colorectal cancer (CRC) is ranked as the third-leading frequent cancer, accounting for 10% of the incidence and 9.4% of the mortality rate (Ferlay et al., 2020;Sung et al., 2021).The American Cancer Society reported that 1,53,020 patients will be diagnosed with CRC and 52,550 individuals will eventually die by 2023, comprising 19,550 incidences and 3,750 mortality cases among individuals under 50 years of age (Siegel et al., 2022).The worldwide incidence of colon cancer between 2020 and 2035 is expected to increase by 43.6% and that of rectal cancer by 39.6% (Ferlay et al., 2020).The 5-year relative survival rate of CRC differs from 14% for distant cancer to 91% for localized-stage cancer (Siegel et al., 2022).
Radiotherapy, chemotherapy and surgery are the conventional approaches for CRC treatment.These treatments can be administered in combination for cancer progression and localization (Schmoll et al., 2012;E. Van Cutsem et al., 2014, 2016;Yoshino et al., 2018).In cases of tumors in an accessible region and at a localized stage, transanal surgery and total mesorectal excision (TME) via laparoscopy are frequent strategies (Bonjer et al., 2015; proficient and microsatellite stable) tumors with the absence of MSI characteristics, dMMR-MSI-H (mismatch-repair-deficient and high-level microsatellite instability) tumors with a greater mutation burden and pMMR-MSI-L (mismatch-repair-proficient and low-level microsatellite instability) tumors with a significantly lower mutation burden (Kawakami et al., 2015).Patients with CRC constitute ~15% of dMMR-MSI-H CRC cases and ~ 85% of pMMR-MSI-L CRC cases (Karuna et al., 2019).In certain cancer types, high tumor mutation burden (TMB) has been identified as a predictive biomarker for immunotherapy response (Chan et al., 2019;Samstein et al., 2019).Despite advancements in immunotherapy, the major challenge remains an ineffective current immunotherapy approach to target pMMR-MSI-L CRCs, which account for a large proportion of metastatic CRC cases (Karuna et al., 2019).Failure of immune cell infiltration and low TMB in CRC have been proposed as causes of immune resistance (Galon et al., 2006;Le et al., 2017).
In recent years, the mechanisms by which tumors alter the intrinsic immune system at the cellular level have become more thoroughly understood (Kim & Cho, 2022).Several factors contribute to the heterogeneous tumor microenvironment of CRC, which includes immunosuppressive cytokines (IL-10 and TGF-β), accumulation of regulatory T-cells, myeloid-derived suppressor cells and tumor-associated macrophages, resulting in tumorinfiltrating lymphocyte exhaustion (Shan et al., 2022).Multiple approaches to trigger immunity against cancer have been investigated.Furthermore, immunotherapy has been the most intriguing paradigm shift in cancer treatment over the last few years (Grierson et al., 2017;Stein et al., 2018).Cancer immunotherapy approaches are classified into five major subcategories: immunomodulators (cytokines, agonists, adjuvants and immune checkpoint inhibitors), targeted antibodies (antibody-drug conjugates, bispecific antibodies and monoclonal antibodies), adoptive T-cell therapy (engineered T-cell receptor therapy, chimeric antigen receptor T-cell therapy, tumor-infiltrating lymphocyte therapy and natural killer cell therapy), oncolytic virus therapy and cancer vaccines (dendritic cells, peptides, DNA, RNA, viruses and tumor cell vaccines).Owing to extensive epigenetic heterogeneity in CRC (Fearon, 2011), the success of monotherapy is low (Pecci et al., 2021).Analyzing the limitations of monotherapy treatment efficacy, recent clinical trials have focused on utilizing combination therapies (integrated immunotherapy or immunotherapy in combination with conventional treatments such as chemotherapy and radiotherapy) to synergistically induce significant tumor regression in CRC (Golshani & Zhang, 2020;Karuna et al., 2019;Pecci et al., 2021).

Immunomodulators
Immune checkpoint inhibitors (ICIs) have been extensively investigated as cancer therapies among the immunomodulatory subtypes.The maintenance of immune tolerance is regulated by immune checkpoints through co-inhibitory signaling pathways, but cancer cells modulate this pathway to evade immunosurveillance (Chen & Flies, 2013;Pardoll, 2012).ICIs are designed to inhibit co-inhibitory signaling pathways to promote antitumor immune response against malignant diseases (Figure 1) (Sharma & Allison, 2015a, 2015b).Nevertheless, the efficacy of ICI treatment is determined by MSI level in a particular CRC patient (Menter et al., 2019;Rodriguez-Salas et al., 2017).The predominant immune checkpoint targets are programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) and programmed cell death receptor-1 (PD-1) because of their abundance and overexpression in cancer (Seidel et al., 2018).Furthermore, different immune checkpoints are being studied to explore their prospective role in the cancer-immunity cycle such as T-cell immunoglobulin-3 (TIM-3), T-cell immunoglobulin and ITIM domain (TIGIT) and lymphocyte activation gene-3 (LAG-3) (Joller & Kuchroo, 2017).ICIs targeting PD-1 and CTLA-4 are more effective in MSI-H CRC patients because of their high TMB (D. Le, Uram et al., 2015;Snyder et al., 2018).Previous studies on CRC have identified a high neoantigen load as a genomic determinant of immune cell infiltration (Giannakis et al., 2016).
The advancement of ICI treatment in combination with other therapies resulted in a significant antitumor response through the upregulation of immune system activity at various phases of the cancer-immunity cycle (Zhu et al., 2021).In the last five years of clinical studies (Table 1), the clinical efficacy and safety profile of immunostimulator pixatimod in combination with nivolumab (humanized IgG4 mAb) (clinical trial identifier: NCT05061017) evaluated in a recent phase I study that included 33 CRC patients showed a disease control rate of 44% and an objective response rate of 12% (Lemech et al., 2023).Subsequent correlative analysis showed increased cytokine IP-10 levels, improved infiltration of T-cells and dendritic cells, and enhanced proliferation of CD4 + and CD8 + T-cells (Lemech et al., 2023).

