Combination Therapies for HPV-Associated Malignancies.

Human papillomavirus (HPV)-associated malignancies cause almost all cases of cervical cancer in women, and a significant percentage of head and neck cancer, together totaling almost 5% of the global cancer burden, and representing an important public health issue. The approval and use of two prophylactic HPV vaccines, Gardasil® and Cervarix®, have significantly decreased infections with HPV, but unfortunately, prophylactic vaccination does not treat established infections or malignancies resulting from HPV. Therefore, therapies for HPV-associated malignancies are necessary to improve the quality of life and survival in patients with these diseases. This review will detail new combinations of therapies in clinical development for HPV-associated malignancies.

Subversion of Host Innate Immunity by Rickettsia australis via a Modified Autophagic Response in Macrophages.

We recently reported that the in vitro and in vivo survivals of Rickettsia australis are Atg5-dependent, in association with an inhibited level of anti-rickettsial cytokine, IL-1ß. In the present study, we sought to investigate how R. australis interacts with host innate immunity via an Atg5-dependent autophagic response. We found that the serum levels of IFN-γ and G-CSF in R. australis-infected Atg5flox/flox Lyz-Cre mice were significantly less compared to Atg5flox/flox mice, accompanied by significantly lower rickettsial loads in tissues with inflammatory cellular infiltrations including neutrophils. R. australis infection differentially regulated a significant number of genes in bone marrow-derived macrophages (BMMs) in an Atg5-depdent fashion as determined by RNA sequencing and Ingenuity Pathway Analysis, including genes in the molecular networks of IL-1 family cytokines and PI3K-Akt-mTOR. The secretion levels of inflammatory cytokines, such as IL-1α, IL-18, TNF-α, and IL-6, by R. australis-infected Atg5flox/flox Lyz-Cre BMMs were significantly greater compared to infected Atg5flox/flox BMMs. Interestingly, R. australis significantly increased the levels of phosphorylated mTOR and P70S6K at a time when the autophagic response is induced. Rapamycin treatment nearly abolished the phosphorylated mTOR and P70S6K but did not promote significant autophagic flux during R. australis infection. These results highlight that R. australis modulates an Atg5-dependent autophagic response, which is not sensitive to regulation by mTORC1 signaling in macrophages. Overall, we demonstrate that R. australis counteracts host innate immunity including IL-1ß-dependent inflammatory response to support the bacterial survival via an mTORC1-resistant autophagic response in macrophages.

Characterization of recombinant gorilla adenovirus HPV therapeutic vaccine PRGN-2009.

There are approximately 44,000 cases of human papillomavirus-associated (HPV-associated) cancer each year in the United States, most commonly caused by HPV types 16 and 18. Prophylactic vaccines successfully prevent healthy people from acquiring HPV infections via HPV-specific antibodies. In order to treat established HPV-associated malignancies, however, new therapies are necessary. Multiple recombinant gorilla adenovirus HPV vaccine constructs were evaluated in NSG-ß2m-/- peripheral blood mononuclear cell-humanized mice bearing SiHa, a human HPV16+ cervical tumor, and/or in the syngeneic HPV16+ TC-1 model. PRGN-2009 is a therapeutic gorilla adenovirus HPV vaccine containing multiple cytotoxic T cell epitopes of the viral oncoproteins HPV 16/18 E6 and E7, including T cell enhancer agonist epitopes. PRGN-2009 treatment reduced tumor volume and increased CD8+ and CD4+ T cells in the tumor microenvironment of humanized mice bearing the human cervical tumor SiHa. PRGN-2009 monotherapy in the syngeneic TC-1 model also reduced tumor volumes and weights, generated high levels of HPV16 E6-specific T cells, and increased multifunctional CD8+ and CD4+ T cells in the tumor microenvironment. These studies provide the first evaluation to our knowledge of a therapeutic gorilla adenovirus HPV vaccine, PRGN-2009, showing promising preclinical antitumor efficacy and induction of HPV-specific T cells, along with the rationale for its evaluation in clinical trials.

Therapeutic Vaccines for HPV-Associated Malignancies.

