Immunotherapy for precision medicine in colorectal cancer.

Introduction

Colorectal cancer (CRC) is one of the most common and lethal cancers [ 1 ]. The widespread adoption of early diagnostic screening tests for preventive care has allowed for the early detection of CRC and pre-cancerous lesions. This has contributed to a steady decline in overall CRC incidence and mortality in the past 30 years [ 2 ]. However, as of 2024, CRC remains the fourth most common cause of cancer and second leading cause of cancer-related death in the United States [ 1 ]. Importantly, the 5-year relative survival rate for CRC in patients with distant metastases is less than 20%. There are thus significant gaps in care for advanced disease [ 2 ]. Furthermore, in contrast to the drastic drop in CRC incidence among older people (≥65 years old), the incidence of CRC among younger adults (<55 years old) has doubled between 1995 and 2019, with a steady increase in the mortality rate [ 2 ]. Thus, there is a significant need to improve therapeutic approaches for CRC in younger adults and for advanced stage CRC [ 3 ].

The mainstay curative approach for local, early-stage CRC is surgery. However, the management of patients with advanced stage CRC is more complex and challenging [ 4 , 5 ]. The most commonly used systemic chemotherapeutics in CRC, as single agents or in combinations, are oxaliplatin, 5-fluoropyrimidine, irinotecan [ 6 – 10 ]. These agents inhibit tumor growth, but systemic toxicity due to their low specificity limits their usage in patients with comorbidities and compromises patients’ quality of life. Moreover, clinical outcomes for patients with high-risk or metastatic CRC remain poor [ 4 , 5 ].

While traditional chemotherapeutics treat all patients the same way, precision medicine focuses on a particular trait of the patient, their tumor, or other factors. This mitigates off-target toxicity to normal tissues and/or improves anti-tumor activity. Precision medicine involves tailored methods for detection, prevention, and treatment, with the goal of improving patients’ outcomes. CRC immunotherapies have been extensively investigated in laboratories and clinic trials for decades. Although there are currently a limited number of FDA-approved CRC immunotherapies, incremental progress in our understanding of cancer biology and immunology has led to advances in developing innovative, more precise CRC immunotherapies.

Antibodies were the first major immunological molecules to be explored for precision medicine because of their unique ability to bind specific targets and directly inhibit cancer growth or initiate immune-mediated antitumor activity. The first promising therapeutic monoclonal antibody for CRC, cetuximab, was first described in 1995 and became the first therapeutic monoclonal antibody approved by the FDA as a first-line therapy in 2004 [ 11 – 14 ]. While antibodies can mediate an antitumor immune response, they are not as robust in doing so as CD8 + cytotoxic T cells (CTLs). Therefore, numerous T cell-centered approaches are being developed and investigated for CRC. These approaches include cancer vaccines, adoptive cell therapies (ACT) with tumor-infiltrating lymphocytes (TILs), chimeric antigen receptor-T (CAR-T) cells, T cell receptor-engineered T cells (TCR-T cells), and cell-based therapies utilizing other cell types (e.g. NK cells, NKT cells, and macrophages), with CAR-expressing natural killer cells (CAR-NK cells) being the most prominent.

In this review, we focus on immunological approaches for precision medicine in CRC. We discuss modalities of precision immunotherapy, including antibody-based targeted therapies, immune checkpoint blockade (ICB), cancer vaccines, and adoptive cell therapies. We also discuss a variety of targets that are being actively investigated in clinical trials, including EGFR [ 15 – 17 ], VEGF and VEGF receptor [ 18 – 21 ], GUCY2C [ 22 – 24 ], HER2 [ 25 – 27 ], CEA [ 28 – 30 ], and NKG2D ligands [ 31 , 32 ].

Immunotherapy modalities

Antibody-based targeted therapy

Traditional chemotherapy is associated with substantial systemic toxicity [ 33 – 35 ]. Therapeutic monoclonal antibodies (mAbs), first used in 1988 for cancer treatment, mitigate systemic toxicity by virtue of their increased specificity for tumor cells [ 36 ]. Using mAbs, patient care can be personalized to target the abnormalities on a patient’s tumor cells, allowing for increased antitumor activity and decreasing the risk of adverse effects [ 37 , 38 ]. Therapeutic mAbs can kill cancer cells through multiple mechanisms. For the majority of mAbs, there are two effector mechanisms: 1) mAbs bind receptors or their ligands and block downstream signaling pathways that support cancer cell growth and survival [ 39 – 42 ]; 2) mAbs coat cancer cells, allowing effector immune cells to recognize and eliminate cancer cells via engagement of Fc receptors by the Fc regions of antibodies, a mechanism known as antibody-dependent cellular cytotoxicity (ADCC) [ 43 – 46 ].

Decades of mAb research and advancements in chemistry and gene engineering have allowed the production of mAb variations, such as immunotoxins (ITs) and bispecific antibodies. ITs are mAbs or fragments of mAbs that are chemically conjugated with potent toxins to deliver them precisely to tumor cells [ 47 – 49 ]. Bispecific antibodies are a class of antibodies that simultaneously bind two different epitopes [ 50 , 51 ]. Within the class of bispecific antibodies, there are two main subclasses: 1) antibodies that block multiple signaling pathways [ 52 – 54 ]; 2) bispecific T-cell engagers (BiTEs) that physically engage and activate host cytotoxic T cells to kill cancer cells [ 52 , 55 , 56 ].

Following the FDA approval of cetuximab for CRC in 2004, five therapeutic mAbs were approved over the next 20 years for CRC. Numerous targets and variations of antibody-based therapies are being studied in clinical trials and preclinical settings. We discuss prominent tumor-associated antigens (TAAs) used as targets to direct therapeutic mAbs, bispecific antibodies, and ITs for CRCs.