Targeted antibodies
Monoclonal antibody (mAb) therapy is an efficient therapeutic approach for cancer patients (Mould & Meibohm, 2016).Monoclonal antibodies specifically bind to tumor-specific antigens (TSA) or tumor-associated antigens (TAA) present on the surface of malignant cells (Figure 2) (Bubeník et al., 1973;Hollinshead et al., 1985).Compared to traditional chemotherapeutic agents, mAbs have high target specificity (Lu et al., 2020).Furthermore, its versatility is exploited in next-generation antibody-based treatments such as bispecific/ multispecific antibodies, natural killer cells, antibody-drug conjugates and chimeric antigen receptor T-cells (Weiner et al., 2010).Immunoglobulin IgG is frequently used in mAb therapy due to the IgG interaction with Fc γ-receptors present on effector immune cells  (Weiner et al., 2010).IgG specifically binds to tumor cells, which allows effector immune cells such as dendritic cells, neutrophils, mononuclear phagocytes and NK cells to recognize target tumor cells (Weiner et al., 2010).IgG1 and IgG3 induce complement-dependent  cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) (Weiner et al., 2010).In addition, mAbs inhibit PD-1/PD-L1 interaction, thus suppressing PD-1 regulatory signals to activate tumor-infiltrating T-cells (Nguyen & Ohashi, 2015).Some of the comprehensively investigated overexpressed antigens in CRC are RAS-related protein (Rab-1A), coiled-coil domain containing 34 (CCDC34), human EGF receptor type-2 (HER2) and epidermal growth factor receptor (EGFR) (Geng et al., 2018;Janani et al., 2022;Siena et al., 2018;Wang et al., 2018).A prospective study concluded that KRAS, NRAS, BRAF (Hsu et al., 2016) andPIK3CA (Xu et al., 2017) mutations act as predictors of the extent of resistance to anti-EGFR mAbs in CRC.However, further investigation is required to identify the different mutations in CRC associated with resistance to anti-EGFR mAb therapy, which may contribute to ineffective clinical outcomes.This should be further explored in future clinical trials.
A rise in the trend of employing combination therapeutic strategies is because of their efficiency in synergistically enhancing antitumor immune responses and overcoming ineffective clinical outcomes.It has been hypothesized that radiotherapy and ICIs in combination with mAb therapy can further potentiate such immune responses.A recent single-arm, non-randomized phase II trial was conducted to investigate whether the combination of dual ICIs nivolumab (anti-PD-1) and ipilimumab (anti-CTLA-4) mAbs, along with radiation therapy (clinical trial identifier: NCT03104439), efficiently augmented the immune response in CRC.This promising clinical trial demonstrated an objective response rate of 15%, median progression-free survival (PFS) of 2.5 months, strong disease control rate of 37% and median overall survival (OS) of 10.9 months (Parikh et al., 2021).Transcriptome analysis validated that higher expression of NK cells indicates a potential biomarker for immunotherapy (Table 1) (Parikh et al., 2021).In an exploratory NICHE randomized study, humanized IgG1 kappa mAb ipilimumab (anti-CTLA-4) plus humanized IgG4 mAb nivolumab with or without celecoxib (anti-COX-2) (clinical trial identifier: NCT03026140) was used to examine the feasibility, clinical efficacy, safety and immunological correlates in 40 CRC patients (Chalabi et al., 2020).The treatment group exhibited significantly augmented CD8 + T-cell infiltration and T-cell receptor clonality in dMMR compared to pMMR CRC cases (Table 1) (Chalabi et al., 2020).Considering the changes in responder pMMR cases after post-treatment, an increase in FOXP3 + T-cells, CD3 + T-cells and tertiary lymphoid structures was observed compared to non-responder pMMR (Chalabi et al., 2020).A phase II trial evaluated the fusion of the chemotherapeutic trifluridine/tipiracil oral agent with anti-PD-1 nivolumab mAb (clinical trial identifier: NCT02860546) in microsatellite-stable metastatic CRC patients (Patel et al., 2021).Tumor regression was not observed and the median PFS was 2.8 months (Patel et al., 2021).The study failed to demonstrate clinical benefits owing to insensitive microsatellite-stable tumors to immunotherapy and did not proceed further to the second stage (Table 1).
The first clinical study to investigate the treatment efficiency of combining immunomodulation and DNA demethylation in solid tumors demonstrated pembrolizumab (anti-PD -1) plus azacitidine (DNA methyltransferase inhibitor) (clinical trial identifier: NCT02260440) in 30 chemorefractory CRC patients (Kuang et al., 2022).The results revealed a median PFS of 1.9 months, overall response rate of 3% and median OS of 6.3 months (Kuang et al., 2022).Tumor biopsy analysis showed decreased gene promoter methylation and increased intratumoral CD8 + TIL density (Table 1) (Kuang et al., 2022).
A multicenter, single-arm, phase Ib trial was conducted to investigate the safety profile and clinical efficacy of combining pembrolizumab mAb with a conventional chemotherapy approach in CRC patients (clinical trial identifier: NCT02375672) to explore predictive immune biomarkers in the circulating blood (Herting et al., 2021).The trial showed a complete response rate of 6.7%, median PFS of 8.8 months and a partial response rate of 50% (Herting et al., 2021).Analysis of immune biomarkers revealed significantly augmented CXCL10, upregulated circulating granzyme B, high CD8 + T-cell proliferation, increased FoxP3 + CD4 + T-cells levels, below-baseline levels of TGF-α, and decreased Tc17 and Tc1 cells (Table 1) (Herting et al., 2021).
