HPV-directed Therapeutics for HPV-associated Oropharyngeal Cancer

Abstract

Despite the availability of prophylactic human papillomavirus (HPV) vaccines, there is a growing incidence of HPV-associated head and neck squamous cell carcinomas (HPV-HNSCC) worldwide. The viral etiology of HPV-HNSCC provides an opportunity to develop personalized immune-based therapies, which target the unique viral- or tumor-specific proteins. Novel HPV-targeted immunotherapeutic approaches in clinical development are reviewed. Early results from these trials highlight new opportunities and potential challenges ahead. Immunotherapies for HPV-associated HNSCCs will require a tailored combinatorial approach based on pre-existing mechanisms of host immune resistance. As the field continues to identify the relevant HPV 16/18 immunogenic epitopes that are presented by diverse HLA Class I alleles, improved HPV targeted biologics and clinical monitoring tools can be developed and applied to a broader cancer patient population.

Epidemiology of HPV-associated Head and Neck Cancers

Head and neck squamous cell carcinomas (HNSCCs) are a worldwide public-health problem, with more than 900,000 cases and over 400,000 deaths reported annually¹. In 2021, it is estimated there were 66,630 incident cases and 14,620 deaths attributed to head and neck cancer in the United States (U.S.)². Traditionally, the incidence of head and neck cancers has trailed annual cigarette consumption by 10-15 years, with a peak incidence in the 1970s and a downward trend thereafter. However, a distinct departure from this trend has been observed among Caucasian men under the age of 60, in whom the incidence of oropharyngeal cancers has been on the rise³. In the past three decades, significant evidence has established human papillomavirus (HPV) as the causative agent in a growing proportion of incident cases of oropharyngeal cancers^3-6. Annually, HPV infection is estimated to cause approximately 36,500 cancers in the U.S; 14,400 cases are attributed to oropharyngeal cancers followed by 11,100 cases of cervical cancers⁷. Unlike cervical cancer, which can be caused by several high-risk HPV types, one viral type, HPV type 16, is the causative agent in up to 80% of oropharyngeal cancers⁸, which has important implications in the development of HPV-specific immunomodulatory therapies for HPV-HNSCC patients.

Therapeutic Targeting of HPV16 E6 and E7 Oncogenic Proteins

Currently, the treatment options for HNSCC patients include surgery, radiation therapy, chemotherapy, immunotherapy, or a combination of these modalities. The viral etiology of HPV-associated HNSCCs provides an opportunity to develop personalized immune-based therapies, which target the unique viral- or tumor-specific proteins. Many cancer vaccine strategies, such as those targeting pancreatic, renal, and melanoma cancers (for review^9-12, have focused on self-proteins, which may be aberrantly upregulated (such as MAGEA3) or mutated (such as p53). However, in HPV-HNSCC, therapies leverage tumor expression of viral-specific oncoproteins, such as E6 or E7, to enhance specific targeting of the tumor.

In the cellular progression from viral infection to malignant transformation, episomal HPV DNA can integrate into the host genome with a resultant deletion of non-critical viral genes. Late genes (L1 and L2) and some early genes (E1 and E2) are commonly deleted, whereas E6 and E7 are often the only open reading frames (ORFs) consistently expressed in cancer cell lines¹³ and in HPV-associated cancers¹⁴. E6 and E7 are necessary for viral transformation through their inactivation of two human tumor suppressor proteins, p53 and pRb, respectively, resulting in dysregulated cell cycle proliferation, delayed cellular differentiation, increased frequency of spontaneous and mutagen-induced mutations, and increased chromosomal instability¹⁵.

Since E6 and E7 are constitutively expressed in most HPV-associated cancers, they represent promising targets for the development of antigen-specific therapeutic vaccines. There are several advantages to targeting the E6 and/or E7 proteins. They are foreign viral proteins and are therefore more immunogenic than a self-protein that is genetically altered (such as a mutated p53) or aberrantly reactivated (such as the embryonic protein, MAGEA-1). Furthermore, as E6 and E7 are required for the induction and maintenance of the malignant phenotype of cancer cells¹⁶, tumor cells cannot evade a directed immune response through antigen loss of E6 or E7. Lastly, preclinical studies suggest that vaccines targeting these papillomavirus proteins can generate therapeutic as well as protective effects in vivo^16-19. Therefore, the E6 and E7 proteins represent logical targets for developing antigen-specific immunotherapies or vaccines against head and neck cancers. Various forms of vaccines, such as vector-based vaccines, tumor-based vaccines, DNA-based vaccines, and protein/peptide-based vaccines, have been described in experimental systems targeting the HPV-16 E6 and/or E7 proteins^16-19. Although HPV-16 E7 immune responses are better characterized in preclinical models than E6 responses, both E6 and E7 genes and proteins have been in development for vaccine therapies and other immunotherapies. Recent studies have demonstrated endogenous host immune responses against HPV-16 E2 and E5 in HPV+ HNSCC patients, which supports therapeutic strategies that elicit HPV-specific immune responses across multiple HPV antigens ²⁰ (See Figure 1).