Immune checkpoint blockade (ICB)

Immune checkpoint blocking (ICB) antibodies are mAbs, but rather than blocking tumorigenic signaling pathways or engaging ADCC, they disrupt adaptive immune evasion processes employed by immunogenic tumors [ 57 – 59 ]. Established ICB approaches target PD-1/PD-L1 and CTLA-4, and emerging ICBs for CRC target CD47 and others.

PD-1 and CTLA-4 are inhibitory receptors expressed on the surface of immune cells. PD-1 is a transmembrane protein expressed on activated innate and adaptive immune cells, including T cells, natural killer cells, B cells, macrophages, and dendritic cells [ 60 – 64 ]. PD-L1 is the ligand for PD-1 and, upon binding to PD-1, activates downstream signaling pathways that inhibit T cell activation and survival [ 62 , 63 ]. CTLA-4 is expressed on activated T cells and was classically thought to compete with CD28 to bind CD80/CD86 and block co-stimulatory signaling for T cell activation, though additional mechanisms have recently emerged [ 65 – 68 ]. Under normal physiological conditions, PD-1 and CTLA-4 engagement mitigates autoimmunity [ 66 , 67 , 69 , 70 ]. However, PD-L1 expression on tumor cells and CTLA-4 expression on tumor-associated T cells prevent conventional host effector T cells from recognizing and eliminating cancer cells with abundant neoantigen loads due to highly mutated genomes [ 71 – 75 ]. Therefore, blocking PD-1 and CTLA-4 prevents CRCs with high neoantigen loads, such as those with high microsatellite instability (MSI) and mismatch repair deficiency (dMMR), from escaping host immune surveillance and elimination.

Pembrolizumab and nivolumab, two different anti-PD-1 mAbs, were approved by the FDA as first-line therapies in 2017 for patients with advanced or metastatic MSI or dMMR CRC. Clinical trials evaluating the antitumor activity of pembrolizumab for MSI CRC patients measured efficacy using objective response rate (ORR), the percentage of patients who achieved partial or complete response with treatment within a time span, as the primary endpoint. In two clinical trials, significant ORRs were achieved with the pembrolizumab treatment [ 76 , 77 ]. Similarly, a phase II clinical trial evaluating nivolumab efficacy for MSI and dMMR CRC patients achieved a significant ORR [ 78 ]. Significant ORR was also achieved in a phase II trial testing the combination of ipilimumab, an anti-CTLA-4 mAb, and low-dose nivolumab for advanced MSI high and dMMR CRC patients with manageable safety profiles [ 79 , 80 ]. In 2025, results from the phase III CheckMate 8HW trial demonstrated that nivolumab plus ipilimumab significantly improved progression-free survival (PFS) compared to nivolumab alone in MSI high and dMMR CRC patients, with acceptable safety profiles [ 81 ].

CD47 is a transmembrane protein, and the interaction of its extracellular domain with SIRPα on macrophages induces ‘don’t eat me’ signaling [ 82 , 83 ]. CD47 expression by normal tissues prevents healthy cells from being phagocytosed and cleared by macrophages [ 84 ]. Elevated CD47 expression on cancer cells allows them to evade macrophage surveillance and eventual cytotoxic T-cell recruitment by inhibiting phagocytosis and antigen presentation by dendritic cells [ 85 ]. Moreover, blocking CD47 signaling within cancer cells also promotes cell death and reduces cell migration [ 86 – 90 ]. Therefore, anti-CD47 mAbs were developed to overcome these intrinsic cancer cell and immune evasion processes. Magrolimab is an anti-CD47 mAb that was studied for use against hematologic malignancies and solid tumors, including CRC [ 91 ]. However, because an independent data monitoring committee found that a phase III trial of magrolimab increased the risk of sudden death, the FDA put a full clinical hold on all trials investigating magrolimab in 2024. Nonetheless, other studies targeting CD47 are being carried out to further study this novel therapeutic target. Novel approaches currently under investigation include the targeting of a more selective subset of CD47 and SIRPα interactions, alternative drug delivery approaches, and creating bispecific antibodies [ 92 , 93 ].

ICB disturbs one of the physiological mechanisms maintaining tolerance to self-antigens, which leads to toxicities [ 94 ]. Adverse events associated with ICB, typically known as immune-related adverse events (irAEs) can affect almost all tissue types, with variable severity and timing [ 95 ]. A meta-analysis of 112 clinical trials of PD-1/PD-L1/CTLA-4 blocking antibodies with 19,217 patients concluded the fatality rate to be 0.36% for anti-PD-1, 0.38% for anti-PD-L1, 1.08% for anti-CTLA-4, and 1.23% for combined PD-1/PD-L1 plus CTLA-4 blockade, with the most common cause of deaths being cardiac and neurological toxicities [ 96 ]. Beyond fatalities, irAEs are a significant barrier to ICBs, especially combinations. Investigation of mechanisms underlying beneficial antitumor efficacy and deleterious irAEs is a major focus of the ICB field to identify strategies that decouple efficacy from toxicity.