In the PICCASSO phase I trial, the study focused on investigating the safety profile and clinical effectiveness of combining pembrolizumab mAb (anti-PD-1) and maraviroc (C-C motif chemokine receptor 5 antagonist) (clinical trial identifier: NCT03274804) in refractory pMMR CRC patients (Haag et al., 2022).The results demonstrated a median PFS of 2.10 months, median overall response rate of 5.3% and median OS of 9.83 months (Haag et al., 2022).Furthermore, translational analysis revealed an increase in the antitumor chemokine eotaxin (Table 1) (Haag et al., 2022).A phase I/II clinical trial evaluating the clinical effectiveness of combining anti-PD-1 pembrolizumab (humanized IgG4 mAb) plus Bruton's tyrosine kinase inhibition with ibrutinib (clinical trial identifier: NCT03332498) was initiated for refractory metastatic pMMR CRC patients (Kim et al., 2021).The median PFS was 1.4 months and median OS was 6.6 months, furthermore, stable disease condition was 26% and no objective response was observed (Kim et al., 2021).Moreover, statistically inconclusive evidence for immunological biomarkers showed limited antitumor activity (Table 1) (Kim et al., 2021).
In another phase I trial that utilizes cetrelimab (clinical trial identifier: NCT02908906) for treating advanced CRC patients, the treatment group exhibited a clinical benefit rate of 61.9% and an overall response rate of 23.8%.Subsequent correlative analysis of biomarker expression revealed high IP10 levels and IL-2 expression in treated patients (Table 1) (Felip et al., 2022).
In a recent phase I clinical trial, 42 CRC patients were treated with anti-CD73 IgG1λ mAb oleclumab monotherapy or with humanized IgG1 kappa mAb durvalumab (anti-PD-L1) (clinical trial identifier: NCT02503774).Substantiated median PFS was 1.8 months in both the combination and monotherapy approaches, median OS demonstrated different outcomes for monotherapy of 6.1 months and combination therapy of 5.6 months (Bendell et al., 2023).The immunological features of this study showed that oleclumab reduced CD73 levels on tumor cell exteriors (no evasion of immune surveillance) and restricted CD73 enzymatic activity, in addition to increasing CD8 + T-cell infiltration in tumors (Table 1) (Bendell et al., 2023).MEK inhibition influences the tumor microenvironment by limiting CD8 + T-lymphocyte exhaustion, stimulating CD8 + activation and increasing effector CD8 + T-lymphocyte infiltration into tumors (Ebert et al., 2016;Hu-Lieskovan et al., 2015;Verma et al., 2021).A phase II clinical study evaluated the fusion of trametinib and durvalumab (clinical trial identifier: NCT03428126) in 29 CRC patients (Johnson et al., 2022).The results demonstrated that the overall response was 3.4% and the median PFS was 3.2 months.Further analysis validated the increased expression of PD-1 and Tim3 immune checkpoints on CD8 + T-cells, and no improvement in T-cell infiltration in tumors was observed (Table 1) (Johnson et al., 2022).
In the METADUR phase II trial, the therapeutic efficacy of combining the hypomethylating agent oral azacitidine CC-486 with durvalumab (clinical trial identifier: NCT02811497) to enhance immunogenicity in CRC treatment (Taylor et al., 2020).The median OS was 5 months, median PFS was 1.9 months and diseases control rate was 7.1% (Taylor et al., 2020).Immunophenotyping by flow cytometry analysed peripheral blood mononuclear cells (PBMCs) samples revealed a decrease in circulating monocytes CD14 + T-cells and circulating CD3 + T-cells at C2D15, a subsequent increase in 70% expression of Ki67 in CD8 + T-cells and 47% in regulatory T-cells and CD4 + T-cells, and a lack of DNA demethylation in tumor biopsies (Table 1) (Taylor et al., 2020).However, no evidence of efficient clinical activity was reported because of insufficient DNA demethylation (Taylor et al., 2020).
Only two clinical trials have reported avelumab-containing treatments.The AVETUX phase II trial was designed to investigate the clinical efficacy, safety, feasibility and immunological biomarkers to assess the combination of cetuximab (chimeric human/mouse IgG1 mAb) and mFOLFOX6 with IgG1 mAb avelumab (clinical trial identifier: NCT03174405) in patients with predominant RAS/BRAF wild-type MSS CRC (Stein et al., 2021).The median PFS was 11.1 months, complete response rate was 11%, overall response rate was 81% and diseases control rate was 89% (Stein et al., 2021).Circulating tumor DNA analysis showed rapid tumor regression and elevation in the heterogeneous population of tumorinfiltrating T-cells (Stein et al., 2021).Expression of the PD-L1 K162fs variant causes the loss of PD-L1 function as a consequence of avelumab-induced antitumor effects (NK-mediated antibody-dependent cellular cytotoxicity) (Table 1) (Stein et al., 2021).A phase II trial validated the safety profile and clinical effectiveness of anti-PD-L1 mAb avelumab monotherapy (clinical trial identifier: NCT03150706) in patients with POLE mutations or dMMR-MSI-H CRC (Kim et al., 2020).The median PFS was 3.9 months and the median OS was 13.2 months (Kim et al., 2020).With respect to clinical characteristics, three POLEmutated CRC patients did not respond to monotherapy treatment and 30 dMMR/MSI-H CRC patients benefited from avelumab monotherapy (Table 1) (Kim et al., 2020).
An IMblaze370 phase III randomized trial investigated the safety profile and clinical efficacy in three treatment groups involved atezolizumab monotherapy or cobimetinib, in contrast to regorafenib (clinical trial identifier: NCT02788279) in microsatellite-stable chemorefractory metastatic CRC patients (Eng et al., 2019).The median OS was 7.10 months with atezolizumab monotherapy, 8.87 months with atezolizumab plus cobimetinib and 8.51 months with regorafenib (Eng et al., 2019).Median PFS was 1.94 months with atezolizumab monotherapy treatment, 1.91 months with atezolizumab plus cobimetinib and 2 months with regorafenib (Eng et al., 2019).Moreover, no complete response was reported during the treatment.Despite the involvement of dual PD-L1 and MEK pathway inhibitors, this study failed to demonstrate an antitumor immune response in chemorefractory metastatic CRC patients (Table 1).