Human papillomavirus (HPV)-related malignancies are responsible for almost all cases of cervical cancer in women, and over 50% of all cases of head and neck carcinoma. Worldwide, HPV-positive malignancies account for 4.5% of the global cancer burden, or over 600,000 cases per year. HPV infection is a pressing public health issue, as more than 80% of all individuals have been exposed to HPV by age 50, representing an important target for vaccine development to reduce the incidence of cancer and the economic cost of HPV-related health issues. The approval of Gardasil® as a prophylactic vaccine for high-risk HPV 16 and 18 and low-risk HPV6 and 11 for people aged 11-26 in 2006, and of Cervarix® in 2009, revolutionized the field and has since reduced HPV infection in young populations. Unfortunately, prophylactic vaccination does not induce immunity in those with established HPV infections or HPV-induced neoplasms, and there are currently no therapeutic HPV vaccines approved by the US Food and Drug Administration. This comprehensive review will detail the progress made in the development of therapeutic vaccines against high-risk HPV types, and potential combinations with other immunotherapeutic agents for more efficient and rational designs of combination treatments for HPV-associated malignancies.

Immunomodulation to enhance the efficacy of an HPV therapeutic vaccine.

BACKGROUND: While prophylactic human papillomavirus (HPV) vaccines will certainly reduce the incidence of HPV-associated cancers, these malignancies remain a major health issue. PDS0101 is a liposomal-based HPV therapeutic vaccine consisting of the immune activating cationic lipid R-DOTAP and HLA-unrestricted HPV16 peptides that has shown in vivo CD8+ T cell induction and safety in a phase I study. In this report, we have employed the PDS0101 vaccine with two immune modulators previously characterized in preclinical studies and which are currently in phase II clinical trials. Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domains of the transforming growth factor-ß receptor type II (TGFßRII) fused to a human IgG1 monoclonal antibody blocking programmed cell death protein-1 ligand (PDL1), designed both as a checkpoint inhibitor and to bring the TGFßRII 'trap' to the tumor microenvironment (TME). NHS-interleukin-12 (NHS-IL12) is a tumor targeting immunocytokine designed to bring IL-12 to the TME and thus enhance the inflammatory Th1 response. METHODS: We employed TC-1 carcinoma (expressing HPV16 E6 and E7 and devoid of PDL1 expression) in a syngeneic mouse model in monotherapy and combination therapy studies to analyze antitumor effects and changes in immune cell types in the spleen and the TME. RESULTS: As a monotherapy, the PDS0101 vaccine generated HPV-specific T cells and antitumor activity in mice bearing HPV-expressing mEER oropharyngeal and TC-1 lung carcinomas. When used as a monotherapy in the TC-1 model, NHS-IL12 elicited antitumor effects as well as an increase in CD8+ T cells in the TME. When used as a monotherapy, bintrafusp alfa did not elicit antitumor effects or any increase in T cells in the TME. When all three agents were used in combination, maximum antitumor effects were observed, which correlated with increases in T cells and T-cell clonality in the TME. CONCLUSION: These studies provide the rationale for the potential clinical use of combinations of agents that can (1) induce tumor-associated T-cell responses, (2) potentiate immune responses in the TME and (3) reduce immunosuppressive entities in the TME.

The Use of a Humanized NSG-ß2m-/- Model for Investigation of Immune and Anti-tumor Effects Mediated by the Bifunctional Immunotherapeutic Bintrafusp Alfa.