Figure 1: Opportunities for Targeted Immunotherapy in HPV-associated Head and Neck Cancers.

Both viral and non-viral antigens are immunotherapeutic targets in HPV-associated head and neck cancers. HPV-infected epithelial cells express the viral associated proteins, E6, E7, E2 and E5, which support viral replication and cellular transformation. Antigen presenting cell populations, such as dendritic cells, in the tumor microenvironment (TME) (left) phagocytose apoptotic/necrotic HPV-infected cells and present HPV viral epitopes to CD4+ and CD8+ T cells that then get activated to recognize and kill HPV-infected cells (right, upper arrow). Viral-induced cellular transformation can result in genetic instability leading to cellular re-expression of cancer testis antigens (CTA) and increase the tumor mutational burden (TMB) leading to neoantigen (Neo) expression. These non-HPV antigens can also serve as immunotherapeutic targets. Based on the expression of multiple immunogenic antigens, both foreign and mutated, by the HPV-transformed cell, multiple tumor resistance mechanisms are activated in HPV-associated head and neck cancers (right, lower arrow), including the downregulation of antigen presenting and processing machinery (red X) and recruitment of immune suppressive immune cells, such as myeloid suppressor cells and FoxP3⁺ CD4⁺ (Tregs).

Unique tumor microenvironment of HPV+ HNSCCs

HPV-associated HNSCC (HPV+ HNSCC) arise from the crypts within lymphoid tissue of the tonsil and base of tongue, and most of these cancers can be distinguished from tobacco-related head and neck cancers by the characteristic infiltration of lymphocytes within the stroma and tumor nests^21-23. Nevertheless, despite this profound inflammatory response within the tumor microenvironment (TME), the virus and its associated cancer cells can evade immune surveillance, persist, and grow. Various mechanisms have been proposed for the resistance of human solid tumors to immune recognition and obliteration, including the recruitment of regulatory T cells (Tregs), myeloid derived suppressor cells (MDSC), and local secretion of inhibitory cytokines. Recent evidence suggest that tumors co-opt physiologic mechanisms of tissue protection from inflammatory destruction through the upregulation of immune inhibitory ligands and has provided a new perspective for understanding tumor immune resistance. Antigen-induced activation and proliferation of T cells are regulated by the temporal expression of both co-stimulatory and co-inhibitory receptors and their cognate ligands. Coordinated signaling through these receptors modulates the initiation, amplification, and subsequent resolution of adaptive immune responses. In the absence of co-inhibitory signaling, persistent T cell activation can lead to excessive tissue damage in the setting of infection as well as autoimmunity. However, in the context of cancer, in which immune responses are directed against antigens specifically or selectively expressed by tumor cells, immune checkpoints can represent major obstacles to the generation of clinically meaningful anti-tumor immunity. Therefore, efforts have been made in the clinical arena to investigate blockade of immune checkpoints as novel therapeutic approaches to cancer.

PD1 upregulation in HPV+ HNSCCs: Rationale for combinatorial strategies with anti-PD1 therapy

Our group reported that the PD-1:PD-L1 immune checkpoint pathway facilitates persistent HPV infection and subsequent development of HPV-HNSCCs²⁴. Our findings support a model in which the PD-1:PD-L1 immune checkpoint pathway becomes induced as an adaptive immune resistance mechanism of tumor against host. A multi-institutional first-in-human (FIH) phase I study evaluating blocking anti-PD-1 therapy in HNSCC patients demonstrated single agent activity in HPV-HNSCC²⁵. Subsequently, Phase III clinical trials were successfully completed^26-28. KEYNOTE-048 demonstrated that the combination of chemotherapy with pembrolizumab improved overall survival (OS) compared with chemotherapy plus cetuximab in the total population (13 months vs 10.7 months; HR, 0.77; p=0034). Among patients with PD-L1 combined positive score (CPS) ≥20, OS was superior at 14.9 months as compared to 12.3 months, for pembrolizumab monotherapy compared with chemotherapy plus cetuximab. Among all patients treated with pembrolizumab, the inclusion of chemotherapy led to a higher objective response, with a higher benefit observed in the HPV+ HNSCCs²⁹. In the CheckMate 141 trial, p16+ cancers demonstrated a higher OS survival benefit with nivolumab as compared to investigator’s choice chemotherapy (9.1 months versus 4.4 months; HR 0.56) as compared to the ] p16 negative cancers (OS of 7.5 months versus 5.8 months; HR 0.73)²⁸. These clinical trials highlight that the PD1:PDL1/PDL2 axis is upregulated in HPV+ cancers as compared to HPV negative cancers and additional benefit \may be observed in the combinatorial setting.

