Immunotherapy for HPV Malignancies

Abstract

Owing to the presence of known tumor-specific viral antigens, human papillomavirus (HPV)-associated cancers are well suited for treatment with immunotherapy designed to unleash, amplify or replace the T cell arm of the adaptive immune system. Immune checkpoint blockade designed to unleash existing T cell immunity is currently Food and Drug Administration approved for certain HPV-associated cancers. More specific immunotherapies such as therapeutic vaccines and T cell receptor-engineered cellular therapy are currently in clinical development. Such therapies may offer more specific immune activation against viral tumor antigens and decrease the risk of immune-related adverse events. Current and planned clinical study of these treatments will determine their utility in the treatment of patients with newly diagnosed advanced stage or relapsed HPV-associated cancer.

Introduction:

Immunotherapy has become a practical and widely accepted treatment modality for relapsed cancer. The foundation of modern immunotherapy can be traced back to the 1960s and 1970s with the discovery and characterization of immune cell types that ultimately were proven to play a role in the detection and elimination of cancer cells.¹ Today, immunotherapy is Food and Drug Administration (FDA)-approved for multiple forms of relapsed cancer, including several cancers associated with chronic Human Papillomavirus (HPV) infection.^2,3 The basis of all FDA-approved and investigational immunotherapies for HPV-associated cancer is the function of T cells.⁴ These immunotherapies can be conceptually organized into three main categories (reviewed in Figure 1):

Figure 1 – Categories of immunotherapy for HPV-associated cancer.

Broad categories of immunotherapy that may be useful for activating anti-tumor immunity against HPV-associated cancer (red cancer cells on bottom of diagram). A, immune checkpoint blockade blocks signaling through the programmed death receptor (PD-1) to turn off negative T cell activation signals, unleashing the activity of existing HPV-specific T cell responses. B, therapeutic vaccines, designed to deliver HPV nucleic acid or protein to antigen presenting cells, may prime new or expand existing HPV-specific T cell responses within lymph nodes. C, replacing or supplementing T cells within patients with T cells engineered to express a TCR specific for an HPV antigen may overcome the need for antigen processing and presentation and mediate immediate anti-tumor immunity.

1. Immune checkpoint blockade, designed to unleash existing anti-HPV T cells;

2. Therapeutic vaccines, designed to prime and expand anti-HPV T cells; and

3. Cellular therapies designed to supplement or replace the body’s natural anti-HPV T cell response.

Here, we review the basics of how T cells identify and eliminate HPV-infected cancer cells and summarize FDA-approved and investigational immunotherapies in each of these three categories. Only immune checkpoint blockade is currently FDA-approved for the treatment of metastatic or relapsed HPV-associated cancers, but new HPV-specific immunotherapies in clinical development hold great promise. As data demonstrating the safety and efficacy of immunotherapy for patients with relapsed HPV-associated cancer grows, such treatments will be studied in the “up front” treatment setting in an attempt to improve initial cancer treatment efficacy for newly diagnosed HPV-associated cancer, decrease rates of relapse and improve disease-free survival.

The immune system and HPV:

High-risk HPV (HPV 16 primarily) is a ubiquitous pathogen with tropism for epithelial cells. Many people have been exposed to high-risk HPV, but few develop chronic infections.⁵ Immunity against HPV begins with a humoral immune response that involves the production of antibodies by B cells. Anti-HPV antibodies bind free HPV virions before they infect epithelial cells, leading to their elimination by other components of the immune system. Preventative vaccination against HPV with a multivalent vaccine, such as Gardasil®, boosts anti-HPV antibody titer levels and leads to enhanced protection from HPV infection.⁶ In some patients who have not been vaccinated, the level of natural anti-HPV antibodies is insufficient to prevent infection. Once an epithelial cell is infected with HPV, antibodies no longer play a role. Based on contemporary understanding of anti-viral immunity, the major cell type that works to detect and eliminate an epithelial cell infected with an intracellular virus such as HPV is the T cell.