Cancer vaccines

While immune checkpoint blockade (ICB) seeks to invigorate endogenous immune responses, cancer vaccines seek primarily to stimulate de novo antitumor immune responses [ 97 – 99 ]. Cancer vaccines may contain tumor-associated antigens (TAAs), DNA or RNA encoding said antigens, whole cancer cells or cancer cell lysates, or antigen-presenting cells (APCs) [ 100 ]. After cancer vaccine administration, delivered antigens are acquired by APCs, in particular dendritic cells, via mechanisms such as phagocytosis and micropinocytosis [ 101 ]. The antigens are processed via the endocytic pathway, which involves their cellular entry via vesicular transport and subsequent degradation by acidic proteases. After processing, antigen fragments are loaded onto major histocompatibility complex (MHC) class I and class II molecules. In addition to antigen processing and loading onto MHC, dendritic cell maturation is also required [ 102 ]. In the context of cancer vaccines, these maturation signals are adjuvants (i.e. substances that mimic pathogen-associated molecular patterns and damage-associated molecular patterns) [ 103 ]. As dendritic cells mature, they secrete cytokines (e.g. IL-12, TNFα) and upregulate their expression of co-stimulatory molecules (e.g. CD80, CD86, CD40) critical for T-cell and other immune cell activation. Once fully mature and activated, dendritic cells are guided by chemokine gradients and adhesion molecule expression changes to the lymph nodes [ 104 ]. In the lymph nodes, activated dendritic cells interact with naïve T cells [ 105 ]. Recognition of antigen presented by MHC class I or II complexes on dendritic cells by T cell receptors (TCRs) on CD8 + cytotoxic T cells or CD4 + helper T cells, respectively, is critical for T-cell activation. Moreover, co-stimulatory receptor activation mediated by interactions between CD80/CD86 and CD40 on dendritic cells and CD28 and CD40L on T cells, respectively, are also critical for T-cell activation [ 106 , 107 ]. Dendritic cells also secrete various cytokines and chemokines that enhance innate and adaptive immune responses [ 108 ]. Once activated, CD4 + helper T cells proliferate and release cytokines that stimulate CD8 + cytotoxic T cells [ 109 ]. These CD8 + cytotoxic T cells seek out and kill cancer cells displaying the target antigen recognized by their TCRs. CD4 + helper T cells also support B-cell activation, promoting the production of antibodies targeting tumor antigens [ 110 ]. As cancer vaccines aim to stimulate de novo immune responses, MSI CRCs, which harbor a higher mutation burden and a stronger immunogenic tumor microenvironment compared to MSS CRCs, are likely to generate a more potent response [ 111 , 112 ].

Cancer vaccines can be prophylactic or therapeutic [ 113 , 114 ]. While prophylactic cancer vaccines (e.g. human papillomavirus vaccines to prevent cervical, vaginal, vulvar, and anal cancer) have been very successful, clinical trials for therapeutic cancer vaccines have mostly yielded lackluster results [ 97 , 114 , 115 ]. The first and only FDA-approved therapeutic cancer vaccine is sipuleucel-T for asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer [ 116 ]. Sipuleucel-T targets prostatic acid phosphatase (PAP), an antigen expressed in normal prostate and prostate cancers. The Sipuleucel-T registration trial demonstrated significantly improved overall survival with 512 total patients enrolled, of which two-thirds received Sipuleucel-T and one-third received placebo [ 117 ]. While there are not yet FDA-approved cancer vaccines for CRC, many targets have been explored, including carcinoembryonic antigen (CEA), mucin-1 (MUC-1), EpCAM, KRAS, epidermal growth factor receptor (EGFR), HER2, claudins, and guanylyl cyclase C (GUCY2C) [ 118 – 122 ].

Cancer cell vaccines

Cancer cell vaccines are composed of whole cancer cells or cancer cell lysates [ 123 ]. They are made using the patient’s own tumor cells (i.e. autologous vaccines) or antigens derived from other sources (i.e. allogeneic vaccines). Irrespective of the source of tumor cells/antigens, the goal of cancer cell vaccines is to introduce a broad range of antigens and stimulate a multifaceted, robust antitumor immune response. Some of the earliest CRC vaccines were cancer cell vaccines. An autologous irradiated cancer cell vaccine with the bacillus Calmette-Guérin (BCG) vaccine bacterium was studied in a series of clinical trials [ 124 – 126 ]. The efficacy in treating CRC was limited, with some improvements in survival and disease-free survival seen in patients with colon cancer. GVAX, an allogeneic, whole-cell, GM-CSF-secreting immunotherapy, is another cancer cell vaccine studied in clinical trials [ 127 ]. Although GVAX may modulate an antitumor immune response, no objective responses were achieved in patients with advanced MMR-proficient (pMMR) CRC.

Antigen vaccines

Antigen-targeted vaccines are designed to induce adaptive immune responses targeting the antigen(s) of interest. They may consist of peptides, polypeptides, or DNA or RNA - containing vectors or particles [ 98 – 100 , 113 , 114 ].

Peptide and polypeptide vaccines

Peptide and polypeptide vaccines are designed to contain epitopes that generate a robust immune response. Depending on their composition, peptide antigen vaccines are categorized as full-length or truncated. Full-length peptide vaccines contain the entire peptide sequence of the antigen, allowing for its entire sequence to be processed via the endocytic pathway [ 101 ]. This leads to the loading of a wide range of epitopes onto MHC. Truncated peptide vaccines contain shorter, specific segments of antigens that are usually selected for their strong immunogenicity. Polypeptide antigen vaccines contain multiple peptide chains and are often designed to contain the epitopes of several antigens [ 128 ]. Examples of peptide and polypeptide vaccines currently in clinical trials for CRC include PolyPEPI1018, ATP128, SLP mKRAS vaccine, ELI-002 7P, and CIMAvax-EGF ( Table 1 ) [ 129 – 133 ].

Vector vaccines

Viral and bacterial vectors have been a common approach to cancer vaccines. Typically, genes encoding antigens of interest are inserted into the vector genome. Administration of the recombinant vector results in antigen expression and delivery to APCs. Moreover, PAMPs and DAMPs from the vector provide innate immune activation leading to APC maturation and induction of robust adaptive immune responses to the vector and the tumor antigen. However, the vector immunogenicity is also a limitation. Preexisting or vaccine-induced immune responses to the vector can prevent tumor antigen gene delivery, expression, and immunity in vivo [ 134 ]. However, novel strategies are being developed to overcome both preexisting [ 135 ] and vaccine-induced [ 136 ] vector immunity, allowing for repeated vaccine administration and indication of robust and persistent immune responses. Vectored vaccines targeting CEA, GUCY2C, MUC-1 and others have shown promising immunity and are in active clinical development ( Table 1 ) [ 29 , 137 , 138 ].

