Inactivated Cowpea Mosaic Virus in Combination with OX40 Agonist Primes Potent Antitumor Immunity in a Bilateral Melanoma Mouse Model

Abstract

Viral immunotherapies are being recognized in cancer treatment, with several currently approved or undergoing clinical testing. While contemporary approaches have focused on oncolytic viral therapies, our efforts center on the development of plant virus-based cancer immunotherapies. In a previous work, we demonstrated the potent efficacy of the cowpea mosaic virus (CPMV), a plant virus that does not replicate in animals, applied as an in situ vaccine. CPMV is an immunostimulatory drug candidate, and intratumoral administration remodels the tumor microenvironment leading to activation of local and systemic antitumor immunity. Efficacy has been demonstrated in multiple tumor mouse models and canine cancer patients. As wild-type CPMV is infectious toward various legumes and because shedding of infectious virus from patients may be an agricultural concern, we developed UV-inactivated CPMV (termed inCPMV) which is not infectious toward plants. We report that as a monotherapy, wild-type CPMV outperforms inCPMV in mouse models of dermal melanoma or disseminated colon cancer. Efficacy of inCPMV is less than that of CPMV and similar to that of RNA-free CPMV. Immunological investigation using knockout mice shows that inCPMV does not signal through TLR7 (toll-like receptor); structure−function studies indicate that the RNA is highly cross-linked and therefore unable to activate TLR7. Wild-type CPMV signals through TLR2, -4, and -7, whereas inCPMV more closely resembles RNA-free CPMV which signals through TLR2 and -4 only. The structural features of inCPMV explain the increased potency of wildtype CPMV through the triple pronged TLR activation. Strikingly, when inCPMV is used in combination with an anti-OX40 agonist antibody (administered systemically), exceptional efficacy was demonstrated in a bilateral B16F10 dermal melanoma model. Combination therapy, with in situ vaccination applied only into the primary tumor, controlled the progression of the secondary, untreated tumors, with 10 out of 14 animals surviving for at least 100 days post tumor challenge without development of recurrence or metastatic disease. This study highlights the potential of inCPMV as an in situ vaccine candidate and demonstrates the power of combined immunotherapy approaches. Strategic immunocombination therapies are the formula for success, and the combination of in situ vaccination strategies along with therapeutic antibodies targeting the cancer immunity cycle is a particularly powerful approach.

In Vivo Fate of Cowpea Mosaic Virus In Situ Vaccine: Biodistribution and Clearance

“…CPMV, a plant virus in the Secoviridae family, measures 30 nm in diameter and consists of 60 copies each of large (L) and small (S) coat protein subunits . In previous research, we considered CPMV, genome-free CPMV (termed empty CPMV or eCPMV), as well as UV-inactivated CPMV ,,, and determined that the wild-type CPMV is the most potent ISV formulation. eCPMV-ISV being the least potent of the three formulations still demonstrates high antitumor potency in mouse models and canine patients; − however, direct comparison with chemically inactivated CPMV or native CPMV highlights the importance of the nucleic acid cargo for immune cell signaling through Toll-like receptor (TLR)-7. , Mechanism of action studies revealed that CPMV primes innate immune activation through MyD88-dependent pathways and signals through TLR-2, -4, and -7 .…”

“…eCPMV-ISV being the least potent of the three formulations still demonstrates high antitumor potency in mouse models and canine patients; − however, direct comparison with chemically inactivated CPMV or native CPMV highlights the importance of the nucleic acid cargo for immune cell signaling through Toll-like receptor (TLR)-7. , Mechanism of action studies revealed that CPMV primes innate immune activation through MyD88-dependent pathways and signals through TLR-2, -4, and -7 . UV-inactivated CPMV (with heavily cross-linked RNA and protein components) and eCPMV do not signal through TLR-7 and therefore lack type I interferons, which may be a critical stimulation in potentiating antitumor immunity. , For these reasons, we propose native, live CPMV for translational development.…”

“…33 UV-inactivated CPMV (with heavily cross-linked RNA and protein components) and eCPMV do not signal through TLR-7 and therefore lack type I interferons, which may be a critical stimulation in potentiating antitumor immunity. 33,37 For these reasons, we propose native, live CPMV for translational development.…”

Inactivated Cowpea Mosaic Virus for In Situ Vaccination: Differential Efficacy of Formalin vs UV-Inactivated Formulations

“…10 UV-CPMV induction of much less IL-6 in normal mice agrees with our previous findings. 14 Type-I interferons are key signatures of TLR7 signaling; therefore, we analyzed IFN-α secretion. CPMV signals through TLR7 and induces IFN-α secretion in wild-type mice, which is lost in TLR7 −/− knockout mice and confirms our previous data.…”

Injectable Hydrogel Containing Cowpea Mosaic Virus Nanoparticles Prevents Colon Cancer Growth

“…In the preclinical setting, we have shown that in situ vaccination immunotherapy using immunostimulatory cowpea mosaic virus (CPMV) nanoparticles is a promising strategy for cancer therapy: CPMV is a 30 nm-sized icosahedral, nonglycosylated plant virus with a bipartite RNA genome thatwhile being noninfectious toward mammalstriggers potent innate immune activation through recognition by pattern recognition receptors (PRRs) . We have demonstrated that CPMV elicits potent systemic and durable antitumor immunity through priming the innate immune system; efficacy has been reported in mouse models of colon cancer , and other tumor models , and in canine patients . In recent work, we also demonstrated that CPMV targeted to the lungs primes innate immune activation and therefore protects from the onset of lung metastasis .…”