T cells identify abnormal or infected cells through recognition of specific peptides, called antigens, presented on the surface of cells via human leukocyte antigen (HLA) class I and II molecules. T cells are initially “educated” and primed for antigen-specific responses by specialized antigen presenting cells (APCs) such as dendritic cells. Epithelial cells (or carcinoma cells) are constantly breaking down intracellular proteins (including HPV proteins if infected), and some fraction of these protein breakdown byproducts are loaded onto HLA class I molecules for presentation to T cells. Portions of the HPV 16 genome that are integrated and highly expressed serve as sources of tumor-specific T cell antigens. Numerous, validated T cell antigens from HPV 16 E6 and E7 with different HLA class I restriction elements have been identified.^7–14

Importantly, the formal identification of HPV 16 E6 and E7 T cell antigens validates the role of T cells in identifying and clearing epithelial cells infected with HPV. Despite the presentation of naturally processed and presented T cell antigens derived from HPV gene products, and the presence of HPV-specific T cells educated by APCs, some patients are unable to clear infected cells and develop a chronic HPV infection. The reasons why some people are unable to mount a T cell immune response sufficient to clear an initial HPV infection are unclear.

Immunosuppression may predispose individuals to developing a chronic HPV infection and subsequent HPV-associated cancer.¹⁵ Yet, most patients who develop HPV-associated cancer are not immunosuppressed.¹⁶ In addition to the known oncogenic roles of HPV 16 E6 and E7, HPV gene products themselves may play a role in allowing HPV infected cells to escape T cell immunity. Critical to the initiation of an anti-HPV immune response is the production of interferon that occurs when a cell “detects” intracellular virus. HPV 16 E6 and E7 both are able to dampen or prevent interferon signaling, essentially turning off a cell’s ability to activate immunity.^17,18 Additionally, the E5 from HPV 16 can interact with and prevent the ability of HLA molecules to present T cell antigens.¹⁹ These tumor-cell intrinsic mechanisms of immune suppression driven by HPV gene products exist in addition to a number of physical mechanisms of immunosuppression that exist within tumor such as hypoxia, low pH and elevated interstitial pressures.²⁰

Despite this immunosuppression, patients with HPV-associated cancer are thought to be prime candidates for T cell-based immunotherapy. T cells can detect and eliminate virally infected cells; the presence of T cell antigens derived from “essential” HPV gene products that are required for oncogenesis suggests that T cells should be able to recognize these cancer cells as abnormal. Although non-specific immune activation with immune checkpoint blockade has certainly demonstrated some degree of clinical success, the presence of known tumor-specific antigens makes the development of HPV-specific immunotherapy, such as therapeutic vaccines or TCR-engineered cellular therapy, a possibility.

Immune Checkpoint Blockade:

Use of monoclonal antibodies to disrupt immune checkpoint signaling, or immune checkpoint blockade, has gained FDA-approval for a variety of cancers following demonstration of safety and efficacy in large-scale clinical studies. Physiologically, programmed death receptor-1 (PD-1) exists as a cellular brake and prevents uncontrolled T cell responses. PD-1 is expressed on T and B cells upon activation; the ligand for PD-1, programmed death ligand 1 (PD-L1), is expressed on tumor, stromal and myeloid cells in response to inflammatory cytokines such as interferon γ (IFN-γ). Upon ligation by PD-L1, PD-1 transmits a negative costimulatory signal to attenuate T cell activation.²¹ Therefore, in the setting of cytolytic and effector T cell function, the PD-1/PD-L1 axis ensures that responses remain within a physiological range.²² Cancers are able to pirate this system as a mechanism to evade T cell killing. Therefore, blockade of the PD-1/PD-L1 axis with monoclonal antibodies (mAb) allows the body to unleash the existing T cell repertoire to nonspecifically target and kill cancer cells.²²

Pembrolizumab, a monoclonal antibody that blocks the PD-1/PD-L1 axis, is approved as monotherapy or in combination with chemotherapy for the first line treatment of recurrent and metastatic head and neck squamous cell carcinoma (HNSCC), including HPV-associated oropharyngeal SCC.² Pembrolizumab is also approved for the treatment of advanced, PD-L1 positive (CPS ≥1) cervical cancer with disease progression on or after chemotherapy.³ The Combined Positive Score, or CPS, is how PD-L1 positivity is measured histologically and is calculated by dividing the total number of PD-L1 positive cells by the total number of all viable cells (then multiplying by 100) within a given tumor section. Blockade of cytotoxic T lymphocyte antigen-4 (CTLA-4) is another form of immune checkpoint blockade, but lacks FDA approval for use in HPV associated cancers largely due to increased toxicity and lack of proven increased efficacy beyond that observed with PD-1 blockade.