INCAGN01949, a fully human immunoglobulin G1 kappa mAb, has been studied for its therapeutic efficacy against OX40 co-stimulatory receptors in CRC patients (Davis et al., 2022).A recent phase I/II trial conducted to examine INCAGN01949 (anti-OX40 agonist mAb) (clinical trial identifier: NCT02923349) safety and tolerability concluded that INCAGN01949 monotherapy did not significantly enhance the antitumor immune response (Table 1) (Davis et al., 2022).Furthermore, the potential of INCAGN01949 to evaluate anti-OX40 agonism in combination with other therapies is required.
Three clinical trials in this five-year period have evaluated the efficacy of tremelimumabcontaining treatments.A pilot clinical trial used mAbs to investigate the dual immune checkpoint blockers durvalumab and tremelimumab (anti-CTLA-4) (clinical trial identifier: NCT02754856) in 23 CRC patients (Kanikarla et al., 2021).The study results demonstrated that the median relapse-free survival was 9.7 months and the median OS was 24.5 months (Kanikarla et al., 2021).Post-treatment tumor tissue analysis revealed evidence of T-cell and B-cell activation (Table 1) (Kanikarla et al., 2021).Another similar experimental objective with an additional radiotherapy regimen (clinical trial identifier: NCT03122509) in the treatment of a phase II clinical trial evaluated the enhancement of immunity with the reduction of non-irradiated tumors in CRC patients (Segal et al., 2021).The results reported a median OS of 11.4 months, median PFS of 1.8 months and an objective response rate of 8.3% (Segal et al., 2021).Analysis of PBMC samples revealed CD8 + and CD4 + T-cells proliferation and activation, along with rehabilitation of exhausted CD8 + T-cells as indicated by Ki-67 marker expression and an increase in effector memory CD8 + T-cells (Table 1) (Segal et al., 2021).Similarly, a randomized phase II trial evaluated the improvement in 119 CRC patients treated with tremelimumab plus durvalumab (clinical trial identifier: NCT02870920) (Chen et al., 2020).The median PFS was 1.8 months and the median OS was 6.6 months (Chen et al., 2020).Circulating cell-free DNA analysis revealed improvements in the OS of patients with CRC, in addition to a plasma TMB of 28 variants per megabase or more (Table 1) (Chen et al., 2020).
In a recent phase I/Ib study, QL1706 (MabPair) single bi-functional engineered mAbs (consisting of anti-CTLA-4 IgG1 and anti-PD-1 IgG4) were used to investigate the clinical efficacy in CRC patients (clinical trial identifier: NCT04296994 and NCT05171790) (Zhao et al., 2023).The objective response rate was 7.4% (Zhao et al., 2023).However, this study substantiated the limited QL1706 efficiency for better clinical outcomes in CRC (Table 1).
In the last five years, only two clinical trials have reported targeting EGFR.In a multicenter phase II trial, the study aimed to determine the consequences of gene polymorphisms on the clinical outcomes of KRAS mutated 70 metastatic CRC patients treated with IgG1 mAb cetuximab (clinical trial identifier: NCT01450319) (Borrero-Palacios et al., 2019).The treatment group experienced disease progression in 68 metastatic CRC patients (97.1%); the median PFS was 2.53 months and the median OS was 6.71 months (Borrero-Palacios et al., 2019).Cox regression analysis revealed that improvement in OS after cetuximab treatment was significantly associated with the KIR2DS4 polymorphism (Table 1) (Borrero-Palacios et al., 2019).The MODUL randomized clinical trial was conducted to evaluate the clinical efficacy of biomarker-driven maintenance treatment which included a combination of vemurafenib (BRAF inhibitor) and cetuximab with 5-fluorouracil/leucovorin (thymidylate synthase inhibitor) (clinical trial identifier: NCT02291289) treated 40 CRC patients with BRAF mutation (Ducreux et al., 2023).The median OS was 24 months, median PFS was 10 months, overall response rate was 50% and diseases control rate was 90% (Ducreux et al., 2023).Exploratory genomic analysis of circulating tumor DNA revealed that acquired alteration of MAPK pathway genes, especially MAP2K1 variants was the most prevalent post-treatment mutation (Table 1) (Ducreux et al., 2023).
First-in-human phase I clinical trial of humanized IgG1 mAb NEO-201 (anti-core 1 O-glycans) administered in treatment-refractory 11 CRC patients (clinical trial identifier: NCT03476681) (Cole et al., 2023).No partial or complete response was observed; stable response was the best outcome observed in four CRC patients with RAS-mutated genes (Cole et al., 2023).NEO-201 treatment may reduce regulatory T-cells in the tumor microenvironment and increase cytokine levels (TNF-α and IL-10) after 24 hours of dose infusion (Table 1) (Cole et al., 2023).
To illustrate the clinical efficacy of HuMax-IL8, a humanized IgG1 kappa mAb that inhibits the function of chemokine interleukin-8 (IL-8), which is involved in evading immunosurveillance, tumor growth, recruitment of myeloid-derived suppressor cells and epithelial-mesenchymal transition, this phase I clinical trial illustrated the tolerability and safety profile of HuMax-IL8 treatment (clinical trial identifier: NCT02536469) in CRC patients (Bilusic et al., 2019).Immune assay analysis revealed a decrease in serum IL-8 after HuMax-IL8 treatment (Table 1) (Bilusic et al., 2019).However, the sample size for CRC patients is limited and should be explored in future clinical studies in combination with other immunotherapy strategies.