DNA vaccines

DNA vaccines introduce DNA sequences encoding antigens. DNA vaccines must pass through the APC’s cell membrane, migrate to its nucleus, and ensure the initiation of transcription using the APC’s cellular machinery. After transcription and translation of the coding sequence, the antigens are processed via the endogenous pathway, which involves their degradation by cytosolic proteasomes, the transport of antigen fragments by TAP into the endoplasmic reticulum, and eventual presentation on MHC class I [ 139 ]. Although DNA vaccines are easy to design, they have poor immunogenicity [ 140 ]. While there are methods to increase DNA vaccine immunogenicity (e.g. use of adjuvants), and DNA vaccines continue to undergo clinical testing in CRC ( Table 1 ) [ 29 , 138 ], the field has seen a momentum shift toward mRNA vaccines.

RNA vaccines

RNA vaccines introduce mRNA sequences encoding tumor antigens. Like DNA vaccines, RNA vaccines are processed in APCs via the endogenous pathway [ 139 ]. However, mRNA vaccines have several advantages over DNA vaccines [ 141 ]. Most importantly, mRNA vaccines have stronger immunogenicity and better delivery to APCs due to their packaging in lipid nanoparticles (LNPs). Moreover, the ability of mRNA sequences to be quickly adjusted makes mRNA vaccines an excellent platform for personalized antitumor immunotherapy. Advances in genomic sequencing (e.g. next-generation sequencing, single-cell sequencing) and genomic analysis allow for the rapid determination of a patient’s specific tumor genome. In turn, mRNA vaccines can be quickly manufactured for expedient treatment and modified as the patient’s tumor genome changes. An example of mRNA vaccines currently in clinical trials for CRC is GRT-C901/GRT-R902, the personalized vaccine that targets 20 tumor-specific neoantigens ( Table 1 ) [ 142 ].

Adoptive cell therapy (ACT)

Adoptive cell therapy (ACT) is an innovative and promising strategy to robustly target cancer cells for elimination. Specific ACTs include chimeric antigen receptor (CAR)-expressing T-cell (CAR-T) and NK cell (CAR-NK) therapies, tumor-infiltrating lymphocytes (TILs), and TCR-engineered T-cell (TCR-T) therapy ( Figure 1 ). FDA-approved CAR-T therapies targeting CD19 or BCMA have revolutionized the management of refractory B cell-derived hematologic malignancies [ 143 – 148 ]. There is hope that ACTs will also revolutionize the management of CRC.

Tumor infiltrating lymphocytes (TILs)

Tumor infiltrating lymphocytes (TILs) are tumor-infiltrating and tumor-reactive lymphocytes that are extracted from the tumor and its microenvironment [ 149 , 150 ]. TILs were pioneered as an antitumor therapy by Steven Rosenberg and his colleagues at the National Cancer Institute, where they demonstrated the efficacy of TILs for treating metastatic melanoma [ 151 ]. TILs are extracted from surgically resected tumors and enriched by stimulation with cancer cells and IL-2 stimulation. They are then expanded for 2 to 3 weeks and infused into to the patient from which they were isolated [ 152 , 153 ]. Lymphodepletion with cyclophosphamide and fludarabine before infusion increases the antitumor efficacy of TILs [ 154 ]. In 2024, Amtagvi ® (lifileucel) was FDA approved for metastatic melanoma, based on clinical trials showing that about one-third of participants experienced a reduction in tumor size, several with complete remission, and 40% of the participants experienced no progression 1 year after a single injection ( NCT02360579 ) [ 155 ].

TIL infiltration strongly correlates with disease progression and disease outcomes in CRC patients undergoing conventional therapies with surgery ± adjuvant chemotherapy. Although a clinical trial conducted between 1995 and 1998 in Italy showed no efficacy of IL-2 expanded TILs in 47 enrolled CRC patients [ 156 ], more recent clinical trials have shown more promising results. In 2010, a group in Sweden demonstrated that four out of nine stage IV CRC patients who received TILs achieved complete tumor regression, with extended median survival time, without toxicity [ 157 ]. In 2015, a group in China demonstrated improved 24-month survival and median overall survival in 25 stage IV CRC patients [ 158 ]. In 2016, Rosenberg’s team expanded and infused KRAS-mutant-specific CD8 + T cells from a lung metastasis of a CRC patient and demonstrated regression of all lung metastases [ 159 ]. Multiple ongoing clinical trials are investigating TILs in combination with immune checkpoint blockade for CRC ( Table 2 ).

T cell receptor-engineered T (TCR-T) cell therapy

T cell receptor-engineered T (TCR-T) cell therapy utilizes genetically engineered autologous T cells expressing TCRs that recognize a tumor antigen peptide presented by MHC class I [ 160 , 161 ]. In contrast to TILs, which contain a heterogenous population of T cells recognizing different antigens and epitopes presented by MHC-I, high-affinity TCRs toward the most desired antigens are selected to be sequenced and subsequently engineered into autologous T cells [ 162 ]. In August 2024, the FDA approved Tecelra ® (afamitresgene autoleucel), a TCR-T therapy targeting MAGE-A4, for metastatic synovial sarcoma. Notably, Tecelra ® was the first TCR-T therapy to receive FDA approval. Its approval was based on a phase I clinical trial conducted at the MD Anderson Cancer Center, where 19 out of 44 patients showed overall responses with acceptable toxicity profiles [ 163 ]. TCR-T therapy has been explored for CRC in multiple clinical trials; safety has been the primary hindrance to obtaining FDA approval. For example, in a clinical trial concluded in 2011 at NIH, CEA-directed TCR-T therapy for metastatic CRC-induced tumor regression in 3/3 patients. However, this TCR-T therapy was associated with severe colitis in all three patients [ 164 ]. This study demonstrated the high potency of TCR-T cells, emphasizing the need to carefully select target antigens to minimize toxicity risk.