Based upon current understanding of the mechanisms behind anti-tumor immunity activated by immune checkpoint blockade, this form of immunotherapy is non-specific.²³ Immune checkpoint blockade unleashes the activity of existing T cell clones with the expectation that T cells specific for tumor antigens exist. Whether immune checkpoint blockade unleashes the activity of T cell clones specific for HPV-derived antigens or other tumor antigens is unclear.²⁴ It is also currently unclear whether PD-1/PD-L1 blockade has differential efficacy in HPV positive and HPV negative tumors. HPV associated cancers display high levels of PD-L1, driven in part by E7 from HPV and in part by an inflamed microenvironment high in interferons.^25,26 Histologically, HPV-associated cancers are infiltrated by a significant number of PD-1 positive effector T cells.²⁷ Tumor infiltrating lymphocytes that express high levels of PD-1 are more likely to respond to PD-1 blockade.

PD-1 immune checkpoint blockade is effective in treating relapsed head and neck cancer regardless of HPV status. KEYNOTE 048 led to first line FDA approval of pembrolizumab alone or in combination with chemotherapy for recurrent and metastatic HNSCC. In this study, different degrees of benefit to pembrolizumab monotherapy were stratified by CPS score, with the greater the CPS score the greater the chance of benefit. Pembrolizumab monotherapy increased median overall survival when compared to standard of care in patients with CPS ≥20 (14.9 vs. 10.7 months) or CPS≥1 (12.6 vs 10.3 months). Patients with CPS<1 demonstrated less benefit to pembrolizumab monotherapy, however pembrolizumab with chemotherapy increased median overall survival (13.0 vs. 10.7 months, p=.003) when compared to standard of care in the total population.² In a large phase III trial of Nivolumab for second line treatment of recurrent or metastatic HNSCC, the hazard ratio for death was significantly decreased for HPV-associated but not HPV-negative HNSCC patients.²⁸ This serves as preliminary evidence that patients with HPV-associated tumors may experience a greater clinical benefit as compared to patients with HPV-negative tumors when treated with PD-1 blockade. Similarly, clinical study of the PD-L1 mAb durvalumab for recurrent and metastatic HNSCC demonstrated a 29.4% response rate in patients with HPV-associated cancer compared to 10.8% for patients with HPV-negative cancer, again implying patients with HPV-associated cancer may experience greater clinical benefit following PD-1 axis immune checkpoint blockade.²⁹ Currently, the HPV status of relapsed oropharyngeal cancers does not alter the standard-of-care treatment with immune checkpoint blockade.^30,31

PD-1 blockade has also demonstrated clinical benefit in patients with HPV-associated cervical cancer. KEYNOTE 158 demonstrated an overall response rate of 14.6% among 98 patients with CPS ≥1 following administration of pembrolizumab, and has led to FDA-approval for the second-line treatment of relapsed disease.³ Several ongoing trials are studying PD-1 blockade in the first line setting for cervical cancer. KEYNOTE-826 is a phase III trial evaluating chemotherapy with or without pembrolizumab for the first line treatment of persistent, recurrent, or metastatic cervical cancer (NCT03635567).³² ENGOT-cx11/KEYNOTE-A18 is a phase III trial evaluating chemoradiotherapy with or without pembrolizumab in treatment naive patients with locally advanced cervical cancer (NCT04221945).³³ Preliminary results from these studies are expected soon.

While PD-axis immune checkpoint blockade has shown promising data in HPV-associated anal SCC (SCCA), it has not yet received FDA approval. NCI9673 was a phase II, single arm study testing nivolumab monotherapy in patients with treatment-refractory, metastatic SCCA. Out of 37 patients, two complete and seven partial responses were observed for an overall response rate of 24%.³⁴ KEYNOTE 028 evaluated the efficacy of pembrolizumab in PD-L1 positive (>1% of all cells) advanced SCCA. Four partial responses were observed among 24 patients for an overall response rate of 17%.³⁵ As a result of these two trials, nivolumab and pembrolizumab were included as options for second line treatment of metastatic anal cancer in the most recent NCCN guidelines.³⁶ Currently, nivolumab is being studied for use after combined modality therapy in patients with high risk stage II-IIIB SCCA (NCT03233711) or in combination with ipilimumab for metastatic refractory SCCA (NCT02314169). Pembrolizumab is also being studied in a phase II trial of metastatic SCCA (NCT02919969).