HPV-targeted Immunotherapies

There are several on-going clinical efforts that leverage the host’s immune system to treat HPV+ HNSCC. Clinical trials with nucleic acid-based and peptide/protein-based vaccines, pathogen-derived delivery platforms, and cellular therapies, including genetically modified or chimeric antigen receptor T cells (CAR-T) that target the HPV viral proteins, E6 and E7, are summarized in Table 1. Given that there are comprehensive reviews on DNA and peptide therapeutic vaccines (reviewed in ^30-33), this review will provide a brief summary of HPV cancer vaccines and focus on innovative HPV-targeted biologics.

Table 1:

HPV targeted HNSCC clinical trials¹

DNA Vaccines
Drug(s)	Device/ Delivery route/	Phase	# Of Participants	Title	Sponsor	Start Date	Completion Date2	NCT#
INO-3112	CELLECREA/Intramuscular (i.m.)3	I and II	22	Prospective Study of HPV Specific Immunotherapy in Participants with HPV Associated Head and Neck Squamous Cell Carcinoma	Inovio Pharmaceuticals	13 Aug 2017	Jan 2017	NCT02163057
MEDI0457 w Durvalumab	CELLECTRA 2000/i.m.	Ib and IIa	35	A Phase 1b/2a, Multi-Center Open-Label Study to Evaluate the Safety and Efficacy of Combination Treatment with MEDI0457 (INO-3112) and Durvalumab (MEDI4736) in Patients with Recurrent/Metastatic HPV Associated Head and Neck Squamous Cancer	Medimmune LLC	26 June 2017	March 2021	NCT03162224
GX-188E and GX-17 w Nivolumab	TriGrid Delivery System/i.m.	II	21	HPV 16-positive and/or HPV 18-positive Recurrent and/or For Patients with Metastatic Head and Neck Cancer to Evaluate GX-188E DNA Vaccination, GX-I7 and Nivolumab Combination Therapy Open-label Phase 2 Clinical Trial with Lead-In Safety Cohort (TRINITY)	Yonsei University	April 2022	March 2026	NCT05280457
GX-188E and GX-17 w Pembrolizumab	TriGrid Delivery System /i.m.	II	25	Open-label Phase II Trial of the Combination of GX-188E Vaccination, GX-I7 and Pembrolizumab in Patients with Advanced, Non-Resectable HPV Type 16 and/or 18 Positive Head and Neck Cancer (GENUINE)	Yonsei University	April 2022	Dec 2026	NCT05286060
UCPVax w Atezolizumab	CELLECTRA/i.m.	II	47	A Phase II Study Evaluating the Interest to Combine UCPVax a CD4 TH1-inducer Cancer Vaccine and Atezolizumab for the Treatment of Human Papilloma Virus Positive Cancers (VolATIL)	Centre Hospitalier Universitaire de Besancon with Roche Pharma AG	18 Feb 2020	Feb 2023	NCT03946358
RNA Vaccines
Drug(s)	Device/ Delivery route/	Phase	# Of Participants	Title	Sponsor	Start Date	Completion Date	NCT#
BNT113 w Pembrolizumab	Intravenous (i.v.)	II	285	AHEAD-MERIT An Open Label Phase II Randomized Trial of BNT113 in Combination with Pembrolizumab Versus Pembrolizumab Monotherapy as a First Line Therapy in Patients with Unresectable Recurrent, or Metastatic Head and Neck Squamous Cell Carcinoma (HNSCC) Which is Positive for Human Papilloma Virus 16 (HPV+) and Expresses PD-L1 (AHEAD-MERIT)”	BioNTech SE	07Jan 2021	May 2025	NCT04534205
BNT113 Plus Anti-CD40	Intradermal, (i.d.)	I and II	44	Therapeutic HPV Vaccine Trial +/− Anti-CD40 in HPV-driven Squamous Cell Carcinoma (HARE-40)	University of Southampton	11 April 2017	Oct 2024	NCT03418480
Peptide or Protein Vaccines
Drug(s)	Device/ Delivery route/	Phase	# Of Participants	Title	Sponsor	Start Date	Completion Date	NCT#
MAGE-A3 and HPV16	Subcutaneous (s.c.)	I	17	A Phase 1 Open Label, Dose Escalation Study to Evaluate the Effect of Four Doses of MAGE-A3/HPV16 Trojan Peptides 0001 and 0002 Administered Subcutaneously in Combination with Montanide and GM-CSF on Immunological Response, Safety, Tolerability, and Preliminary Efficacy in Patients with Squamous Cell Carcinoma of the Head and Neck	University of Maryland and NIDCR	Nov 2005	Oct 2012	NCT00257738
ISA101b (w IMRT, Pembrolizumab, Cisplatin)	Subcutaneous/s.c	II	50	Phase II Study Evaluating HPV-16 Vaccination (ISA101b) and Pembrolizumab Plus Cisplatin Chemoradiotherapy for "Intermediate Risk" HPV-16 Associated Head and Neck Squamous Cell Carcinoma (HNSCC)	University of Pittsburgh Medical Center Merck, ISA Pharma	06 July 2020	June 2024	NCT04369937