Chimeric antigen receptor-T (CAR-T) cell therapy

Chimeric antigen receptor-T (CAR-T) cell therapy employs engineered autologous T cells expressing chimeric antigen receptors (CARs) that recognize surface tumor antigens, independent of MHC [ 145 ]. MHC I peptide presentation is a complicated cellular process, and reliance on MHC I presentation for tumor antigen recognition leaves ample opportunities for escape, such as downregulated expression of MHC I and mediators in the peptide processing pathways [ 165 ]. Indeed, in the Rosenberg study described above, a CRC metastatic lesion escaped TIL therapy by eliminating expression of the HLA molecule presenting the KRAS neoepitope recognized by the TILs [ 159 ]. Moreover, TCRs employed in TCR-T therapies are restricted by HLA (MHC) type, resulting in limited treatable patient populations – the most common MHC-I molecules are present in ~20% of the United States population. CARs overcome this limitation by using extracellular antigen-recognition domains composed of single-chain variable fragments (scFvs) from the heavy and light chains of immunoglobulins to allow MHC-independent antigen recognition. Intracellular domains of CARs, connected to the extracellular domain with a hinge region for flexibility and a transmembrane domain, contain critical activators of TCR signaling, including CD3ζ, CD28, and 4–1BB [ 166 , 167 ].

CAR-T therapy has revolutionized therapy for patients with certain end-stage hematologic malignancies following the first FDA approval in May 2018. Since then, there have been several approved CAR-T therapies directed against the surface antigens CD19 and BCMA. These approved CAR-T therapies are highly efficacious in controlling an array of B cell leukemias, B cell lymphomas, and multiple myeloma [ 144 , 147 ]. Although CAR-T therapies for solid malignancies is a rigorously active field of research, none have obtained FDA approval thus far. Various approaches are being pursued to improve the efficacy and safety of CAR-T therapies for solid malignancies, including cytokine-secreting CAR-T cells [ 168 ], combined therapies with immune checkpoint blockade [ 169 ], and logic-gated CARs [ 170 ]. Moreover, allogeneic CAR-T therapy is also being investigated as advances in genetic engineering continue [ 171 , 172 ]. Allogeneic CAR-T therapy allows the use of any healthy donor’s T cells for manufacture, which not only overcomes the barrier of compromised T cell compartments in cancer patients but also bypasses the wait time of manufacturing CAR-T cells for patients. For CRC, many targets have been explored for CAR-T therapy, including CEA, EGFR, GUCY2C, NKG2D ligands MICA/B, and HER2.

Chimeric antigen receptor-natural killer cell (CAR-NK) therapy

NK cells are a major subset of immune cells with cytotoxic capabilities and roles in antiviral and antitumor immunity via adaptive and innate immunity [ 173 ]. As such, NK cells express a complex repertoire of inhibitory and activating receptors, and the disturbed combination of inhibitory and activating signals can activate them for killing. Once activated, NK cells secrete cytolytic granules and induce apoptosis. CAR-NK therapy is attractive because, unlike canonical T cells, allogeneic donor NK cells can be administered to unrelated recipients. Furthermore, the cytokines secreted by NK cells are less likely to induce cytokine release syndrome and neurotoxicity than those secreted by T cells [ 172 – 177 ]. Major challenges to successful CAR-NK therapy for solid malignancies include the short lifespan of NK cells, the inhibitory effects of the tumor microenvironment, and low tumor infiltration [ 178 ]. Nonetheless, multiple clinical trials are investigating CAR-NK cells for CRC. These include CAR-NK cells engineered to target NKG2D ligands ( NCT05213195 ), MUC-1 ( NCT02839954 ), and TROP2 ( NCT06358430 ).

CRC immunotherapy targets

Epidermal growth factor receptor (EGFR)

EGFR is a well-studied transmembrane glycoprotein overexpressed in 25–80% of CRC tumors [ 15 , 17 , 179 – 183 ]. EGFR is a receptor tyrosine kinase expressed on cell membranes, and its downstream signaling cascades regulate cell proliferation, survival, migration, and differentiation in normal and cancer cells [ 184 – 187 ]. A litany of ligands bind the extracellular domain of EGFR, including epidermal growth factor, transforming growth factor, betacellulin, epiregulin, epigen, and amphiregulin [ 188 ]. Upon ligand binding, EGFR dimerizes and becomes activated, leading to the auto-phosphorylation of tyrosine residues in the cytoplasmic region [ 189 ]. Phosphorylated tyrosine residues activate various essential cell signaling pathways, including RAS, AKT, and STAT [ 184 , 185 ].

Cetuximab and panitumumab are anti-EGFR mAbs approved by the FDA in 2004 and 2006, respectively. Both bind the extracellular domain III of EGFR [ 190 – 192 ]. The binding of anti-EGFR mAbs to tumor cells not only engages host immune cells to eliminate tumors through the canonical ADCC mechanism [ 193 ] but also inhibits downstream signaling cascades essential for cancer cellular functions by preventing ligand binding and inducing receptor internalization for degradation [ 194 – 196 ]. Cetuximab achieved FDA approval in 2004 after the BOND trial demonstrated that cetuximab, given alone or with irinotecan, improved progression-free survival (PFS) in chemotherapy-refractory CRC patients [ 11 ]. Four clinical trials in 2007, 2009, 2011, and 2012 investigated cetuximab in combination with the chemotherapy regimens FOLFIRI and FOLFOX, revealing no improvement in overall survival (OS) and mixed results for PFS improvements [ 13 , 14 , 197 , 198 ]. However, subsequent studies (PARADIGM, FIRE-3, and CALGB-80405) established blockade of EGFR and VEGFR (below) as first-line treatment with chemotherapy in left-sided, KRAS/NRAS/BRAF wild-type CRC [ 199 – 201 ]. In terms of using cetuximab for maintenance therapy, a 2018 trial showed that PFS was not negatively impacted, and the number of adverse events was less if cetuximab alone is given after combination therapy of FOLFOX with cetuximab, compared to additional FOLFOX [ 12 ]. Panitumumab, the other FDA-approved anti-EGFR mAb for first-line CRC treatment, differs from cetuximab in that it is a fully humanized monoclonal antibody with a lower risk for hypersensitivity reactions [ 191 ]. A phase III trial in 2014 demonstrated that neither cetuximab nor panitumumab is superior or inferior to each other [ 202 ].