Recent, promising research has studied “next generation” immune checkpoint immunotherapy combining PD-axis blockade with disruption of other immunosuppressive signals. M7824 is bifunctional fusion protein composed of a monoclonal antibody targeting PD-L1 fused to the extracellular domain of the human transforming growth factor β (TGF-β) receptor II, which acts as a TGF-β trap.³⁷ TGF-β pathway dysregulation is hypothesized to play a critical role in the development of HPV associated cancers.³⁸ Preliminary results from a phase I study of M7824 in relapsed HPV associated cancers demonstrated a total clinical response rate of 38.9% in a cohort of 36 checkpoint-naive patients.³⁹ Based on these results, a larger phase II study is underway investigating M7824 in HPV associated malignancies (NCT03427411).

Therapeutic Cancer Vaccines:

Knowledge about antigens that are often integrated into the host genome and critical for oncogenesis (HPV 17 E6 and E7) in HPV-associated malignancies as well as the identification of specific immunogenic epitopes allows for the development of HPV-specific immunotherapies.^40,41 One form of such HPV-specific immunotherapy is therapeutic vaccination. Differentiated from preventative vaccines designed to induce a humoral (antibody) response to prevent viral infection of epithelial cells, therapeutic vaccines are designed to induce T cell responses that could lead to the elimination of cells already infected with HPV.

Although there are many different types of therapeutic vaccines (discussed individually below), a common goal is shared. Therapeutic vaccines aim to deliver an immunogenic payload to antigen presenting cells. Within antigen presenting cells, immunogenic protein is naturally processed through the proteasome and peptides are loaded onto MHC class I or II. If a peptide-HLA complex elicits a T cell response, the peptide is an antigen. Recognition of antigen-HLA complexes can lead to the priming of new, antigen specific CD8 and CD4 T cell responses which may control and eradicate HPV infected cells.^42–44 Whereas immune checkpoint blockade unleashes the activity of existing T cell clones; therapeutic vaccines may be able to prime and activate new antigen-specific T cell clones. Although no therapeutic HPV vaccines have gained FDA-approval, many are being studied clinically. Table I summarizes therapeutic HPV vaccines in clinical development.

Table I.

Summary of active clinical trials assessing the safety and/or clinical efficacy of therapeutic vaccines for HPV-associated cancer.

Name	Identifier	Phase	Description	Setting
PRGN2009	NCT04432597	I	Gorilla adenovirus vector-based vaccine containing multiple CTL agonist epitopes of E6 and E7	Alone or in combination with M7824
HB-201	NCT04180215	I/II	Arenavirus vector-based vaccine expressing the inactivated fusion protein HPV 16 E7E6	Monotherapy or in combination with an immune checkpoint inhibitor
TG4001	NCT03260023	Ib/II	Vaccina vector-based vaccine engineered to express HPV 16 E6 and E7	In combination with Avelumab
ADXS11-001	NCT02853604, NCT02002182, NCT02291055, NCT01266460	III	Live attenuated Listeria monocytogenes-based vaccine engineered to secrete a tLLO-HPV- 16-E7 fusion protein	Administered following CCRT
TA-CIN	NCT02405221	I	Fusion protein vaccine containing HPV 16 L2, E6, and E7	Monotherapy
GL-0810	NCT00257738	I	Vaccine of EIPV 16 antigens	Monotherapy
ISA101a	NCT04369937, NCT03669718, NCT02426892, NCT04398524, NCT03258008	II	A peptide vaccine containing overlapping synthetic long peptides of HPV 16 E6 and E7	In combination with Pembrolizumab and chemoradiotherapy
MEDI0457	NCT03162224, NCT04001413, NCT03439085	I/II	A DNA based vaccine containing a plasmid for E6 and E7 and a second plasmid for IL-12	In combination with Durvalumab
CUE101	NCT03978689	I	IL-2 and a pMHC complex composed of HLA A*02:01 complexed with a dominant E7 peptide	Monotherapy
MG1-E6E7	NCT03618953	I	Recombinant form of oncolytic rhabdovirus maraba encoding inactive, mutant E6 and E7	Sequential treatment with atezolizumab