DPX-E7	n.d.	Ib and II	11	A Phase Ib/II Trial to Test the Safety and Efficacy of Vaccination with HPV16-E711-19 Nanomer for the Treatment of Incurable HPV16-Related Oropharyngeal, Cervical, and Anal Cancer In HLA-A*02 Positive Patients	Dana Farber	Dec 2016	Dec 2023	NCT02865135
P16_37-63 peptide w Montanide® ISA-51 VG	i.d.	I	11	Pilot Study on Concurrent Cisplatin-based Chemotherapy Combined with Vaccination Therapy with the P16_37-63 Peptide in Patients With HPV- and p16INK4a-positive Cancer (VICORYX-2)	Oryx GmbH & Co KG	June 2015	May 2017	NCT02526316
CUE-101 w Pembrolizumab	Intravenous (i.v.)	I	85	A Phase1, First-in-Human, Open-Label, Dose Escalation and Expansion Study of CUE-101 Monotherapy in Second Line or CUE-101 Combination Therapy with Pembrolizumab in First Line Patients with HPV16+ Recurrent/Metastatic HNSCC (KEYNOTE-A78)	Cue Biopharma with Merck	30 July 2019	Aug 2023	NCT03978689
PDS0101 w Pembrolizumab	na	II	95	A Phase II, Open-Label, Multi-Center Study of PDS0101 and Pembrolizumab (KEYTRUDA®) Combination Immunotherapy in Subjects with Recurrent and/or Metastatic HNSCC and High-Risk HPV16 Infection (VERSATILE002)	PDS Biotechnology Corp with Merck	29 March 2021	July 2024	NCT04260126
PepCan	na	I/II	20	A Phase I/II Clinical Trial of PepCan in Head and Neck Cancer Patients	University of Arkansas	Jan 2019	Oct 2021	NCT03821272
Pathogen-derived Vaccines
Drug(s)	Device or Delivery Route	Phase	# Of Participants	Title	Sponsor	Start Date	Completion Date	NCT#
HB-201, HB-202	i.v.	I and II	200	A Phase I/II Study of TheraT® Vector(s) Expressing Human Papillomavirus 16 Positive (HPV 16+) Specific Antigens in Patients with HPV 16+ Confirmed Cancers	Hookipa Biotech	11 Dec 2019	June 2025	NCT04180215
ADXS11-001 (ADXS-HPV)	n.a.	II	15	Window of Opportunity Trial of Neoadjuvant ADXS 11-001 Vaccination Prior to Robot - Assisted Resection of HPV-Positive Oropharyngeal Squamous Cell Carcinoma	Andrew Sikora w Advaxis Inc	Dec 2013	Aug 2023	NCT02002182
Cell-based Therapies
Drug(s)	Device/ Delivery route/	Phase	# Of Participants	Title	Sponsor	Start Date	Completion Date	NCT#
RTX-321	i.v.	I	63	A Phase 1 Study of RTX-321 for the Treatment of Patients with Advanced Malignancies Associated with Human Papillomavirus-16 Infection	Rubius Therapeutics	08 April 2021	Sept 2023 Sept 2023	NCT04672980
SQZ-eAPC-HPV W Pembrolizumab	i.v.	I and II	60	Phase 1/2, First-in-Human, Multicenter, Open-Label Study of SQZ-eAPC-HPV as Monotherapy and in Combination with Immune Checkpoint Inhibitor(s) in patients w HPV16+ Recurrent, Locally Advanced, or Metastatic Solid Tumors	SQZ Biotechnologies	24 March 2022	March 2024 March 2024	NCT05357898
RPTR-168 (PRIME-102)	i.v	I	24	A Phase 1/2 Open-Label Multi-Center Study to Characterize the Safety and Tolerability of RPTR-168 in Patients with Relapsed/Refractory HPV-16 E6/E7 Positive Tumors and Melanoma	Repertoire Immune Medicines	10 May 2021	Ma May 2024	NCT04762225
Allogeneic CD4	i.v.	I and II	24	A Phase I Clinical Trial Assessing the Safety, Feasibility and Immunologic Correlates of Allogeneic HPV-specific Cluster of Differentiation 4 (CD4)+ T Cells in Advanced HPV16-associated Malignancies	Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins and PapiVax Biotech	15 June 2022	Dec 2024 March 2024	NCT04713046
HPV16-E7	i.v	I and II	180	A Phase I/II Trial of T Cell Receptor Gene Therapy Targeting HPV-16 E7 for HPV-Associated Cancers	NCI	27Jan 2017	Jan 2026	NCT02858310
CRTE7A2-01 TCR-T cell	i.v.	I	12	A Phase I Study to Evaluate the Safety, Tolerance and Efficacy of CRTE7A2-01 TCR-T Cell for HPV16 Positive AdvancedCervical or Head and Neck Cancers	Corregene Biotechnology	Dec 2021	Dec 2024 Dec 2024	NCT05122221
HPV E6-specific TCR-T cells	i.v.	I	20	wo-Arm Open-Labeled Trial of HPV-E6-Specific TCR-T Cells with or without Anti-PD1 Auto-secreted Element in the Treatment of HPV-Positive Head and Neck Carcinoma or Cervical Cancer	Xinqiao Hospital of Chongqing and TCRCure Biopharma Ltd.	01 Sept 2018	Aug 2021	NCT03578406