EGFR-directed CAR-T therapy has been investigated in multiple phase I and II clinical trials for glioblastoma and non-small lung cancer. These trials have all demonstrated clinical safety, yet the efficacy and survival benefits of said therapeutics are thus far ambiguous [ 203 – 205 ]. There are multiple ongoing clinical trials investigating EGFR-directed CAR-T therapy for CRC ( NCT03542799 , NCT03152435 ).

Vascular endothelial growth factor (VEGF)-A and VEGFR-2

VEGF-A and its receptor VEGFR-2 are overexpressed in 90% and 80% of CRC patients, respectively [ 18 , 206 ]. VEGF-A is one of the five ligands of the VEGF family [ 19 , 20 , 207 ], and VEGFR-2 is one of the five receptors of the VEGFR family [ 208 , 209 ]. VEGF-A is the primary ligand for VEGFR-2, a tyrosine kinase receptor [ 20 ]. Upon ligand binding, VEGFR-2 is phosphorylated, leading to the activation of critical downstream signaling pathways, including PLCγ, AKT, and MAPK [ 209 ]. These activated pathways promote cell growth and induce cytoskeleton changes with nitric oxide secretion that promotes vascular formation and permeability [ 210 – 215 ]. Angiogenesis, the formation of new blood vessels, allows sufficient nutrients and oxygen to be delivered for tumor growth and provides routes for cancer cells to migrate and metastasize [ 216 – 219 ].

The anti-VEGF-A mAb bevacizumab was approved in 2004 for metastatic CRC as a first-line therapy 14 days after cetuximab. A phase III trial of 813 patients completed in 2004 comparing bevacizumab alone to a regimen of irinotecan, 5-fluorouracil, and leucovorin showed significantly improved OS and PFS with the bevacizumab alone regimen [ 220 ]. However, five subsequent phase III trials investigating bevacizumab in combination with FOLFOX and FOXFIRI showed mixed results for improved OS or PFS [ 221 – 225 ]. A large observational cohort study in 2008 also demonstrated improved OS with bevacizumab as maintenance therapy compared to no maintenance therapy [ 226 ].

Ramucirumab is an anti-VEGFR-2 mAb that is approved for metastatic CRC patients as a second-line therapy in combination with 5-fluorouracil. A phase III trial concluded in 2015 showed improved OS and PFS by adding ramucirumab to FOLFIRI compared to adding a placebo to FOLFIRI [ 227 ].

Aflibercept, a fusion protein of the extracellular domains of VEGFR-1/2 with the human IgG1 Fc domain, is another FDA-approved VEGF-targeted therapy for metastatic CRC [ 228 , 229 ]. The phase III VELOUR study, which concluded in 2012, demonstrated that adding aflibercept to FOLFIRI significantly improved OS and PFS, compared to FOLFIRI alone [ 230 ].

Cancer vaccines inducing immunologic responses to VEGFR for CRC patients have also been investigated in clinical trials [ 21 ]. In a phase I clinical trial concluded in 2013, a peptide cancer vaccine targeting VEGFR1, VEGFR2, RNF43, and TOMM34, in combination with oral chemotherapy medications was investigated for advanced or recurrent colorectal cancer [ 231 ]. In this trial of 10 patients, one patient had a partial response, seven had stable disease, and two had progressive disease based on tumor imaging after one course of administration [ 231 ]. However, a phase II trial of 96 chemotherapy-naïve advanced CRC patients did not show improved efficacy endpoints with VEGFR1, VEGFR2, RNF43, TOMM34, and KOC1 peptide vaccine combined with oxaliplatin-based chemotherapy [ 232 ].

Guanylyl cyclase C (GUCY2C)

GUCY2C is a transmembrane glycoprotein expressed exclusively on the apical side of normal intestinal epithelium [ 233 ], select parts of the brain [ 234 , 235 ], and throughout gastrointestinal tumors [ 236 , 237 ]. GUCY2C can be activated by endogenous ligands, guanylin and uroguanylin [ 238 ], and bacterial heat-stable enterotoxins [ 239 ]. Upon activation, GUCY2C converts GTP to cGMP, which then initiates multiple critical downstream signaling pathways, including those maintaining homeostasis [ 240 ], genomic integrity [ 241 ], limiting cell proliferation [ 241 ], and protection against reactive oxygen species [ 234 ]. GUCY2C is expressed in nearly all CRCs [ 242 , 243 ], and its restricted expression on the apical aspect of enterocytes allows it to be a safe therapeutic target [ 134 , 243 – 245 ]. Although none of the GUCY2C-targeting mAb therapies have thus far achieved FDA approval [ 243 , 246 – 254 ], multiple modalities targeting GUCY2C are being explored in clinical trials ( NCT04111172 , NCT05287165 , NCT04171141 , NCT05779917 ).

Given that multiple studies have demonstrated that GUCY2C expression is retained in tumors [ 236 , 237 , 242 , 245 ] and that GUCY2C undergoes receptor endocytosis upon binding with ligands and antibodies [ 253 , 255 , 256 ], GUCY2C has been utilized as a promising target for immunotoxins (ITs). The first anti-GUCY2C IT tested in clinical trials was TAK-264 [ 253 ], an anti-GUCY2C mAb conjugated to monomethyl auristatin E (MMAE). MMAE is a cytotoxic agent that inhibits cell division by blocking tubulin polymerization, and it is one of the most commonly used cytotoxins [ 257 ]. TAK-264 showed promising efficacy in preclinical models and manageable safety profiles in a phase I clinical trial with 35 CRC patients [ 252 ]. Phase II clinic trials for TAK-264 were initiated for only gastric and pancreatic cancer, and both were terminated due to unsatisfactory efficacy [ 247 ]. The second generation of anti-GUCY2C IT is TAK-164, which utilizes the same anti-GUCY2C mAb but is conjugated to DGN549 instead of MMAE [ 253 ]. DGN549 is a highly toxic alkylator that causes DNA damage to interfere with cell proliferation [ 258 ]. TAK164 showed promising efficacy in preclinic models and manageable safety profiles in a phase I clinical trial concluded in 2023 with 25 CRC patients [ 248 ]. However, the recommended phase II dose determined by the study is considered too low to render clinic benefits.

