**5. Intratumoral delivery of repurposed vaccines**

Success with T-vec and other immune-boosting viruses have prompted various groups to search among routinely available attenuated viral vaccines to find other therapeutic options. The advantage of repurposing such approved and marketed agents is that clinical development would be much simplified, based on wellestablished safety records [58].

Commercially available attenuated rotavirus vaccines are preparations of double-stranded RNA attenuated strains. They are very potent stimulators of the nuclear factor kappa-light-chain-enhancer of activated B cells and type I interferon pathways. Interestingly, this stimulation is independent from the innate Toll-like immune receptors but dependent on RIG-I, which is able to detect intracytoplasmic dsRNA. Furthermore, rotavirus exerts cytocidal effects on adult and pediatric cancer cell lines in culture with features of immunogenic cell death. Intratumoral delivery to mouse bearing transplantable tumors, including pediatric syngeneic neuroblastoma models, elicited clear therapeutic effects mediated by natural killer (NK) cells and CD4 and CD8 T cells. In models of tumors refractory to checkpoint inhibitors, intratumoral rotavirus enabled to overcome resistance. Prevaccination of mice prior such intratumoral virotherapy did not spoil its efficacy [59].

A vaccine based on the 17D strain of the yellow fever virus, commonly used for travelers and dwellers in endemic areas, was demonstrated cytocidal for a large panel of human and mouse tumor cell lines. Its intratumoral administration was able to delay tumor progression by activating CD8 T cell-mediated immunity and some measurable effect could be observed against non-injected tumor lesions [60]. Additive effects with systemic immunostimulatory monoclonal antibodies directed to anti-PD1 or anti-CD137 were demonstrated. Very importantly, efficacy was potentiated by previous vaccination against the same virus in a manner dependent on T-cell antiviral acquired immunity [61].

Intratumoral injections of anti-influenza vaccines were also demonstrated to elicit immune-mediated antitumor activity in melanoma, in a series of experiments with

**167**

**Figure 4.**

**Figure 4**.

*Repurposing Infectious Pathogen Vaccines in Cancer Immunotherapy*

syngeneic transplantable tumor model [62]. Most surprisingly, only unadjuvanted inactivated influenza vaccines were able to generate such antitumor efficacy. Indeed, squalene-based adjuvanted influenza vaccines were losing their antitumor activity because adjuvants were recruiting interleukin-10-secreting B regulatory cells [62]. The detrimental role of adjuvants was observed in another seminal study when analyzing the cause of a lack of therapeutic enhancement of anti–CTLA-4 monotherapy by concurrent vaccination with gp100 peptide in incomplete Freund's adjuvant (IFA) [63]. Genetically engineered poliovirus vaccine antitumor activity was studied in mice a few years ago [64]. It has later been moved to a phase I clinical trial for recurrent glioblastoma with interesting results [65]. In this study, patients were pre-immunized with the vaccine against poliomyelitis and then treated intratumorally with the genetically engineered virus. The role of previously developed immunity was important for successful activation of immunity against tumors treated

In an older phase I-II trial, a recombinant nontoxic diphtheria protein

(CRM197), used in many common vaccines, was used to treat a variety of accessible tumors by local delivery. Response was observed in patients that had an already developed immunization (measured both by IgG titer and delayed type hypersensi-

Since immunosuppression mechanisms are in place in the tumor microenvironment [67], from these examples it is clear that an effective immunity developed outside tumors could enable a better response when antigens are later delivered intratumorally. The fact that developing immunity outside the tumor microenvironment is a valuable strategy has been also demonstrated in the case of a new neoantigen vaccine formulation. In fact, the biomaterial-based vaccine prevented the engraftment of AML cells when administered as a prophylactic and when combined with chemotherapy, and eradicated, established AML even in the absence

As a last example, a recent Report in JAMA Dermatology suggested that Gardasil®9 might be employed for cancer treatment. Cutaneous basaloid squamous cell carcinoma (BSCC) was eradicated by intratumoral administration of the vaccine. Preventive systemic immunization was performed by a standard initial dose and a booster one, followed by intratumoral delivery of the same vaccine into just a few of the largest lesions, injected monthly over the next months. During this relatively long period, even tumors that had not been injected went into complete regression. Notably, no recurrence was observed in the follow-up period (18 months). This report first presents clinical evidence that a prophylactic antiviral

All mentioned studies point out to the value of a therapeutic strategy outlined in

vaccine may be used as an effective immunotherapy for cancer [69].

*Schematic diagram of the neoadjuvant intratumoral delivery of repurposed vaccines.*

*DOI: http://dx.doi.org/10.5772/intechopen.92780*

locally [65].

tivity) against diphtheria [66].

of a defined vaccine antigen [67, 68].

#### *Repurposing Infectious Pathogen Vaccines in Cancer Immunotherapy DOI: http://dx.doi.org/10.5772/intechopen.92780*

*Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

phase I against melanoma (NCT04197882).

established safety records [58].

spoil its efficacy [59].

on T-cell antiviral acquired immunity [61].