Footnotes:

¹ClinicalTrials.gov search criteria included the following terms: Carcinoma of Head and Neck, Neoplasm, Human papilloma virus and was performed on May 19, 2022.

²Estimated study completion date.

³Intramuscular (i.m.), subcutaneous (s.c.), intradermal (i.d.), intravenous (i.v.)

⁴not disclosed, n.d.

Nucleic acid vaccines

DNA and RNA-based HPV therapeutic vaccines (Table 1) use non-replicating DNA or RNA that express HPV16 or 18 E6 and/or E7 sequences. Nucleic vaccines depend on the hosts’ cells to translate the delivered DNA sequence into proteins that are then processed, presented, and recognized by the host immune system as foreign, to generate systemic HPV-specific T cell responses. Nucleic acid vaccines have several advantages over peptide or protein vaccines, among these are prolonged antigen expression, ability for repeat dosing, and feasible and cost-effective manufacturability of plasmid DNA. To date however, DNA vaccines have demonstrated modest therapeutic efficacy, which may be due, in part, to low transfection efficiency in vivo and their inherent inability to replicate. Thus, pre-clinical research and clinical trials have focused on strategies to enhance DNA vaccine potency, such as diversifying immunogenic antigens, co-administration of adjuvant immune-enhancing agents like cytokines, and costimulatory molecules, and investigating routes and methods of vaccine delivery.

Strategies to generate more robust tumor-specific cytotoxic T lymphocyte (CTL) responses thematically align with immune synapse signaling between two cell types, T cells and antigen-presenting cells (APC). These temporally sequenced signaling pathways are commonly referred to as Signals 1, 2, and 3. Antigenic peptides presented on major histocompatibility complexes (MHC) initiate Signal 1. Viral peptides and proteins derived from E6 and E7 have been the primary focus of HPV cancer vaccines, but the delivery of additional antigens, such as tumor-associated antigens (TAAs) or neoantigens, in combination with E6 and E7, has the potential to generate a broader and more diverse anti-tumor T cell response. MAGE-A3 (NCT00257738), universal cancer (UCP) peptides, and telomerase reverse transcriptase (TERT)-derived peptides are some examples of non-HPV peptides and proteins currently being developed as vaccines. UCPVax (NCT03946358, VolATIL) and TERT-targeting trials are focused to elicit CD4 helper T cell responses³⁴ to facilitate stronger tumor-specific CD8 T cell responses.

The addition of costimulatory molecules and/or cytokines that enhance T cell activation are also being evaluated. For example, a combination of DNA plasmids, one with HPV16 E6 and E7 and one with HPV18 E6 and E7 sequences (VGX-3100) together with a DNA plasmid encoding human IL-12 (INO-9012) is under development. Medimmune LLC completed a Phase 1b/2a trial to evaluate the safety and efficacy of INO 3112/ MEDI0457 (NCT05280457) in combination with an anti-PD-L1 monoclonal antibody. Genexine Inc (GEX-188) has combined a DNA plasmid expressing HPV16 E6 and E7 Fms-like tyrosine kinase-3 ligand (FLT3L) with a long-acting interleukin IL-7 (GX-17, huIL-7 hyFc™). Cytokines, such as IL-12 or IL-7, directly or indirectly, activate antigen-specific T cells, as well as other immune cell types, like Natural Killer (NK) cells.