Peptide- and protein-based vaccines

Peptide- and protein-based vaccines rely on the uptake of peptides from the extracellular environment by APCs for presentation on MHC II and cross presentation on MHC I. These vaccines in general are less immunogenic than other delivery systems such as live vector-based vaccines.^45,46 Whole proteins included in a vaccine must be naturally processed and presented by APCs via the patient’s native MHC molecules to serve as an antigen. Identification of candidate antigens may be challenging as the peptide repertoire presented by each HLA allele is different. Therefore, vaccines containing only minimal epitopes (antigens) may be limited to patients with certain HLA alleles. Whole protein vaccines are not HLA restricted, but are otherwise limited because whole proteins are inefficiently transported into APCs from the extracellular environment in a way that facilitates efficient antigen processing and presentation.⁴⁷ A possible compromise is to use sets of overlapping peptides of length 15-35 that span the entire protein, such as in the ISA101a vaccine (Table I). The upside to peptide and protein-based vaccines is that they are safe and easy to produce. In fact, identification, validation, and production of these vaccines is feasible for creating personalized vaccines in small cohorts of patients.⁴⁸ Three peptide/protein vaccines administered peripherally are currently under clinical study. TA-CIN is made of a fusion protein containing HPV 16 L2, E6, and E7; GL0810 consists of several selected HPV 16 antigens; and ISA101 is composed of overlapping synthetic long peptides of the HPV 16 E6 and E7 genes (Table I).

ISA101 is one of the most studied and most promising therapeutic HPV vaccines. In a landmark study of ISA101 in vulvar intraepithelial neoplasia, durable, complete remission was observed in nine of 19 patients at two years. Patients’ clinical responses correlated well with antigen specific T cell responses.⁴⁹ Importantly, viral clearance was observed in all but one patient with histological clearance.⁵⁰ However, ISA101 alone did not have a significant treatment effect on advanced cervical cancer even though a broad and robust T cell response against the E6 and E7 peptides was detected in the blood of patients.⁵¹ This suggested that immunosuppression in the local tumor microenvironment prohibited effective T cell responses. Therefore, the combination of ISA101 and PD-L1 immune checkpoint blockade was tested in patients with HPV related cancer with the reasoning that inhibiting mechanisms of immunosuppression could increase the efficacy of ISA101. A response was observed in eight of 24 patients for an overall response rate of 33% and a median duration of response of 10.3 months at the time of trial publication.⁵² Additionally, preclinical studies identified that the combination of cisplatin based chemotherapy and ISA101 worked synergistically because cisplatin stimulated tumor infiltration of APCs expressing T cell costimulatory ligands.⁵³ A clinical trial of ISA101 given during standard of care chemotherapy in recurrent or metastatic cervical cancer patients produced tumor regressions in 31 of 72 patients (43%). Importantly, study of tissue and blood samples showed that the chemotherapy regimens decreased systemic and tumoral immunosuppression by myeloid cells, which was associated with the detection of spontaneously induced HPV16 specific T cells.⁵⁴ ISA101 is currently in several phase II trials investigating its use in combination with immune checkpoint blockade and/or chemotherapy (Table I).

Nucleic acid-based vaccines

DNA and RNA based vaccines rely on APC uptake of nucleic acid from the extracellular environment, translation into protein, and subsequent presentation of naturally processed and presented antigens on MHC class I and II molecules. As such, they share many of the same pros and cons as peptide/protein-based vaccines. They generally have low immunogenicity but are relatively safe. DNA vaccines are easy to produce whereas RNA vaccines are relatively unstable and more difficult to produce in large quantities.^55–59 One possible advantage is that DNA and RNA vaccines ensure natural antigen (protein) expression prior to processing and presentation, such that any antigen specific response against APCs can be assumed to occur naturally.

Delivery of DNA or RNA into APCs can occur under multiple methods. Transfection and electroporation are commonly used methods of delivery. Currently, MEDI0457, a DNA plasmid vaccine encoding E6 and E7, is under clinical study (Table I).⁶⁰

MEDI0457 (formerly INO-3112) is delivered as a combination of two DNA plasmids: one encoding HPV 16/18 E6 and E7 antigens (VGX-3100) and another encoding a recombinant IL12 adjuvant (INO-9012). Both plasmids are delivered by intramuscular injection followed immediately by electroporation with the CELLECTRA device.⁶⁰ A pilot study of VGX-3100 alone in patients with high grade CIN had shown cytolytic activity of HPV specific CD8 T cells.⁶¹ A follow up placebo controlled clinical trial showed histopathological regression and clearance of HPV 16/18 in approximately half of CIN2/3 patients who received VGX-3100 alone.⁶² A recent study of MEDI0457 in HPV associated oropharyngeal SCC provided proof of concept evidence for the clinical activity of MEDI0457 in cancer. In this trial, patients were either treated before and after definitive surgery or after concurrent chemoradiation. Out of 21 patients, 18 showed increased HPV antigen-specific peripheral blood T cell activity. The CD8/FoxP3 ratio was increased in four of five and perforin positive immune infiltrates were increased in five of five evaluable tumors. These data suggest that MEDI0457 can generate durable antigen-specific responses in patients with HPV-associated cancer. Whether this vaccine approach can induce immunity of sufficient magnitude to produce clinical benefit for patients with HPV-associated cancer is unclear at this time. Further studies of MEDI0457 in combination with immune checkpoint blockade are underway (Table I).

