*3.4. Limitations of Immunotherapy*

The previously described immunotherapy strategies (Figure 11) have some limitations and face different challenges.

**Figure 11.** Summary of the different immunotherapies described above. (1) Cytokines, (2) monoclonal antibodies (mAb), (3) CAR-T cells, (4) onco-vaccines and (5) ICIs [173].

Although cytokines were the first approach for immunotherapy introduced in the clinic, they also have some drawbacks. Cytokine treatments consist of high-dose injections, as their half-life is short, resulting in vascular leakage and cytokine release syndrome. In addition, cytokines can promote the survival of regulatory T cells and induce death in stimulated T cells, resulting in autoimmunity against healthy tissues [174].

As for agonist antibodies, they have dose-limiting toxicities, as do cytokines, since they can induce activity on unwanted immune cell subtypes, and immune activity towards healthy cells. In addition, some of these antibodies induce regulatory activity on T cells [175]. Therefore, it is necessary to evaluate dose-associated toxicities and develop delivery platforms. One example is anti-4-1BB antibodies, which—when anchored to liposomal NPs—have a higher intra-tumoral accumulation and lower toxicity than antibodies released freely in mouse models [176].

In the case of CAR-T cells, unlike other treatments, they are unique therapies and the cells can maintain their activity for several years after injection. Despite this, the long-term effects of therapy with CAR-T cells are still being investigated [177]. Other disadvantages of this therapy are that the production of CAR-T cells is expensive, technically complex and time-consuming. In certain tumours, especially solid tumours, depending on their microenvironment, the infused cells do not persist and need combination therapies and new drug delivery systems to improve T-cell survival [175].

CAR-T cells and TCR cells can cause cytokine release syndrome and neurotoxicity [177]. Another problem is making these modified cells effective in solid tumours. One of the solid tumours that has been successfully treated with CAR-T cells is glioblastoma [58], but it expresses the target antigen (EGFRvIII) at much higher levels in the tumour cells than normal cells, which is unusual. As for T cells with high affinity TCR, their toxicity is difficult to predict [178].

In the case of vaccines, those based on DCs have demonstrated high safety profiles, while in clinical trials they have shown a lack of efficacy [179]. The efficacy could be improved by identifying subsets of dendritic cells expressing high levels of specific antigens and by improving the supply to the lymph nodes [180]. As for DNA- or RNA-based vaccines, the former have been tested in clinical trials but are often not successful due to nuclear supply barriers and immunogenicity [181]. mRNA vaccines also have some drawbacks, such as the fact that mRNA can be degraded by nucleases and not internalized into cells. The use of delivery pathways to mediate intracellular internalization may be a good option [182]. Neoantigen vaccines cover an unlimited number of neoantigens, but delivery platforms can improve their efficacy by increasing the stability of the encapsulated molecules and by housing several neoantigens within one platform to treat heterogeneous cancers [175].

For ICIs administered by the systemic route, they can have serious side effects in several organs [183,184]. In addition, many patients do not respond to this treatment, which may be due to a low number of tumour-infiltrating T cells, dysregulation of the checkpoint axes or adapted resistance to checkpoint inhibition [185]. Different tumour microenvironments also have different mechanisms of immunosuppression that require new approaches for effective treatment.

The TME, in the case of solid tumours, is a challenge in the implementation of the above-mentioned immunotherapies. The TME of these tumours can be classified as immunologically "hot" (high immunogenicity) or "cold" (low immunogenicity), with high or low levels of cytotoxic lymphocyte infiltration, respectively. "Hot" tumours have better responses to ICIs than "cold" tumours; then, delivery technologies might be exploited to modulate immunogenicity for "cold" tumours [186].

Another drawback of immunotherapies is related to the systemic toxicity, which can be reduced by delivery platforms by limiting drug exposure in specific tissues, thus allowing for the delivery of otherwise highly toxic combination therapies [187]. The study by Wantong Song et al. shows that NPs allow for the administration of combination immunotherapy treatments, making "cold" tumours susceptible to immunotherapy [188]. Nanomedicines can be designed to respond to the tumour microenvironment and increase site penetration in both "hot" and "cold" solid tumours, overcoming the limitations of immunotherapy [189].

Immunotherapies that require intracellular administration, such as genetic vaccines, must overcome extra- and intracellular barriers with minimal systemic toxicity [190]. Administration and delivery technologies, such as NPs, would allow for the therapeutic burden of such immunotherapies to be encapsulated and protected until they can be released into the cytosol of the target cells [191,192].

### **4. Nanomedicine and Immunotherapy: Synergy Combination**

In order to improve the effectiveness and minimize the toxicity associated with cancer immunotherapy, new strategies have been attempted, including the use of nanomaterials to increase host immunity. Nanomedicine can play a role in improving both active and passive immunotherapy, depending on the functions for which the different NPs have been designed and the processes in which they participate (Figure 12). These NPs can be designed as delivery platforms for immunotherapy, i.e., as delivery vehicles which allow for more efficient and specific transport of immunostimulatory agents, which we will call passive nanomedicine; or they can be designed with nanomaterials which have intrinsic immunomodulatory properties that help to increase anti-tumour immune responses by selectively regulating signalling pathways in different immune cell populations, called active nanomedicine.