Optimizing the route of nucleic acid delivery systems have also led to improved T cell responses. Inovio delivery platform consists of intramuscular needle injection followed by electroporation with the CELLECTRA™ device (NCT02163057). Intramuscular administration delivery routes are commonly utilized for prophylactic vaccines but may not be the most effective route for therapeutic vaccines, which aim to generate tumor specific CTLs rather than neutralizing antibodies. In a preclinical study, our group compared intramuscular (IM) and intratumoral (IT) routes with the same E7 vaccine formulation in the TC-1 preclinical tumor model. IT vaccine administration delayed tumor growth more effectively than IM delivery, which correlated with higher frequencies of activated E7-specific CD8 T cells and evidence of epitope spreading beyond the vaccine targeted E7 antigen¹⁹.

For nucleic acid vaccines, advancements are also being explored with the delivery method, including electroporation devices used for gene transfer into tumor cells. Preclinical pipelines are, for example, developing techniques and devices that are more effective in delivering DNA plasmids by improving delivery, dosage, and limiting local tissue damage. One area of improvement in electroporation is to assess the specific electrochemical properties of the tumor and then tailor the electronic pulses using impedance spectroscopy in real time³⁵. This delivery approach is particularly germane for IT vaccine delivery, when electroporating tumors composed of heterogeneous cell type admixtures. Syngeneic murine tumor models are useful models for immunotherapy development because of their intact immune system, but the tumors are homogeneous at the cellular level, which mimics the complex cellularity of human cancers.

RNA vaccines

Prophylactic SARS-CoV2 vaccine helped pave the regulatory approval pathway for therapeutic mRNA vaccine platforms. Therapeutic mRNA cancer vaccines were under development when BioNTech redirected their resources to meet the unprecedented medical need for COVID vaccines. Although an HPV trial (BNT-113) recently started enrolling, interim data on the mRNA FixVac vaccine demonstrated that the vaccine was well tolerated, generated anti-tumor CD4 and CD8 responses to the different mRNA-encoded antigens resulting in durable objective responses in checkpoint inhibitor experienced patients with unresectable melanoma³⁶. To enhance delivery to antigen presenting cells (APC), the BioNTech therapeutic platform encapsulates mRNA in specially formulated liposome compositions with a specific charge and diameter to increase trafficking to dendritic cells³⁷ after systemic intravenous injection. BNT-113, a mRNA-based vaccine with HPV16 E6 and E7 oncoprotein sequences, is being evaluated in patients with HPV16+ head and neck cancers in combination with anti-CD40. (NCT03418480). Agonist CD40 antibodies stimulate immunostimulatory cell surface receptors on dendritic cells resulting in APC maturation and promoting anti-tumor T cell responses.

Peptide or protein vaccines

Therapeutic HPV vaccines composed of peptides, proteins or fusion proteins derived from HPV16 and 18 E6 and E7 have been investigated for over two decades³⁸. Antigens, peptides or proteins, present in the cytoplasm of dendritic cells lead to the generation of antigen-specific CD8 T cells, while extracellular antigens are processed and presented via different pathways to generate antigen-specific CD4 T cells. Ideally therapeutic vaccines generate both cytotoxic CD8 T cells and CD4 helper T cells. The immunogenicity and efficacy of peptide or protein vaccines depends on many factors, including the different antigen processing pathways in APCs, which is a critical aspect of effective signaling from the peptide-MHC : T-cell receptor (TCR) complex. To improve tumor-specific T cell responses, different therapeutic approaches are being developed to enhance bi-directional signaling at the immune synapse, including engaging the innate immune system via adjuvants, and inclusion of co-stimulatory molecules or cytokines to enhance anti-tumor T cell effector function.

The delivery of a vaccine composed of synthetic long peptides (SLP) from HPV16 E6 and E7 with the adjuvant, Montanide ISA-51 (NCT04369937), has shown limited efficacy in advanced cancers, potentially due to inability to elicit an adequate response in the immunosuppressive HNSCC tumor microenvironment (TME). Logically, several trials utilizing combinatorial strategies have been initiated, including a peptide vaccine with an agonist monoclonal antibody directed to CD137, a costimulatory molecule (i.e., Utomilumab) and checkpoint inhibitory molecules, PD-1/PD-L1 (e.g., nivolumab (NCT0246892)^39,40 or Cemiplimab (NCT04260126)). Recently, the combination of ISA101b with Cemiplimab was given fast track designation for recurrent and metastatic HPV16 positive oropharyngeal cancers. SLP vaccine efficacy depends on the presence of necessary and functional antigen processing and presentation machinery (APM) in the host, such as transporter associated with antigen processing (TAPs) and immunoproteasome components. However, a common immunosuppressive strategy of tumors is to downregulate APM function.