Live vector-based vaccines

Live vector-based vaccines use bacterial or viral vectors to deliver the immunogenic payload directly to APCs. The vectors are highly immunogenic themselves owing to their pathogenic nature and there are a wide range of possible vectors to choose from. These vaccines can be engineered to deliver either nucleic acid or protein payloads to APCs. However, these vaccines come with safety concerns because of their live vector foundation. To circumvent this concern, many vaccines vectors are engineered to lose the ability to replicate. Additionally, there are concerns of possible pre-existing immunity towards the vector leading to quick elimination by the host immune system, potentially limiting vaccine efficacy in the general population.⁵⁶ There are currently four different HPV therapeutic cancer vaccines using live vectors. PRGN 2009 is a gorilla adenovirus based vaccine which contains multiple epitopes of the HPV 16 and 18 E6 and E7 genes.⁶³ Promising pre-clinical data has led to the initiation of a phase 1 clinical trial which is planning to open in summer 2020. HB-201 is based on an Arenavirus vector that expresses a fusion E6+E7 protein; TG4001 is based on a Vaccinia vector that expresses HPV 16 E7 and E7; and ADXS11-001 is based on a Listeria monocytogenes vector that secretes an HPV 16 E7 fusion protein. These three latter vaccines are in various stages of clinical development. MG1-E6E7 is a unique recombinant form of the oncolytic rhabdovirus Maraba that targets cancers via both cancer-specific viral lysis and expression of mutant E6 and E7 proteins (Table I).

Axalimogene filolisbac (ADXS11-001), one of the most studied live vector-based therapeutic vaccines, is a live, attenuated Listeria monocytogenes (Lm)-listeriolysin O (LLO) that has been engineered to secrete a fusion protein of truncated LLO fused to HPV-16 E7 (tLLO-HPV-16-E7).⁶⁴ Phagocytosis of ADXS11-001 results in presentation of E7 antigens on MHC class I and II molecules.⁶⁵ In a phase II study of ADXS11-001 with or without cisplatin for the treatment of recurrent or refractory cervical cancer following prior chemotherapy and/or radiotherapy, ADXS11-001 demonstrated promising safety and efficacy results with a 12 month combined overall survival rate of 34.9% (survival did not differ significantly between groups).

The overall response rate was 15.9%.⁶⁵ In a separate trial of patients with untreated, nonmetastatic anal cancer, ADXS11-001 was given before and after standard of care chemoradiation.⁶⁶ All nine evaluable patients had complete clinical responses by sigmoidoscopy and eight of nine (89%) of patients were progression free at a median follow up of 42 months.⁶⁶ Concerningly, one case of systemic listeriosis was reported during a phase I trial of ADXS11-001 in a patient with HPV-associated oropharyngeal SCC. The trial was immediately put on hold and ultimately terminated. This represents a potential adverse event of ADXS11-001 and live vector vaccines in general. There are currently at least four active clinical trials testing ADXS11-001 in different settings (Table I).

Dendritic cell-based vaccines

Dendritic cell-based vaccines are administered as whole cell products that include dendritic cells that have been pre-loaded with the antigenic payload of interest. These cells are then able to directly prime antigen specific CD4 and CD8 immune responses. These vaccines are highly immunogenic and versatile. Loading antigen into dendritic cells ex vivo allows researchers and clinicians a greater flexibility in antigen choice and loading mechanism.^67–69 However, the process of personalized cell processing is labor intensive and costly. For this reason, dendritic cell vaccines are less often used in clinical studies as compared to other vectors. Dendritic cells pulsed with HPV 16 and HPV 18 E7 and keyhole limpet hemocyanin have been tested in phase I trials of cervical cancer with promising safety and immunogenicity profiles (Table I).⁷⁰

