**6. Conclusions and Perspectives**

The development of anti-cancer drugs has focused on strategies that kill cancer cells directly, such as surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Immunotherapy is based on the recognition of tumour cells as foreign by the immune system. For good results, immunotherapy has to activate and expand tumour-specific T cells. Various approaches have been used for this purpose: direct activation of anti-tumour immunity by means of cancer vaccines (tumour antigens), recombinant cytokines or the infusion of tumour-specific cells. However, these methods did not guarantee that the tumour-specific T cells could nest or perform their function within the tumour. This is due to the existence of tumour-induced immunosuppressive mechanisms in the tumour microenvironment, which prevent the breakdown of immune tolerance to cancer. With the approval of the checkpoint inhibitors, it was demonstrated that an anti-tumour immune response can be initiated by targeting the immune system to break the tolerance to cancer. In contrast, a significant clinical response was only obtained in a few patients with solid tumours such as melanoma, non-small cell lung cancer, kidney or bladder cancer. This is because the response to this treatment depends on the presence of pre-existing tumour-specific CD8 T cells, which correlates with the presence of neoantigens derived from tumour mutations. Because of this relationship, different approaches have

been tested that allow for the expansion of anti-tumour T cells along with checkpoint inhibition. These combination therapies have been successful in clinical trials, but the current focus is on targeted therapies to target neoantigens derived from tumour mutations, as the number of mutations in each tumour correlates directly with the efficacy of checkpoint inhibitors. Therefore, it is necessary to establish combined therapies in cancer, among which small molecule inhibitors also stand out. These combination approaches are key to understanding the relationship between the established tumour and the immune system [381]. Combination therapies are useful in inhibiting tumour growth and changing or restoring the TME. Among the most explored are combinations of checkpoint inhibitor (anti-PD1) and targeted (antibody or small molecule) therapies. Rapid lysis of tumour cells with targeted therapies can generate an environment of acute inflammation that enhances tumour immunity, making these therapies additive [73].

On the other hand, programmed physiological cell death, usually in the form of apoptosis, has always been considered a non-immunogenic or even tolerable process. In contrast, the concept of "programmed cell death" (PCD) has assigned immunogenic capabilities to apoptosis. This type of apoptosis is characterised by the ability of dying cells to trigger adaptive immune responses against the altered autoantigens/neo-epitopes derived from cancer, in the case of tumour cells. In addition to antigenicity, adjuvancy, conferred by DAMPs, is necessary.

The pathways that induce ICD can be used to design new therapeutic tools in immunotherapy, to reduce the tumour burden and improve the immunogenic capacity of dying tumour cells, provoking adaptive immune responses in the long term. Various immune-based therapies can benefit from ICD, such as antibody-based therapies, adoptive cell therapy (TIL, NK cell or CAR-T), checkpoint inhibitors, tumour vaccines and combination immunotherapy strategies [256]. One of the most promising strategies is to exploit the ICD concept to obtain highly immunogenic antigen sources for the development of "next generation" DC-based vaccines [382]. ICD inducers can be used to generate immunogenicity in dying tumour cells and to load DC, enhancing their ability to stimulate effector cells and improve T-cell responses to cancer in vivo [99]. This may improve general immunity or create an immune-friendly tumour microenvironment [256]. A number of chemotherapeutic agents are ICD inducers, meaning that many therapeutic strategies have known immunomodulatory or immune-stimulatory effects that should be further investigated to determine if they are associated with the release of DAMPs. The characterisation of new DAMPs may open up new therapeutic targets for targeted chemotherapy [383]. Understanding the molecular pathways involved in these processes would allow for the identification of a new set of potential prognostic biomarkers, but more research is needed to understand the true impact of ICD therapy and exposure to DAMPs [256].

The immune system of the cancer patient detects abnormalities in structure, function, intracellular location, and other cellular alterations during tumorigenesis, which may manifest themselves in humoral or cellular immune responses, which may be the earliest sign of carcinogenesis. The current aim is to use cancer autoantibodies as diagnostic biomarkers, but there is also the possibility that they may be used as monitors of the therapeutic response. If an anti-TAA antibody is detected in the patient, changes in the levels of these antibodies may reflect the status of the tumour, its changes or its tumour load in relation to therapy [367].

Cancer immunotherapy relies heavily on the use of peptide antigens derived from amino acid sequences of tumour antigens and modulates the response of T cells. The problem here is that the peptides selected must be strongly immunogenic and induce a T cell response. Therefore, it would be important to identify the regions of the TAAs that can be recognised by the patient's immune system, which would confirm that they are real targets in vivo and allow for the design of immunotherapies directed to these auto-epitopes. Such auto-epitopes must be able to be identified and isolated from MHC class I molecules [367].

In this sense, T lymphocytes αβ detect alterations in the host's cellular components, which may be induced by infectious pathogens, chemical or physical damage or oncogenic transformation. The T-lymphocytes generated in the thymus each have a clonally restricted T cell receptor (TCR) [384]. Human tumours contain a high number of somatic mutations, and if peptides containing these mutations occur on MHC class I molecules they may be immunogenic and recognised by the adaptive immune system, which recognizes them as "non-self" neoantigens. Mutant peptides can serve as T cell epitopes [385]. During immune surveillance, each T cell receptor recognizes a different foreign peptide attached to MHC molecules. MS technology allows for the identification of epitopes relevant to different tumours. Molecular CCR cloning methods allow for the molecular quantification of TCR–pMHC interactions [384,386]. This is a major challenge, since MHC-bound peptidoma consists of thousands of different peptides with relevant non-self-antigens often embedded in low numbers, among them the self-peptides, which occur in a greater order of magnitude [384].