A recent first-in-human phase I trial was conducted to evaluate the safety profile, clinical efficacy and tolerability of administering a humanized IgG4 MK-1248 mAb targeting glucocorticoid-induced tumor necrosis factor receptor (GITR) alone or in combination with another humanized IgG4 kappa mAb pembrolizumab (anti-PD-1) (clinical trial identifier: NCT02553499) in CRC patients (Geva et al., 2020).With respect to clinical characteristics, only one patient with stable disease was reported to have the best response with monotherapy (Table 1) (Geva et al., 2020).However, this study cannot be considered further because of the limited sample size of CRC patients.

Adoptive T-cell therapy
Adoptive T-cell therapy enhances antitumor immunity by utilizing cells from other donors (allogeneic transfer) or patient (autologous transfer) to eradicate cancer (Rosenberg et al., 2008).The main effector immune cells are CD8 + T-cells (cytotoxic T lymphocytes) and CD4 + T-cells (helper T lymphocytes).CD4 + T-cells promote an adaptive immune response, predominantly via cytokine secretion, to initiate the antitumor response of cytotoxic T lymphocytes (CTLs) (Ahrends & Borst, 2018).CD8 + T-cells are stimulated by recognizing TAA exhibited by major histocompatibility complex (MHC) class I and effectively mediate cytotoxicity (Durgeau et al., 2018).The αβ T-cell receptor (TCR) heterodimer is expressed by most peripheral T-cells (Attaf et al., 2015), although the prominent T-cell population in the gut region expresses the γδ TCR heterodimer (Suzuki et al., 2020).Therefore, this may be an effective immunotherapeutic strategy for CRC.
Cytokine-induced killer (CIK) cells are non-specific immunologic effector cells of adoptive T-cell therapy and are composed of CD3 + CD56 + (NKT cells), CD3 − CD56 + (NK cells) and CD3 + CD56 − (T-cells) populations (Introna, 2017).CIKs cells are produced by ex vivo expansion of PBMCs with interferon-gamma (IFN-γ), recombinant human interleukin-2 (rhIL-2) and anti-CD3 antibodies (Pan et al., 2014;Pan et al., 2020;Zhang et al., 2014).Furthermore, CIK cells are MHC-unrestricted antitumor effector CD3 + T lymphocytes that induce cytotoxicity leading to antitumor activity, thus overcoming the limitation of TCR-MHC-based interactions for effector immune cell activation (Linn & Hui, 2003).CIK cell therapy is widely used as a treatment approach for cancer (Figure 3).A recent study concluded that combination therapy, which includes chemotherapy and sequential infusion of CIKs cells in CRC patients, significantly augmented the overall survival rate (Zhao et al., 2016).A prospective phase I trial investigated the safety profile and clinical efficacy of combining hyperthermia with adoptive T-cell therapy (CIK cells) and either anti-PD-1 pembrolizumab mAb or chemotherapy (clinical trial identifier: NCT03757858) in patients with advanced cancer (Qiao et al., 2019).Recruited CRC patients were divided into two treatment cohorts: hyperthermia + adoptive T-cell therapy + pembrolizumab (n = 4) and hyperthermia + adoptive T-cell therapy + chemotherapy (n = 3).The diseases control rate was 74.30% and the objective response rate was 42.80% (Qiao et al., 2019).Serum cytokine levels determined using cytometric bead arrays revealed an increase in TNF-α, IFN-γ, IL-4 and IL-2 levels in the circulating blood of clinical responder patients (Table 1) (Qiao et al., 2019).Thus, this study demonstrated promising clinical outcomes and serum cytokine levels may serve as predictive biomarkers for determining clinical efficacy.These findings offer a rationale for using CIK cells to treat CRC patients.

Oncolytic virus therapy
Oncolytic viruses are genetically engineered or naturally existing viruses recognized as a distinct therapeutic strategy designed to target cancer cells through preferential infection and replication in the cancer cells, stimulate host antitumor immune responses and cause cell lysis without harming healthy cells (Kaufman et al., 2015).The therapeutic effectiveness of oncolytic viruses is influenced by two mechanisms: first, oncolytic viruses inhibit tumor cell protein synthesis and infect tumor cells by self-replication until cell disintegration; and second, oncolytic viruses trigger tumor-infiltrating immune cells by secreting cytokines and an extensive quantity of tumor antigens (Shi et al., 2020;Twumasi-Boateng et al., 2018).Through viral genome engineering techniques, oncolytic viruses are often constructed to encode transgenes such as pro-apoptotic and immune-stimulatory genes; for example, proapoptotic genes include p53, tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and tumor necrosis factor alpha, while immune-stimulatory genes include IL-4, IL-2, IL-12 and granulocyte macrophage colony-stimulating factor (GM-CSF) to enhance antitumor efficacy (Andreansky et al., 1998;Bai et al., 2014;Han et al., 2007;Heiber &  Barber, 2011; Kim et al., 2006;Parker et al., 2000).These characteristic features are widely associated with oncolytic virus therapy; therefore, they are predicted to have a significant influence on the survival of cancer patients (Figure 4).The therapeutic efficacy of oncolytic viruses extends beyond tumor lysis to involve integrated regulation of the tumor microenvironment and immune system (Ilkow et al., 2015;Martin & Bell, 2018;Zhang et al., 2014).Thus, oncolytic viruses are an effective approach for immunostimulation in the tumor microenvironment and sensitize the tumor to ICI treatment (Shi et al., 2019).
The combination of ICIs with oncolytic virus therapy evaluated in clinical trials has shown synergistic effects in promoting intratumoral T-cell infiltration and enhancing tumor immunogenicity to potentiate the antitumor activity of ICIs (Lan et al., 2020;Oh et al., 2020;Ren et al., 2022;Ribas et al., 2017).Talimogene laherparepvec (T-VEC) is an intralesional injectable genetically modified herpes simplex virus type 1 encodes the GM-CSF which has demonstrated potential for advanced cancer treatment and is also a forefront therapeutic agent for combination immunotherapy (oncolytic virus therapy plus ICIs) treatment (Chesney et al., 2023;Johnson et al., 2015).Recently, few clinical trials have been conducted in the last five years to evaluate the efficacy of oncolytic viruses and ICIs in combination (Table 1).In a recent phase Ib clinical trial, 24 CRC patients were treated with atezolizumab (humanized IgG1 mAb) in combination with talimogene laherparepvec (clinical trial identifier: NCT03256344).The median PFS was 3 months, median OS of CRC patients was 3.8 months and objective response rate was 0%, along with limited evidence of antitumor activity (Table 1) (Hecht et al., 2023).