Vaccine Adjuvants and Combination Therapies

Because some therapeutic vaccine payloads and/or delivery mechanisms suffer from inherently low immunogenicity, vaccine adjuvants are often used to promote local inflammation to increase vaccine efficacy. For example, MEDI0457 is administered as two separate plasmids: one encoding the E6 and E7 proteins, and another encoding IL-12. The use of IL-12 as a molecular adjuvant promotes the maturation and function of T cells, leading to an increased quality and breadth of the antigen specific immune response.⁷¹ Another example is GL-0810, which is administered with Montanide and GM-CSF adjuvants.⁷² The use of such therapeutic vaccine adjuvants may promote dendritic cell migration to the site of vaccination, enhance antigen presentation, and lead to a more robust antigen-specific T cell response.⁷³

Co-expression of molecules important for antigen processing and use of prime/boost administration strategies may also enhance therapeutic vaccine efficacy. Calreticulin is a component of the intracellular protein complex that loads minimal epitope peptides onto MHC class I molecules in the endoplasmic reticulum. Calreticulin also lengthens engagement of the TCR:MHC:peptide complex at the cell surface.⁷⁴ Delivery of an HPV E7 DNA vaccine that encodes calreticulin as an adjuvant results in the expression of a fusion protein CRT/E7.⁷⁵ This results in better protein homing to the endoplasmic reticulum and therefore improved antigen processing and presentation in pre-clinical studies.⁷⁶ Other adjuvants including the anti-apoptotic molecule BCL-xL and interferon stimulating gene 15 have also been shown to enhance HPV therapeutic vaccine efficacy in preclinical models.^77,78 Another strategy to enhance the potency of therapeutic HPV vaccines is using a prime-boost regimen. Preclinical studies have shown that boosting with live vector based vaccines after administration of a HPV 16 E6/E7 DNA vaccine may significantly enhance the immune response.^79,80 Boosting with a live vector based vaccine can also enhance the immune response following RNA or protein based vaccines.^81,82

Lastly, many cancer vaccines have been combined with other treatments to augment their effects. Radiation therapy may sensitize tumors to HPV vaccines because radiation induced cancer cell death results in release of tumor antigens and inflammatory cytokines which can act as a natural adjuvant to vaccines.⁸³ Chemotherapy may also sensitize tumors to therapeutic HPV vaccines through its effects on the local immune microenvironment.^84,85 The combination of therapeutic HPV vaccines with immune checkpoint blockade also serves as a rational approach because immune checkpoints such as PD-1/PD-L1 may restrain the activity of T cells that would otherwise be elicited by vaccination.⁵²

Cellular Therapy

A third category of immunotherapy involves the adoptive transfer of T cells to replace or augment a patient’s endogenous T cell immune response. Adoptive T cell therapy is the infusion of autologous tumor specific T cells that can recognize and eradicate target cancer cells. Pioneering work in adoptive T cell therapy began at the Surgery Branch of the National Cancer Institute where Steven Rosenberg and colleagues observed that administration of IL-2, which acts to increase proliferation and survival of T cells, resulted in durable tumor responses in a small subset of melanoma patients.^86–88 Given the clear ability of autologous T cells to induce an anti-tumor response, adoptive transfer of tumor infiltrating lymphocytes (TIL) was tested. T cells were cultured and expanded from resected metastatic melanoma deposits and infused into patients, resulting in high response rates and even some cures.⁸⁹ Since then, TIL therapy has moved from melanoma into solid tumors, including HPV associated tumors.^8,90,91

Initial results of using TIL cultured from tumor to treat patients with relapsed HPV-associated malignancies were promising, with a 28% objective response rate in patients with cervical cancer and an 18% response rate in patients with oropharyngeal and anal cancer.^91,92 Two patients with relapsed cervical cancer demonstrated complete responses. Reactivity of the infused cultured TIL to HPV correlated with clinical responses, but 30% or less of cultured TIL were found to be specific for HPV.⁹⁰ This data suggested that approaches to enhance HPV reactivity of adoptively transferred T cells could enhance responses and clinical benefit.

One promising strategy that expanded on the foundations of TIL therapy was to administer autologous T cells that have been genetically engineered to express a T cell receptor (TCR) specific for a minimal epitope derived from HPV that is expressed via a specific HLA molecule. While T cells engineered to express a chimeric antigen receptor can be targeted to cancer cells that express a specific surface target, a TCR is needed to target T cells to an intracellular antigen such as HPV. Two such therapeutic anti-HPV TCRs have been discovered, described, and clinically studied at the National Cancer Institute.^10,93 Two of 12 patients experienced an objective tumor response in a phase I trial of E6 TCR engineered T cells in patients with metastatic HPV-associated cancer.⁹³ Four of 12 patients experienced an objective tumor response in a similar trial of E7 TCR engineered T cells.⁹⁴ These data provide proof of concept evidence that TCR engineered T cells can clinical benefit as a monotherapy in patients with relapsed HPV-associated cancer.