The lack of serial biopsies in patients with a range of carcinomas has been one obstacle in our understanding of the mechanism of action of immuno-oncology agents as well as the elucidation of mechanisms of resistance to these novel therapeutics. While much information can be obtained from studies conducted with syngeneic mouse models, these models have limitations, including that both tumor and immune cells being targeted are murine and that many of the immuno-oncology agents being evaluated are human proteins, and thus multiple administrations are hampered by host xenogeneic responses. Some of these limitations are being overcome by the use of humanized mouse models where human peripheral blood mononuclear cells (PBMC) are engrafted into immunosuppressed mouse strains. Bintrafusp alfa (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-ßRII to function as a TGF-ß "trap" fused to a human IgG1 antibody blocking PD-L1. A phase I clinical trial of bintrafusp alfa showed promising anti-tumor efficacy in heavily pretreated advanced solid tumors, and multiple clinical studies are currently ongoing. There is still much to learn regarding the mechanism of action of bintrafusp alfa, including its effects on both human immune cells in the periphery and in the tumor microenvironment (TME), and any temporal effects upon multiple administrations. By using the NSG-ß2m-/- mouse strain humanized with PBMC, we demonstrate here for the first time: (a) the effects of bintrafusp alfa administration on human immune cells in the periphery vs. the TME using three different human xenograft models; (b) temporal effects upon multiple administrations of bintrafusp alfa; (c) phenotypic changes induced in the TME, and (d) variations observed in the use of multiple different PBMC donors. Also discussed are the similarities and differences in the data thus far obtained employing murine syngeneic models, from clinical trials, and in the use of this humanized mouse model. The results described here may guide the future use of this agent or similar immunotherapy agents as monotherapies or in combination therapy studies.

Activation of ASC Inflammasome Driven by Toll-Like Receptor 4 Contributes to Host Immunity against Rickettsial Infection.

Rickettsiae are cytosolically replicating, obligately intracellular bacteria causing human infections worldwide with potentially fatal outcomes. We previously showed that Rickettsia australis activates ASC inflammasome in macrophages. In the present study, host susceptibility of ASC inflammasome-deficient mice to R. australis was significantly greater than that of C57BL/6 (B6) controls and was accompanied by increased rickettsial loads in various organs. Impaired host control of R. australis in vivo in ASC-/- mice was associated with dramatically reduced levels of interleukin 1ß (IL-1ß), IL-18, and gamma interferon (IFN-γ) in sera. The intracellular concentrations of R. australis in bone marrow-derived macrophages (BMMs) of TLR4-/- and ASC-/- mice were significantly greater than those in BMMs of B6 controls, highlighting the important role of inflammasome and these molecules in controlling rickettsiae in macrophages. Compared to B6 BMMs, TLR4-/- BMMs failed to secrete a significant level of IL-1ß and had reduced expression levels of pro-IL-1ß in response to infection with R. australis, suggesting that rickettsiae activate ASC inflammasome via a Toll-like receptor 4 (TLR4)-dependent mechanism. Further mechanistic studies suggest that the lipopolysaccharide (LPS) purified from R. australis together with ATP stimulation led to cleavage of pro-caspase-1 and pro-IL-1ß, resulting in TLR4-dependent secretion of IL-1ß. Taken together, these observations indicate that activation of ASC inflammasome, most likely driven by interaction of TLR4 with rickettsial LPS, contributes to host protective immunity against R. australis These findings provide key insights into defining the interactions of rickettsiae with the host innate immune system.

The multi-functionality of N-809, a novel fusion protein encompassing anti-PD-L1 and the IL-15 superagonist fusion complex.

Here we describe a novel bifunctional fusion protein, designated N-809. This molecule comprises the IL-15/IL15Rα superagonist complex containing the Fc-domain of IgG1 (N-803, formerly designated as ALT-803) fused to two single chain anti-PD-L1 domains. The fully human IgG1 portion of the N-809 molecule was designed to potentially mediate antibody dependent cellular cytotoxicity (ADCC). The studies reported here show that N-809 has the same ability to bind PD-L1 as an anti-PD-L1 monoclonal antibody. RNAseq studies show the ability of N-809 to alter the expression of an array of genes of both CD4+ and CD8+ human T cells, and to enhance their proliferation; CD8+ T cells exposed to N-809 also have enhanced ability to lyse human tumor cells. An array of genes was differentially expressed in human natural killer (NK) cells following N-809 treatment, and there was increased expression of several surface activating receptors; there was, however, no increase in the expression of inhibitory receptors known to be upregulated in exhausted NK cells. N-809 also increased the cytotoxic potential of NK cells, as shown by increased expression of granzyme B and perforin. The lysis of several tumor cell types was increased when either NK cells or tumor cells were exposed to N-809. Similarly, the highest level of ADCC was seen when both NK cells (from donors or cancer patients) and tumor cells were exposed to N-809. These studies thus demonstrate the multi-functionality of this novel agent.