In another phase I non-randomized clinical trial, administration of enadenotucirev in combination with nivolumab treatment (clinical trial identifier: NCT02636036) validated significant improvement in 45 CRC patients, with a prolonged median OS was 16 months, median PFS of 1.6 months and objective response rate of 2% (Fakih et al., 2023).Patients who benefited from the combination therapy reported an increase in the infiltration of intratumoral CD8 + T-cells in 12 CRC patients and 7 patients had increased biomarker expression of CD8 + T-cells cytolytic activity (Table 1) (Fakih et al., 2023).These results indicate that the enadenotucirev oncolytic virus encodes immunostimulatory payloads and may enhance immune cell infiltration to improve the efficacy of nivolumab by overcoming anti-PD-1 resistance (Fakih et al., 2023).Based on these findings, further investigation is ongoing to design an enadenotucirev for reprograming the tumor microenvironment by encoding immune-enhancer transgenes.

Cancer vaccines
Antitumor immune responses were stimulated in patients by utilizing TSA in cancer vaccines (Figure 5).Two specific approaches to cancer vaccine treatment are therapeutic and prophylactic, where therapeutic cancer vaccines target existing malignancies and prevent tumor progression; and prophylactic cancer vaccines target the reduction of cancer mortality, incidence and morbidity (Lollini et al., 2015;Schlom et al., 2014).Overall, in previous studies, cancer vaccines have not demonstrated improved survival benefits compared with placebo or standard therapy (Hazama et al., 2014;Lazoura & Apostolopoulos, 2005;Okuno et al., 2014;Schulze et al., 2009).However, recent studies have used cancer vaccines in combination with ICIs to induce a stronger immunogenic activity in CRC (Table 2).
Only two clinical trials targeting the mucin 1 peptide have been reported in the last five years.In a phase II trial, double-blind, multicentre, placebo-controlled randomized study  investigated the efficiency of generating an antitumor immune response by administering a MUC1 peptide vaccine (clinical trial identifier: NCT02134925) targeting transmembrane glycoprotein mucin 1 in colorectal adenoma patients (Schoen et al., 2023).At week 12, 25% of MUC1 peptide vaccine recipients showed a ≥ 2-fold increase in anti-MUC1 IgG levels (Schoen et al., 2023).This study validated that MUC1 vaccine-treated patients with immune activity at week 12 showed a 38% reduction in colorectal adenoma recurrence compared with placebo (Schoen et al., 2023).Analysis of serum cytokines revealed that IL-6 and IL-8 levels increased in non-responders CRC (Table 2) (Schoen et al., 2023).Multispectral imaging of the immune infiltrate showed a significantly high level of myeloid-derived suppressor cell population in MUC1 vaccinated non-responder patients, and colorectal adenoma recurrence was observed in 27.3% of the treatment responders group (Schoen et al., 2023).However, the immune response to MUC1 is limited, and there is a need to further improve the trial design to obtain better clinical benefits.Tecemotide is another antigen-specific cancer vaccine that induces an immune response against the MUC1 antigen in CRC.This placebo-controlled, double-blind, multicenter, randomized phase II clinical trial was designed to examine the safety profile and clinical efficacy of tecemotide administration in patients with metastatic CRC (clinical trial identifier: NCT01462513) (Schimanski et al., 2020).The median OS was 62.8 months with an estimated 3-year OS rate was 69.1%, recurrence-free survival was 6.1 months (treatment group) and 11.4 months (placebo group) (Schimanski et al., 2020).However, this trial was insufficient to support the evidence of better clinical outcomes in metastatic CRC patients (Table 2) and did not validate the correlation between MUC1 expression status and clinical outcome.
Another recent phase IIb clinical study was based on the multi-peptide vaccine PolyPEPI1018 (clinical trial identifier: NCT03391232) to induce T-cell responses against cancer testis antigens expressed in colorectal tumors (Hubbard et al., 2022).The extended period of median PFS was 12.5 months in multiple vaccination recipients compared to a single dose (Hubbard et al., 2022).Using an in vitro stimulated ELISpot assay that assessed the induced CD8 + and CD4 + T-cell responses and hence the recruitment of cytotoxic tumor-infiltrating lymphocytes to tumor region, converting "cold" tumor into "hot" tumor (Table 2) (Hubbard et al., 2022).In addition, host human leukocyte antigen genotyping analysis has revealed PEPI-specific CD8+ T-cell responses as a predictor of efficient clinical outcomes (Hubbard et al., 2022).These developments highlight the significance of peptide-based cancer vaccines in CRC treatment.
Heat shock protein 105 (HSP105)-derived peptide vaccine is another peptide-based cancer vaccine targeting overexpressed HSP105 (anti-apoptotic) biomarker in CRC, but HSP105 is absent in healthy tissues except in adult testis (Kai et al., 2003).A recent phase  et al., 2019) I clinical trial evaluated the immunological effectiveness of an HSP105-derived peptide vaccine for the treatment of 30 advanced CRC patients (Shimizu et al., 2019).At the postvaccination stage, after 1 month, 16 patients showed progressive disease and 11 patients had stable disease.Ex vivo IFN-γ ELISPOT assay analysis reported an increase in the frequency of HSP105 peptide-specific CTL in PBMCs in 37% of patients and in vitro IFN-γ ELISPOT results indicated that 20% of patients had activated HSP105 peptide-specific CTLs (Table 2) (Shimizu et al., 2019).The study indicated a decrease in HSP105 expression levels in tumors after vaccination (Shimizu et al., 2019).