GUCY2C has also been explored as a target for bispecific T-cell engagers (BiTEs) containing two antigen-recognizing domains: one against GUCY2C and one against CD3 [ 259 ]. With BiTEs, T cells without tumor-specificity can be redirected to target GUCY2C-expressing tumor cells to mediate conventional effector T cell functions through CD3 signaling independent of the canonical TCR-MHC binding [ 259 ]. PF-07062119 is the first anti-GUCY2C BiTE to enter clinical testing. A total of 79 patients were recruited in a phase I trial, and the study was completed in November 2023, but the results from this trial are still pending [ 259 ].

GUCY2C is an attractive target for cancer vaccines and CAR-T therapy as well. A phase I clinical trial in 10 early-stage CRC patients demonstrated the safety and immunogenicity of an adenovirus-based GUCY2C cancer vaccine [ 260 ]. The safety and efficacy of a 2nd-generation GUCY2C cancer vaccine is currently being evaluated in a phase II clinical trial ( NCT04111172 ). GUCY2C has also been demonstrated as a safe and effective CAR-T target in preclinical models [ 23 , 244 ], and a phase I dose-escalation study of GUCY2C-directed CAR-T therapy has demonstrated an acceptable safety profile and 15.4% (2/13) overall response rate for the low dose level and 50% (4/8) for the high dose level investigated in 21 metastatic CRC patients [ 261 ].

HER2

HER2 is a receptor tyrosine kinase in the same family as EGFR [ 183 , 186 , 262 ]. It dimerizes and autophosphorylates upon ligand binding, and it shares many downstream tumorigenic signaling pathways with EGFR [ 25 ]. HER2 is overexpressed in approximately 2% of total and 5% of KRAS-wild type metastatic CRCs [ 262 ]. The HER2-directed therapeutic mAb, Herceptin ® (trastuzumab), is FDA approved for breast and gastric cancers. HERACLES and MyPathway studies established the use of HER2-directed mAb or inhibitor for CRC in single-arm phase II in the late 2010s [ 26 , 27 , 263 ]. In 2023, the Mountaineer study demonstrated clinically meaningful antitumor efficacy and safety of tucatinib, a tyrosine kinase inhibitor that blocks phosphorylation of HER2, plus trastuzumab for HER2-positive, RAS wild-type, unresectable or metastatic CRC [ 264 ]. This study facilitated accelerated FDA approval of this HER2-directed therapy for CRCs. In 2024, promising efficacy and safety results from DESTINY-PanTumor02 and -CRC studies facilitated FDA approval for tissue-agnostic use of trastuzumab deruxtican for HER2 over-expressing/amplified solid tumors, including CRC [ 265 , 266 ].

HER2 is one of the first CAR-T therapy targets to be explored for metastatic CRC. This is in large part due to Herceptin’s efficacy and safety profile in other solid malignancies. However, the first clinical trial testing a HER2-directed CAR-T therapy employing the Herceptin-derived scFv was terminated after the death of a CRC patient due to respiratory distress induced by the infusion of CAR-T cells [ 267 ]. In the past decade, attenuating the toxicity of HER2-directed CAR-T therapy has been an active area of research. The FRP5 scFv fragment, which has a lower affinity compared to the Herceptin-derived scFv, combined with only the CD28 co-stimulatory domain, has been shown to be safe in two clinical trials for glioblastoma and sarcoma [ 268 , 269 ]. While these safety data are encouraging, significantly more work is needed to establish HER2-directed CAR-T therapy for CRC.

Carcinoembryonic antigen (CEA)

CEA is a glycoprotein belonging to the family of carcinoembryonic antigen cell adhesion molecules (CEACAM) family. CEA was thought to be an oncofetal target expressed during embryonic development and overexpressed in cancers, including CRC, but it is now known to be expressed in the adult intestine as well [ 270 , 271 ]. In 2017, a clinical trial of CEA-directed CAR-T therapy for metastatic CRC patients in China demonstrated tumor reduction in 2/10 patients and tumor control in 7/10 patients, with none experiencing severe adverse events [ 28 ]. In 2024, another group in China demonstrated that with 24 CRC patients enrolled for intravenous (i.v.) administration and 16 for intraperitoneal (i.p.) administration of hypoxia-responsive CEA CAR-T therapy, the disease control rate was 88% in the i.p. group and 67% in the i.v. group [ 30 ]. While TCR-T therapy directed to CEA was unacceptably toxic [ 164 ], CEA-directed CAR-T therapy may be safe and effective for CRC patients.

NKG2D ligands

NKG2D is a type C lectin-like receptor that is expressed by NK cells, CD8 + and CD4 + αβ T cells, and γδ T cells [ 31 , 32 ]. There are two primary types of ligands that activate NKG2D in humans: the MHC-related proteins MICA and MICB and the ULBP/RAET family of proteins. NKG2D ligands have a very low basal expression in normal tissue, and they are upregulated upon infection, DNA damage, cell transformation, and other forms of ‘stress’ [ 32 ]. Interestingly, NKG2D ligands are drastically overexpressed in many solid tumor malignancies, including CRC.

Unlike most other CAR-T therapies in which the extracellular domain relies on scFvs derived from monoclonal antibodies to recognize antigens, NKG2D CAR-T cells are engineered to express NKG2D or NKG2D ectodomain fused to conventional CAR signaling domains to recognize NKG2D ligands on CRCs. A phase I clinical study of NKG2D CAR-T therapy for metastatic CRC concluded in 2019 and demonstrated no severe adverse events [ 31 ]. However, no efficacy data has been disclosed thus far. In addition, multiple ongoing clinical studies are exploring NKG2D CAR-T therapy in combination with ICB (NC T04991948), as well as NKG2D CAR-NKtherapy ( NCT05213195 ).