Like the potential specificity of cancer vaccines, cell transfer of TCR engineered T cells is promising because the expression and presentation of T cell antigens from viral E6 and E7 proteins provide an ideal, tumor-specific target. However, correlative studies suggest that viral epitopes may not be the main target antigens following adoptive cellular therapy designed to target HPV.²⁴ Dominant T cell responses in two patients with metastatic cervical carcinoma who were treated with cultured TIL and experienced complete cancer regression were directed against neoepitopes (derived from mutations) and cancer germline antigens rather than HPV antigens.²⁴ This implies that initiation of anti-tumor immunity with HPV-specific T cells may lead to the activation of T cells targeting other antigens. The results of this study may have broad implications for other forms of T cell-based immunotherapy.

The main limitations of these treatments are HLA restriction and the need for chemotherapy pre-conditioning prior to adoptive transfer. Only HLA-A*02:01 positive patients can potentially benefit from the therapeutic TCRs currently in clinical development. TCRs restricted other HLA alleles may be discovered using high throughout discovery methods to expand the population of candidate patients. Another limitation is toxicity related to the cyclophosphamide and fludarabine conditioning regimen.⁹⁴ This conditioning chemotherapy regimen is needed to prepare the host immune compartment to receive and engraft adoptively transferred T cells. Thus, although TCR engineered T cell therapy is able to induce regression in HPV-associated cancers refractory to multiple prior treatments, a library of TCRs with different HLA restrictions and a less toxic conditioning regimen are likely needed to make this treatment approach feasible and available outside of select research institutions. Several clinical trials studying the adoptive transfer of HPV-specific and non-specific engineered T cells for the treatment of HPV-associated cancers are ongoing (NCT03108495, NCT02379520).

The role of combination radiation and immunotherapy

To date, immunotherapy for HPV-associated cancers has primarily been studied in the relapsed setting. These patients have likely already received definitive radiotherapy. However, with demonstration of the safety and clinical efficacy of immunotherapy in the relapsed setting, there is great interest in the clinical study of immunotherapy combined with definitive standard of care radiotherapy in patients with newly diagnosed cancer. If the combination of immunotherapy and radiotherapy can be administered safely with little toxicity and lead to equivalent or decreased rates of disease relapse compared to surgery plus adjuvant radiotherapy or concurrent chemoradiotherapy, this could serve as another avenue of treatment de-escalation for patients with newly diagnosed, advanced stage HPV-associated cancers.

A large body of pre-clinical data demonstrating the ability of specific doses and fractionation schedules to enhance the efficacy of immunotherapy exists. In general, higher dose, single dose, or hypofractionated radiotherapy enhances antigen-specific T cell immunity through several different mechanisms.^95–97 Conversely, daily fractionated radiotherapy appears to be immunosuppressive in both patients and mice.^98,99 This data has been taken into consideration with the initiation of numerous clinical trials evaluating the ability of primarily high dose, hypofractionated radiotherapy to be combined with immune checkpoint blockade.¹⁰⁰ Whether the addition of immunotherapy to standard, daily fractionated low-dose radiotherapy results in additive or synergistic clinical benefit remains to be seen.

Conclusions:

Immunotherapy holds great promise for the treatment of advanced stage or relapsed HPV-associated cancers. Immunotherapy is already in widespread use for relapsed HPV-associated cancers in the form of immune checkpoint blockade. Numerous new immunotherapies designed to more specifically induce HPV-specific T cell responses, including therapeutic vaccines and TCR engineered T cell therapies, are in various stages of clinical development. It is likely that one or more of these new, more specific immunotherapies will demonstrate safety and clinical efficacy in the setting of relapsed disease sufficient for FDA-approval. The future of immunotherapies for HPV-associated cancer lies in the careful clinical assessment of immunotherapy in the up-front treatment setting, as monotherapy or in combination with standard anti-cancer treatments such as radiation. Through this approach, we may soon be able to offer patients with HPV-associated cancers oncologically equivalent but more HPV specific and less toxic treatment through harnessing the power of the adaptive immune system.