**5. Intratumoral delivery of repurposed vaccines**

TLR9 agonist (CMP-001) in combination with Anti-PD-1 (Nivolumab) is in phase II against melanoma and lymph node cancer (NCT03618641); and TLR8 agonist (VTX-2337) in combination with Anti-PD-1 (Tislelizumab) is in phase I against head and neck cancer (NCT03906526). JX-594 (Oncolytic virus) is in phase II against colorectal carcinoma (NCT01329809); and T-VEC (Oncolytic virus) is in phase II against melanoma (NCT02211131), in combination with Anti-PD-L1 (Atezolizumab) in phase I against breast cancer (NCT03802604), in combination with chemotherapy in phase I/II against breast cancer (NCT02779855), in combination with Anti-PD-1 (Pembrolizumab) in phase II against melanoma (NCT03842943), in combination with BRAF Inhibitor and MEK Inhibitor in phase II against melanoma (NCT03972046), in combination with radiotherapy in phase I/ II against soft tissue sarcoma (NCT02453191), in combination with chemotherapy, radiotherapy, in phase I against rectal cancer (NCT03300544). Rilimogene galvacirepvec (PROSTVAC) in combination with Anti-PD-L1 (Atezolizumab) is in phase II against prostate adenocarcinoma (NCT04020094); GMCI (Adenovirus) in combination with radiotherapy, chemotherapy, is in phase II against pancreatic adenocarcinoma (NCT02446093); and HF10 (Oncolytic virus) in combination with Anti-PD-1 (Nivolumab) is in phase II against melanoma (NCT03259425). OrienX010 (Oncolytic virus) in combination with Anti-PD-1 (Treprizumab) is in

Success with T-vec and other immune-boosting viruses have prompted various groups to search among routinely available attenuated viral vaccines to find other therapeutic options. The advantage of repurposing such approved and marketed agents is that clinical development would be much simplified, based on well-

Commercially available attenuated rotavirus vaccines are preparations of double-stranded RNA attenuated strains. They are very potent stimulators of the nuclear factor kappa-light-chain-enhancer of activated B cells and type I interferon pathways. Interestingly, this stimulation is independent from the innate Toll-like immune receptors but dependent on RIG-I, which is able to detect intracytoplasmic dsRNA. Furthermore, rotavirus exerts cytocidal effects on adult and pediatric cancer cell lines in culture with features of immunogenic cell death. Intratumoral delivery to mouse bearing transplantable tumors, including pediatric syngeneic neuroblastoma models, elicited clear therapeutic effects mediated by natural killer (NK) cells and CD4 and CD8 T cells. In models of tumors refractory to checkpoint inhibitors, intratumoral rotavirus enabled to overcome resistance. Prevaccination of mice prior such intratumoral virotherapy did not

A vaccine based on the 17D strain of the yellow fever virus, commonly used for travelers and dwellers in endemic areas, was demonstrated cytocidal for a large panel of human and mouse tumor cell lines. Its intratumoral administration was able to delay tumor progression by activating CD8 T cell-mediated immunity and some measurable effect could be observed against non-injected tumor lesions [60]. Additive effects with systemic immunostimulatory monoclonal antibodies directed to anti-PD1 or anti-CD137 were demonstrated. Very importantly, efficacy was potentiated by previous vaccination against the same virus in a manner dependent

Intratumoral injections of anti-influenza vaccines were also demonstrated to elicit immune-mediated antitumor activity in melanoma, in a series of experiments with

**166**

syngeneic transplantable tumor model [62]. Most surprisingly, only unadjuvanted inactivated influenza vaccines were able to generate such antitumor efficacy. Indeed, squalene-based adjuvanted influenza vaccines were losing their antitumor activity because adjuvants were recruiting interleukin-10-secreting B regulatory cells [62]. The detrimental role of adjuvants was observed in another seminal study when analyzing the cause of a lack of therapeutic enhancement of anti–CTLA-4 monotherapy by concurrent vaccination with gp100 peptide in incomplete Freund's adjuvant (IFA) [63].

Genetically engineered poliovirus vaccine antitumor activity was studied in mice a few years ago [64]. It has later been moved to a phase I clinical trial for recurrent glioblastoma with interesting results [65]. In this study, patients were pre-immunized with the vaccine against poliomyelitis and then treated intratumorally with the genetically engineered virus. The role of previously developed immunity was important for successful activation of immunity against tumors treated locally [65].

In an older phase I-II trial, a recombinant nontoxic diphtheria protein (CRM197), used in many common vaccines, was used to treat a variety of accessible tumors by local delivery. Response was observed in patients that had an already developed immunization (measured both by IgG titer and delayed type hypersensitivity) against diphtheria [66].

Since immunosuppression mechanisms are in place in the tumor microenvironment [67], from these examples it is clear that an effective immunity developed outside tumors could enable a better response when antigens are later delivered intratumorally. The fact that developing immunity outside the tumor microenvironment is a valuable strategy has been also demonstrated in the case of a new neoantigen vaccine formulation. In fact, the biomaterial-based vaccine prevented the engraftment of AML cells when administered as a prophylactic and when combined with chemotherapy, and eradicated, established AML even in the absence of a defined vaccine antigen [67, 68].

As a last example, a recent Report in JAMA Dermatology suggested that Gardasil®9 might be employed for cancer treatment. Cutaneous basaloid squamous cell carcinoma (BSCC) was eradicated by intratumoral administration of the vaccine. Preventive systemic immunization was performed by a standard initial dose and a booster one, followed by intratumoral delivery of the same vaccine into just a few of the largest lesions, injected monthly over the next months. During this relatively long period, even tumors that had not been injected went into complete regression. Notably, no recurrence was observed in the follow-up period (18 months). This report first presents clinical evidence that a prophylactic antiviral vaccine may be used as an effective immunotherapy for cancer [69].

All mentioned studies point out to the value of a therapeutic strategy outlined in **Figure 4**.

#### **Figure 4.** *Schematic diagram of the neoadjuvant intratumoral delivery of repurposed vaccines.*

Available immunity against pathogen can be checked initially in patients by means of standard serological testing and/or delayed-type hypersensitivity testing. A standard vaccination protocol can be performed when required before starting intratumoral delivery of a corresponding vaccine. Afterward, timely standard delivery of other therapies (i.e., with systemic CIs) follows.