Thus, to more efficiently activate T cells and generate anti-tumor responses, protein complexes that mimic peptide-MHC complexes (pMHC) are being explored. This strategy counters the inherent instability of a peptide-MHC complex, as well circumvents the downregulation of APM by the host. CUE BioPharma has developed a modular fusion protein design, CUE-101, which includes an MHC class I protein, HLA-A*02, and IL-2 variant on a human IgG4 Fc scaffold complexed to the E7 peptide_11-20 - β2 macroglobulin (β2M) fusion protein to activate HPV16 E7-specific CD8 T cells⁴¹. This protein complex aims to enhance multiple signaling components to generate cytotoxic anti-E7 CD8 cells. APC signaling is enhanced by the pMHC- β2M complex, which mitigate issues of high affinity and rapid peptide dissociation, and the selective delivery of IL-2 protein to E7-CD8 T cells in juxtracrine increases proliferation of an antigen-specific T-cell population.

Many HPV targeted therapeutics are also evaluated in combination with immune checkpoint inhibitors, such as Nivolumab, Pembrolizumab, Durvalumab, Atezolizumab or Cemiplimab, which are highlighted in Table 1.

Pathogen- or viral-derived vaccine platforms

The human immune system has developed a multilayered protection schema to defend itself from pathogens, such as bacteria and viruses. To leverage many of these pathogen-derived immune stimulating mechanisms, researchers have utilized pathogen-based mimicry by simultaneously altering pathogens to display and present HPV antigens and disarm their detrimental pathogenic features. Pathogen-derived approaches have an immune advantage because pathogens activate both the innate and adaptive immune systems. Cytokines elaborated from pathogen-triggered innate immune systems can facilitate robust adaptive immune responses. In contrast to DNA and peptide vaccines that augment immunogenicity by expressing or including a single cytokine, pathogen-derived vaccines stimulate a multi-faceted array of cytokines creating a favorable milieu for T-cell activation. Advaxis, for example, developed a genetically modified strain of listeria monocytogenes bacteria that expresses HPV16 protein E7⁴². Bacteria expressing E7 antigen are phagocytosed and immunoproteasome-derived E7 peptides are cross-presented to T-cells in a cytokine storm that is generated by the hosts’ innate immune system providing strong adjuvating stimuli (ADXS 11-001, NCT02002182). Using a different pathogen, Hookipa Biopharma has developed modified viral arenavirus vectors, lymphocytic choriomeningitis (LCM) virus or Pichinde virus (PICV), to infect dendritic cells with HPV16 E6 and E7 fusion proteins. Non-lytic, replication competent LCM vectors infects dendritic cells resulting in their activation and maturation evidenced by upregulation of costimulatory molecules, CD80/86, and expression of inflammatory cytokines and chemokines, and processing and presentation of HPV antigens to CD8 T cells generating anti-HPV CTLs. A potential disadvantage of using modified arenaviruses-derived vectors is the concern that the modified LCM viral vector is capable of replication, albeit to a much lesser extent than wild-type LCMV⁴³.

Cellular therapies

In the last decade, development of cellular based therapies, such as dendritic cell vaccines, adoptive cell therapy (ACT), genetically engineered T cells using chimeric antigen receptors (CAR), and transgenic T-cell receptors (T-TCR) have made significant advances⁴⁴. Autologous therapies utilize a patients’ own immune cells, while allogeneic therapies use cells from a donor; both approaches modify immune cells ex vivo under strict regulatory criteria. The latter approach allows a clinician to treat more than one patient as donor cells are often used to generate a master cell bank. Autologous cell therapies mitigate risk of tissue rejection (e.g., graft vs host disease), but present challenges related to the available quantity and quality of the initial cell product, which reflects a patient’s overall health and stage of disease. The over-arching aim of cellular therapies is to generate a diverse and robust anti-tumor immune response by providing spatially intact signaling APC or TCR juxtracrine complexes to the immune synapse.

Dendritic cell (DC) vaccines, for example, provide improved APC signaling by ex vivo maturation and loading of autologous DCs with HPV antigens and then administering HPV-peptide loaded MHC (pMHC) APCs back to the patient. The goal of this strategy is to broaden the immune repertoire and bolster the quality of tumor-targeting T cells by enhancing APC signaling. The strategy of providing stronger APC signaling has generated several additional novel approaches.

Artificial APC (aAPC) form the basis for one of these approaches. CD34+ stem cells are exposed to cytokines to become red blood cells (RBCs), which are then transduced with constructs to express an HPV16 E7 peptide bound HLA*A02 complex, in addition to 41BBL and membrane-bound IL-12 (RTX-321, NCT04672980). The genetically engineered RBCs express the transgenes after enucleation, even though the genetic modifications are no longer present in the final cell product. This allogeneic therapy provides multiple signals to stimulate and activate T cells, including the E7 bound peptide-HLA*A02 complex, a costimulatory molecule, 4-1BBL and IL-12⁴⁵. Squeeze Bio is developing an autologous approach with a similar theme. A patient’s RBCs are exposed to HPV peptides or proteins under specific microfluidic conditions creating an ‘activating antigen carrier’ (ACC)⁴⁶. “Squeezing” RBCs not only loads the antigenic payload in an MHC context, but also results in flipping phosphatidylserine from the inner to the outer leaflet of the plasma membrane, essentially signaling that the RBCs should be removed from circulation. As result, macrophages/monocytes (APCs) phagocytose the pHPV-MHC expressing ACCs in vivo resulting in antigen processing and presentation, ultimately priming anti-tumor T cell responses.