Adenovirus 5 [E1-, E2b-]-CEA (Ad-CEA) vaccine was recently assessed in a phase II randomized trial combined with avelumab (anti-PD-L1 mAb) plus mFOLFOX6 and bevacizumab (anti-VEGF mAb) (clinical trial identifier: NCT03050814) for the treatment of MSS metastatic CRC patients (Redman et al., 2022).The median OS for combinational therapy was 15.1 months and the median PFS was 10.1 months (Redman et al., 2022).Exploratory analysis of peripheral blood immune cell subsets reported evidence of proliferative marker Ki67 expression in effector CD8 + T-cells, memory CD8 + T-cells, intermediate monocytes and NK cells (Table 2) (Redman et al., 2022).Serum cytokine analysis revealed a reduction in immunosuppressive cytokines sCD40L and TGF-β levels (Redman et al., 2022).This study reported the development of multifunctional CD4 + and CD8 + T-cell mediated immune responses targeting MUC1 and brachyury antigens that were not encoded by the vaccine (Table 2) (Redman et al., 2022).Previous studies have identified overexpressed GUCY2C (receptor synthesizing the second messenger cyclic GMP) as a potential target for vaccine therapy in CRC (Snook et al., 2008(Snook et al., , 2016;;Witek et al., 2015).Recently, in a phase I trial, another adenovirus-based cancer vaccine, Ad5-GUCY2C-PADRE (clinical trial identifier: NCT01972737), was administered to early stage CRC patients to evaluate its immunological efficacy and safety (Snook et al., 2019).Analysis of ELISA and IFNγ-ELISpot assays for the quantification of T-cell response revealed that the GUCY2C-specific CD4 + T-cell response was eradicated by self-tolerance, whereas a GUCY2C-specific CD8 + T-cell response was observed in 40% of patients (Table 2) (Snook et al., 2019).This study emphasizes the significance of split tolerance in the induction of antitumor immunity.Selective elimination of the CD4 + T-cell response due to split tolerance limits the potential clinical efficacy of cancer vaccines.
GVAX is another vaccine utilized to induce an antitumor immune response by secreting GM-CSF as a vaccine adjuvant administered along with the immunomodulator cyclophosphamide (Cy) to inhibit regulatory T-cells (Bever et al., 2021).In this randomized pilot study, Cy/GVAX was initiated in combination with guadecitabine (DNA methyltransferase inhibitor) (clinical trial identifier: NCT01966289) to treat 18 advanced CRC patients (Bever et al., 2021).The median PFS was 50 days and median OS was 393 days.Immunohistochemistry staining analysis showed a rise in the CD68 + :CD8 + cell density ratio, whereas the major immune cell subsets were statistically insignificant (Bever et al., 2021).The study validated that an increase in CD45RO + tumor-infiltrating lymphocytes had no correlation with clinical benefits (Table 2) (Bever et al., 2021).However, this theory should be evaluated with a larger treatment group and well-designed combination therapy in future trials.Additionally, a phase II clinical trial examined the Cy/GVAX vaccine in combination with the PD-1 inhibitor pembrolizumab mAb treatment (clinical trial identifier: NCT02981524) in pMMR metastatic CRC patients (Yarchoan et al., 2020).The median OS was 213 days, diseases control rate was 18% and median PFS was 82 days (Yarchoan et al., 2020).Analysis of immunohistochemistry staining revealed an increase in PD-L1 expression within the tumor region and tumor necrosis observed in serial biopsies (Table 2) (Yarchoan et al., 2020).Based on these results, the study demonstrated evidence of biological activity, but was unsuccessful in demonstrating radiographic clinical responses in pMMR CRC patients.
A prospective randomized controlled clinical trial of pneumococcal 13-valent conjugate vaccine (PCV13) in combination with adjuvant chemotherapy (fluoropyrimidine ± oxaliplatin) regimens was conducted to analyze the serologic response in CRC patients (Choi et al., 2020).Overall, elicited antibody responses to the PCV13 vaccine were sufficient in CRC patients (Choi et al., 2020).During serum sampling, neutropenia was observed in 20 patients as a consequence of cytotoxic chemotherapy, although the serological response to PCV13 was not significantly different (Table 2).
The administration of immune adjuvants such as the Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) in combination with the WT1 peptide as a tumor antigen in cancer vaccine therapy was initiated in a recent phase I trial (Nishida et al., 2019).This clinical study was designed to investigate the immunological effects and safety profile of coadministering immune adjuvants and tumor peptides in advanced cancer patients.Immunological assessment revealed an insignificant transient increase in monocyte and neutrophil counts (Nishida et al., 2019).Subsequent correlative analysis validated that most CRC patients had an invariable median absolute lymphocyte count after vaccination (Nishida et al., 2019).Furthermore, the study investigated the WT1-specific immune response and the results showed that only one CRC patient had enhanced WT1-CTLs immunity (Nishida et al., 2019).Based on the overall results, this study failed to demonstrate conclusive evidence to investigate the immunological and clinical assessment of BCG-CWS co-administered with the WT1 peptide to modulate the antitumor immune response in CRC (Table 2).

Future perspectives and conclusions
CRC is a highly malignant solid tumor.Abnormalities in the tumor microenvironment in CRC significantly contribute to homeostasis imbalance and induce an immunosuppressive microenvironment that promotes tumor progression.Moreover, genetic heterogeneity in CRC, characterized by epigenetic and multiple genetic events, causes tumor phenotypic variation; therefore, major traditional therapeutic strategies remain inefficient in targeting CRC (Burrell et al., 2013).In recent decades, significant advancements in research and development of immunotherapy have focused on overcoming the limitations of past therapeutic approaches.Hence, an indepth understanding of the antitumor immune response induced by modern cancer immunotherapy is necessary as well as to identify predictive biomarkers.
