**4. Strategies beyond ICI**

### **4.1 Adoptive cell transfer (ACT)**

Another revolutionary treatment option aiming to augment the host's immune system is represented by ATCs. The approach consists of transferring the patient's immune cells, which were previously genetically engineered, and expanded to destroy cancer cells. ACT can include tumor-infiltrating lymphocytes (TILs), natural killer (NK), chimeric antigen receptor T-cell therapy (CAR-T), or engineered T cell receptors (TCR).

ACTs have achieved impressive success in several tumor types in the last two decades, especially in hematologic malignancies, like B cell lymphoma and leukemia [43].

ACT usage for cancer treatment originates from the observation of TILs, representing a population of lymphocytes infiltrating the tumor or located at its' margins. TILs represent the host's natural antitumor immune response and can recognize tumor-specific antigens presented by MHC 1 [44].

In 2016, a group of researchers identified in the TILs from CRC metastatic lesions a polyclonal population of CD8+ cells directed against KRAS G12D. After expansion, this TILs population was further reinfused into the patient and eradicated six out of seven lung metastases. So far, harvesting TILs from colorectal tumors has faced many difficulties [45]. One of the concerning issues is the contamination with intestinal flora, which can be overcome by acquiring tumor-specific T cells from tumor-draining lymph nodes [46]. Another ideal source for aseptically harvesting TILs in CRC might be liver metastasis. However, further research is needed to overcome all the impediments to the usage of TILs in CRC and other solid tumors.

CAR-T cell therapies have been extensively studied in hematologic malignancies, with less evidence in solid tumors at the moment. This kind of personalized medicine combines genetic therapy with immunotherapy. It involves T cells harvesting from the patient, which are genetically modified to express a chimeric antigen receptor (CAR) that can recognize a tumor-associated antigen (TAA) [47, 48]. The clinical trials investigating CAR-T cells in CRC treatment targeted various TAAs, including carcinoembryonic antigen (CEA), mesothelin (MSLN), EGFR, HER2, and natural killer group 2 member D (NKG2D) [49]. A phase I clinical trial investigated CAR-T cell therapy targeting CEA in previously treated CEA-positive mCRC patients. Out of the 10 patients included in the study, seven experienced stable disease for longer than 30 months. Moreover, the study reported a sustained decline in CEA serum levels [50]. Apical surfaces of the intestinal epithelium express the membranebound receptor guanylyl cyclase C (GUCY2C). Magee et al*.* tested the efficacy of a GUCY2C-specific CAR-T cell molecule in an mCRC mice model. The result showed that GUCY2C CAR-T cells reduced the number of lung metastasis in mice, lowering morbidity and improving survival [51].

Although ACTs have shown therapeutic potential in many cancer types, there are still many obstacles to their effectiveness in solid tumors, including CRC.

### **4.2 Cancer vaccines**

Cancer vaccines are a form of active immunotherapy thought to enhance the antitumor immune response by evoking TAA in order to be targeted by the immune system. In mCRC, several vaccine types have been studied, including peptides, dendritic cells, autologous tumor cells, and recombinant viral vectors [52]. The vaccine must supply enough tumor antigens to induce a robust immune response and, therefore, to obtain a substantial clinical benefit [53]. Unfortunately, these requests are challenging to be acquired; thus, the clinical trials investigating cancer vaccines reported mixed results.

A benefit of peptide-based vaccines is that they are affordable in terms of production and storage. A recently developed peptide vaccine, PolyPEPI1018 consisting of

12 epitopes derived from seven antigens frequently expressed in mCRC, demonstrated increased CD8+ T cell and CD4+ T cell responses against three antigens after only one dose [54].

Since plasmid DNA encoding influenza nucleoprotein A was discovered to trigger a specific T cell response, DNA vaccines have received much attention. These types of vaccines consist of bacterial plasmids created to provide tumor antigens that will be further presented via MHC proteins and stimulate an immune response [55]. MYB is an oncoprotein abnormally expressed in many tumor types, including CRC. In CRC transgenic mice, MYB-based vaccines showed good therapeutic efficacy. However, several corners about DNA vaccines include poor immunogenicity and potential interactions with the host's genome [56].

RNA-based vaccines, another widely investigated therapeutic and prophylactic form of immunotherapy, consist of a platform that encodes tumor-specific antigens. After the internalization of mRNA transcript by the target cell, the translation takes place in the cytoplasm and is followed by tumor antigen presentation via MHC proteins, triggering a robust immune response. mRNA vaccines offer several benefits, making them appealing therapeutic options. They are nonintegrating molecules, affordable, relatively fast to produce, and easy to modify [57]. A phase I/II trial is currently investigating an mRNA-based vaccine (mRNA 4650) for treating various tumor types, including gastrointestinal, melanoma, genitourinary, and CRC [58]. There are only two anticancer vaccines approved in oncological practice: Provenge (sipuleucel-T) for prostate cancer treatment and Oncophage for kidney cancer [59, 60]. At the moment, cancer vaccines are extensively studied in clinical trials and will hopefully improve treatment strategies for CRC.