To enhance anti-tumor T cell responses, therapies are also being developed to mimic T-cell signaling. Allogenic therapies, for example, obtain donor-derived HPV-specific CD8 T cells from HPV-vaccinated related donors’ PBMCs or bone marrow transplanted material (NCT04713046). HPV16 vaccinated donors will likely have generated a diverse repertoire of HPV16-specific T cells, which will be enriched prior to infusing these cells into a patient. Repertoire Immune Medicines is developing T-cell therapies targeting HPV16 tumors by delivering T-cell targeted IL-12 and T cells primed for E6/E7 that are also decorated with T-cell targeted IL-12. To directly prime anti-tumor T cells and avoid IL-12 toxicity, the two subunits of IL-12 are fused to anti-human CD45⁴⁷, which directs this cytokine to T-cells resulting in stronger immunogenicity. In addition, autologous T cells are primed with tumor-associated antigens (TAAs) (i.e., PRAME, MAGE-A4 and survivin) (RPTR-168 (also known as PRIME-102). Priming T-cells with multiple tumor antigens, in addition to HPV E6 and E7, in concert with IL-12 tethered to the surface of antigen primed T cells will likely broaden the repertoire and enhance the cytotoxic T-cell phenotypes.

Clinical trials using genetically engineered T cells such as chimeric antigen receptor (CAR)-T, and T-cell receptors (TCR) have been successful in hematopoietic cancers but have met limited success in solid tumors ^48,49. In HPV positive cancers, E6 and E7 are the rationale targets of choice as discussed previously. However, E6 and E7 are not extracellular proteins and consequently cannot be targeted with CAR-T cells. Hinrich and coworkers have identified high-avidity TCRs that recognize HPV16 E6_29-38 and HPV16 E7_11-19, presented by HLA*A02^50,51. Data has recently been reported for a subset of patients who received TCR-HPV16 E7 therapy. Six out of 12 patients had partial responses (PR) with complete regression of some tumors demonstrating the potential of HPV engineered TCR therapies. Interestingly, the aggregate translational studies from these two HPV TCR trials provides tremendous insight to tumor resistance mechanisms when metastatic tumors appeared after tumor regression of the primary lesion. In four patients, the metastatic lesions had genomic alterations that impaired the presentation of the target antigen with mutations in HLA*A02, and biallelic alterations in B2M. Interestingly, in the TCR-HPV16-E6 trial, a similar tumor escape mechanism was observed, the loss of HLA*A02, in order to evade the host immune system.

Thus, it appears that in the face of effective TCR therapy, tumor resistance mechanisms evolve specific escape routes. For TCR therapies, the host tumor cells selectively evolve mechanisms that result in defects in peptide presentation, such as mutation in the specific HLA allele necessary to present the HPV E7 peptide. The early successes of HPV-TCR therapies have also paved the way to develop TCR therapies for additional HLA subtypes that bind HPV peptides with high affinity. Diversifying TCR therapies to include additional HPV peptides and HLA alleles have several benefits. First, use of a variety of HLA allelic subtypes allows a TCR treatment modality to include ethnically diverse patients. HLA-A02 is a predominate in Caucasians, and not as prevalent in other ethnicities, which limits broader patient application. Second, a repertoire of TCRs for E6 and E7 presented by different HLAs could allow for combinatorial TCR therapy, or sequential therapy to counter inevitable tumor resistance mechanisms.

Conclusions

HPV immunotherapy trials have expanded rapidly over the last decade, but these approaches are still in early clinical development. Patients with HPV-associated HNSCCs have benefited from treatment with checkpoint inhibitors²². However interim data indicate response rates are less than 20%, indicating other therapeutic modalities are urgently needed. Novel HPV-targeted therapies under development focus on improving current response rates. It is now clear that diverse tumor suppressive mechanisms will need to be simultaneously investigated and mitigated as these mechanisms elicit adaptive immune resistance selective to the applied therapy. To date, novel therapies have leveraged the advantages of targeting the E6 and E7 viral peptides/proteins and the available immune monitoring tools. Identification of high avidity pMHC:HPV16/18 complexes may be one avenue to broaden anti-tumor therapies for ethnically diverse patient populations. Effective immunotherapies for HPV-HNSCC will require a combination of therapeutic approaches, such as those discussed here, to overcome adaptative immune resistance as the tumor continuously evolves with exposure to various therapies.