In immunogenic tumours, the sequencing of the complete exome and transcriptome of individual tumours, together with mass spectrometry, allows for the identification of mutant peptides to develop vaccines on an individual basis for each patient [385]. In non-immunogenic tumours, the induction of the expression of multiple neoepitopes can direct a polyclonal CTL attack against a cancer. One goal of therapeutic antitumour vaccines is the targeting of CTLs on MHC-bound peptides restricted to cancer cells, increasing the CTLs of high avidity at the site of the tumour [386].

In contrast, few mutant epitopes have been described, as it requires the exploration of the patient's tumour infiltrating lymphocytes based on their ability to recognize antigen libraries created after sequencing of the tumour exome. This requires the use of mass spectrometry combined with transcriptomical or exome sequence analysis to identify neo-epitopes [385].

In short, a large part of the antigens that drive the effectual responses of antitumour CD8 T cells remains unknown. These antigens can be classified into tumour-associated autoantigens and antigens derived from tumour-specific mutant proteins. The presentation of autoantigens in the thymus may result in the elimination of highly avid T cells, thus mutant neo-antigens will be more immunogenic. In contrast, these neoantigens evade identification by mass spectrometry because this method relies on sequence clarification with proteomic databases that do not contain patient-specific mutations. By using transcriptomics and exome sequence analysis to identify mutations, together with the use of MHC class I binding prediction algorithms, too many candidate mutant peptides are detected to be evaluated. Mass spectrometry would allow for the selection of peptides with sufficient expression and presentation by MHC class I, which would be the most immunogenic. By combining both tools, it is possible to identify mutated peptides associated with tumours that present in MHC class I [385].

The immunogenicity of neoepitopes is correlated with the affinity for peptide binding by MHC class I, but other factors such as the interaction of the mutated amino acid with the TCR also play a role, as this is essential for the recognition of the mutated peptide as a stranger [387].

The analysis of MHC peptidoma allows for the identification of peptides derived from the proteolysis of proteins that are generally short-lived in tumour tissues and therefore cannot be identified by conventional proteomic methodologies. One of the current challenges is to find differences between MHC peptidomas from healthy patients and cancer patients, since MHC peptidomas have large amounts of different peptides. Most of these peptides will be from the cellular proteome but also small amounts of cancer-related peptides will be present, which will be different even among different patients presenting the disease. Ultimately, such analysis would allow for the identification of thousands of peptides, including some potential biomarkers of disease. Furthermore, these cancer-related MHC peptides could be used to design patient-specific immunotherapeutics. In other words, the final goal would be to use the MHC peptidoma data to personalize treatments. Mass spectrometry analysis would be a good tool for this purpose, as it is becoming less expensive and faster and can be used in clinical diagnosis on a routine basis [358].

In conclusion, the identification of epitopes that induce the immune response in cancer is necessary to understand and manipulate the immune responses of CD8 T cells for clinical benefit. Tumour-specific mutations are important in shaping the antitumour response, but their identification remains a challenge. The identification of neo-epitopes by combining whole exome sequencing, transcriptome and mass spectrometry analysis strategies, together with a structural prediction algorithm to predict peptide

immunogenicity in MHC class I would facilitate the monitoring of tumour-specific T cells, which would be useful in the prognosis of cancer patients, as well as the development of new vaccines [385].

Cancer immunotherapy is undergoing a major transition from traditional approaches that activate systemic immune responses based on understanding the processes of immune activation to more effective and less toxic treatments that target immune normalization in the tumour microenvironment based on tumour-induced immune escape mechanisms [73].

NPs play an important role in these improvements in immunotherapeutic treatments. Nanomaterials applied to nanomedicine would make it possible to increase the effectiveness and reduce the toxicity of practically all the immunotherapeutics described. There are very varied nanoparticle designs that can serve as immunotherapy delivery platforms, allowing for specific and targeted delivery. In addition, NPs can also be designed to enhance the immune response of the host. Due to their great potential, research into these nanomaterials in combination with drugs is necessary to ensure their biosafety and determine their specific functions and applications based on their biocompatibility. Improving these approaches would make it possible to overcome some of the drawbacks of immunotherapy and initiate a new path in cancer treatment.

**Author Contributions:** V.A.-F. and M.F. designed manuscript outline. V.A.-F. designs and prepares figures and tables. All the authors (V.A.-F., A.L.-V., P.J.-V., A.-P.H., A.O.-P., R.M.-R., R.G., M.F.) participate in writing, discussing and prepare manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI17/01930 and CB16/12/00400. Fundación Solórzano FS/38-2017. The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023, of the PE I + D + I 2017-2020, funded by ISCIII and FEDER. A. Landeira-Viñuela is supported by VII Centenario-USAL PhD Program, P. Juanes-Velasco is supported by IBSAL PhD Program, V-Acebes-Fernández is supported by "Retención Talentos" Programm sponsorized by Ayuntamiento Salamanca (Salamanca, Spain).

**Acknowledgments:** We thanks GRG. for her help in language edition.

**Conflicts of Interest:** The authors declare no conflict of interest.