Conclusion

Immunological therapeutics represent promising modalities for precision medicine for CRC. Therapeutic mAbs with anti-tumor efficacy were studied in the 1990s, and several have shown considerable success in improving patient outcomes, achieving FDA approval for CRC. More recently, FDA-approved ICBs have further improved CRC patient outcomes, though this is limited to only a small subset of CRC patients whose tumors have high neoantigen densities (MSI or dMMR). Although no cancer vaccines or adoptive cell therapies have obtained FDA approval yet, both modalities continue to attract attention for preclinical and clinical research. Cancer vaccines and adoptive cell therapies are highly personalized approaches that reprogram or engineer patients’ autologous T cells or other immune components to identify and destroy CRC cells. With a better basic scientific understanding and continuing preclinical development and clinical investigation of these highly complex and delicate immunotherapy modalities, there is great potential to significantly improve survival and quality of life for CRC patients.

Expert opinion

CRC continues to be a substantial burden on public health. Regular colonoscopy screening in the adult population enables the identification of pre-cancerous lesions and early-stage cancers, which are often cured with surgery. However, metastatic CRC is challenging to manage, as demonstrated by the substantial difference in 5-year survival in patients with localized disease (90%) compared to those with distant metastasis (14%). Current therapies available for metastatic CRC patients include surgery, systemic cytotoxic chemotherapies, EGFR and VEGF blocking antibodies, and ICB. The latter three targeted therapies allow for more precise targeting of CRC cells. However, anti-EGFR and anti-VEGF therapeutics are restricted to patients with the target expression, and resistance often develops through 1) target downregulation, 2) downstream signaling pathway mutations that allow ligand-independent activation, and 3) alternative compensatory signaling pathway amplification. ICB benefits an even more selective MSI CRC patient population, and only tumors with checkpoint upregulation and high neoantigen burden are likely responsive to ICB.

Immunotherapies, especially the FDA approved CD19 and BCMA targeted CAR-T therapies, have revolutionized the standard care and outcomes for patients with liquid malignancies. Although no cancer vaccines or adoptive cell therapies have shown sufficient safety and efficacy for CRC, there are ample reasons to be optimistic about the future of these immunological approaches. First, the FDA has approved three cancer vaccine or adoptive cell therapies for solid malignancies: the therapeutic cancer vaccine Sipuleucel-T for castration-resistant prostate cancer in 2010; the TCR-T therapy afamitresgene autoleucel for metastatic synovial sarcoma in 2024; and the TIL therapy lifileucel for metastatic melanoma in 2024. These approvals, especially the two in 2024, are encouraging with respect to the development of CRC immunotherapies. Second, for cancer vaccines, rapid advances in genomic manipulation allow for the production of more personalized therapeutics that target a combination of tumor antigens. These advances are also allowing us to develop a more refined understanding of the immunosuppressive tumor microenvironment. This enables us to better select target antigens and adjuvants that will, in turn, improve therapeutic efficacy. Third, for adoptive cell therapies, a better understanding of barriers to their efficacy in solid malignancies, such as T-cell trafficking, T-cell persistence, and the immune tumor microenvironment, will allow for strategies to overcome those barriers and improve immunotherapy efficacy.

Over the next 5 to 10 years, we expect that an improving understanding of cancer vaccines and adoptive cell therapies will allow for innovative approaches to overcome their challenges. The current known challenges on the tumor cell side remain largely unchanged: target identification, antigen escape, and the immunosuppressive tumor microenvironment. In preclinical models and clinical trials, there are already innovative approaches being proposed to overcome these barriers. For example, CAR-T cell engineering to express cytokines to help with persistence and prevent exhaustion, novel adjuvants to improve immunogenicity of cancer vaccines, and new antigens currently under investigation. With the very first TILs being FDA-approved for melanoma in 2024, it can be reasonably speculated that TILs for CRCs with high mutation burdens may soon be used for CRC. Moreover, combination therapies of adoptive cell therapies or cancer vaccines with ICB may also be promising approaches to improve outcomes in CRC.

Funding

This manuscript was funded by grants from the Department of Defense (W81XWH-19-1-0263, W81XWH-22-1-0207, W81XWH-17-PRCRP-TTSA, W81XWH-22-1-0208), the National Institutes of Health (1R21 CA267087, 1R21 CA286339, 1R01 CA204881, 1R01 CA206026, 1R21 1NS130388, 1R01 DK1388341), the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation, American Parkinson Disease Association, Parkinson’s Foundation Impact Award PF-IMP-1045175, TDT, Inc, and the Courtney Ann Diacont Memorial Foundation and Lorraine and David Swoyer. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Declarations of interest

S.A.W. is a member of the Board of Directors for Targeted Diagnostics & Therapeutics (TDT), Inc., which provided research funding that, in part, supported this work and has a license to commercialize inventions related to this work. A.E.S. reports personal fees from TDT, Inc., and Vittoria Biotherapeutics, Inc., outside the submitted work; pending patents related to the submitted work; and CAR-T-related patents licensed to TDT. B.B. reports consulting or advisory role for: Merck/Eisai, Fate/Ono Therapeutics & KAHR Medical and serves as scientific advisory board for Tempus AI, Inc., and reports research funding to the institution from Amgen, Artios Pharma, Avenzo Therapeutics, Boehringer Ingelheim, Bicycle Therapeutics, Elucida Oncology, Fate Therapeutics, Genentech, Gritstone Bio, Ikena Oncology, Jazz Pharmaceuticals, KAHR Medical, Lyell Immunopharma, Merck, Pionyr Immunopharma, RAScal Therapeutics, Syros Pharmaceuticals, Tarveda Therapeutics. M.C., M.X., and Z.B. have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Ethics statement

This manuscript is based on previously published studies, and the authors did not conduct any new studies involving human participants or animals. Therefore, ethical approval was waived.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

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Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

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