In this review, we analyzed immunotherapy-based clinical trials (monotherapy or in combination with other therapies) conducted in the last five years between 2019 and 2023 to investigate the immunological response reported in CRC patients.This review focused on the selected 38 recent clinical trials, and further evaluation confirmed that majority of the latest clinical trials utilized a combination approach for synergistic effects.Owing to the high TMB in dMMR-MSI-H CRCs, there is an increasing trend for mAbs that block immune checkpoints (clinical trial identifier: NCT03150706) to activate cytotoxic CD8 + T-cell and prevent T-cell anergy.Additional investigations are essential to understand the rationale behind the principle of immunotherapy resistance and identify predictive biomarkers to determine the clinical outcomes in dMMR-MSI-H CRC.However, a significant challenge remains in developing efficient immunotherapy treatments to target pMMR-MSI-L CRC (prevalent CRC cases).Recent immunotherapy approaches have shown temporal heterogeneity of the antitumor immune response with statistically insignificant clinical efficacy and have generally proven ineffective (clinical trial identifiers: NCT03174405 and NCT03050814).With regard to acquired treatment resistance mutations in KRAS, BRAF, NRAS and MAP2KI (clinical trial identifier: NCT02291289) or other pathway genes that restrict immunotherapy efficiency, future treatment strategies must target alternative signaling pathways to overcome resistance.Furthermore, KIR2DS4 gene polymorphisms were significantly correlated with prolonged OS in CRC patients (clinical trial identifier: NCT01450319).
In the past five years, the frequency of adoptive T-cell therapy and oncolytic virus therapy has been reported to be the lowest among other types of immunotherapy (Table 1).With the growing trend of combination therapy and the urgency to find an effective CRC treatment, more investigations of adoptive T-cell therapy and oncolytic virus therapy (monotherapy or in combination approach) are required in order to achieve better clinical outcomes with limited immunosuppression.
In conclusion, cancer immunotherapy has demonstrated gradual progress in inducing an immunological response in CRC with certain limitations.The current study emphasizes the dynamics and broad emerging landscape of clinical trials that enhance immunological responses to identify efficient CRC treatments.We hope that the promising findings in this study will improve CRC treatment strategies in the future to overcome immunotherapy challenges.
the final manuscript editing and supervised the project overall.Both authors volunteered to proofread the manuscript and approved its final version.

Figure 1 .
Figure 1.Mechanisms of immune checkpoint inhibitors based on T-cells activity.T-cell function is initiated through interactions between major histocompatibility complex (MHC) molecules that present tumor peptides to the T-cell receptor (TCR).T-cell activation in the presence of immune checkpoint blockades (CTLA-4 and PD-1 inhibitors) to block respective receptors and initiate a T-cell-mediated antitumor immune response to eliminate cancer cells through apoptosis.Blocking CTLA-4 with inhibitors induces an immune response for tumor regression.

Figure 2 .
Figure 2. Monoclonal antibody (mAb) effector mechanisms in cancer.Anti-PD-1 and anti-PD-L1 mAbs specifically bind to PD-1 and PD-L1 receptors respectively, and inhibition of the PD-1/PD-L1 pathway results in the initiation of a T-cell-mediated antitumor response to eliminate tumor cells.Monoclonal antibodies activate other pathways such as complement cascade-mediated CDC, macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) and NK cell-mediated ADCC to produce an immune response and eradicate tumor cells.

Figure 3 .
Figure 3. Antitumor mechanism of cytokine-induced killer (CIK) cells.Activated CIK cells release granzyme B and perforin to induce caspase cleavage, resulting in tumor cell apoptosis.Additionally, PD-1/PD-L1 signaling pathway inhibited with PD-1 monoclonal antibody treatment to CIK cells.

Figure 4 .
Figure 4. Mechanism of an oncolytic virus triggering an antitumor immune response in synergy with immune checkpoint inhibitor treatment.Upon infection of the tumor with oncolytic viruses, it stimulates an effective antiviral immune response.This response causes the onset of antiviral cytokine production and increases reactive oxygen species (ROS) levels.Released cytokines and ROS from infected tumor cells activate antigen-presenting cells (APC) and CD8 + T-cells.Subsequently, oncolysis is caused by the exit of oncolytic viruses, which release pathogen-associated molecular patterns (PAMPs), tumor-specific antigens and danger-associated molecular pattern signals (DAMPs).Uptake of neoantigens by APC deliver as a signal to activate CD4 + T-cells.DAMPs (host cell proteins) and PAMPs (viral particles) both trigger the immune system by activating Toll-like receptors (TLRs) in APC.Activated CD4 + T-cells secrete IL-2 to trigger CD8 + T-cells.Moreover, treatment with anti-PD-1/PD-L1 antibodies blocks the PD-1/PD-L1 signaling pathway and amplifies the antitumor activity of CD8 + T-cells by promoting T-cell mediated cytotoxicity in tumor cells.Activation of a systemic immune response by oncolytic viruses can change a 'cold' tumor into a 'hot' tumor, resulting in more susceptible to immunotherapy.

Figure 5 .
Figure 5. Mechanism of cancer vaccine treatment.After inoculation with a cancer vaccine, the tumor antigens from the vaccine are processed by dendritic cells and present antigens on MHC I or MHC II (cross-presentation). Activated dendritic cells migrate to the lymph nodes to recruit and activate cytotoxic CD8 + T-cells and helper CD4 + T-cells through interactions between MHC class I on dendritic cells and T-cell receptors (TCR) on T-cells.Activated T-cells proliferate and differentiate into tumorspecific cytotoxic T lymphocytes (CTLs) and infiltrate the tumor microenvironment to promote tumor cell apoptosis.Direct reorganization of tumor antigens by B-cells in lymph nodes promotes B-cells to produce antibodies and initiates tumor elimination via ADCC.

Table 1 .
Immunological responses in recent clinical trials of immunomodulators, targeted antibodies, adoptive T-cell therapy and oncolytic virus therapy for CRC treatment (2019-2023).