Design and delivery strategies of alphavirus replicon-based cervical cancer vaccines
van de Wall, Marie-Nicole Stephanie
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van de Wall, M-N. S. (2018). Design and delivery strategies of alphavirus replicon-based cervical cancer vaccines. Rijksuniversiteit Groningen.
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HPV-Specific Immunotherapy:
Key Role for Immunomodulators
Chapter 4
Stephanie van de Wall1, Hans W Nijman2 and Toos Daemen1
1Department of Medical Microbiology, University of Groningen,
University Medical Center Groningen, The Netherlands
2Department of Obstetrics & Gynecology, University of Groningen,
4
Abstract
Cervical cancer is the second most common malignancy among women worldwide. The prime causal factor of the disease is a persistent infection with human papillomavirus (HPV) with individuals failing to mount a sufficient immune response against the virus. Despite the current success of HPV16- and 18-specific prophylactic vaccination, established HPV infections and associated neoplasia require therapeutic vaccines with the induction of cellular immunity. The sustained expression of early proteins E6 and E7 from major oncogenic HPV genotypes in cervical lesions are ideal targets for the design of immunotherapeutic strategies. These strategies, particularly subunit vaccines, may require additional help from immunomodulators to enhance HPV-specific cellular responses. This review discusses recent studies, published since 2008, relating to immunotherapeutic strategies against HPV that include immunomodulators. These immunomodulators fall within the category of toll-like receptor adjuvants for innate immune activation, adjuvants directly contributing to adaptive immunity, such as cytokines and costimulatory molecules, and those that target tumor-induced immunosuppressive mechanisms. Using a combination of these strategies with delivery-based approaches may be most beneficial for the success of therapeutic vaccines against HPV-induced neoplasia in the clinic.
4
Introduction
Cervical cancer is the second most common malignancy among women worldwide with an estimation of 500,000 new cases and approximately 250,000 deaths annually.1 It is evident that the causal factor of the disease is a persistent infection with human papillomavirus (HPV).2 There are more than 100 different serotypes of this non-enveloped, double-stranded DNA virus with 15 classified as high-risk types implicated in cancers of the cervix, penis, vulva, anus, vagina and oropharynx.3 HPV16 and 18, belonging to the high-risk types, are associated with more than 70% of cervical cancer cases.4
HPV infects basal epithelial cells exposed at the surface of microlesions that facilitate the transmission of the virus. The life cycle of HPV is closely linked to the differentiation program of keratinocytes with viral particles ultimately produced in desquamated epithelial cells.5 Not all infections with the high-risk types result in cancer progression, as most individuals spontaneously reduce HPV-induced lesions upon infection.6 A key factor in epithelial cell transformation is the integration of viral DNA into the genome of the host, resulting in upregulation of two early oncoproteins of HPV: E6 and E7. E6 and E7 bind to p53 and retinoblastoma protein (pRb) respectively, two tumor suppressor proteins contributing to malignant transformation by preventing cell death and promoting cell proliferation.7
The association of HPV with cervical cancer led to numerous prevention strategies. These include the implementation of screening programs for the precursor lesions of cervical cancer, cervical intraepithelial neoplasia (CIN), using Papanicolaou (Pap) smear tests and other methods such as liquid-based cytology.8 However, primary prevention using prophylactic vaccination seems the most straightforward approach. Prophylactic vaccines against HPV consist of the virus capsid protein L1 assembled into recombinant virus-like particles (VLPs) and prevent almost 100% of infection through induction of neutralizing antibodies against the HPV types employed in the vaccine. Cervarix® (GlaxoSmithKline) prevents infection by HPV16 and HPV18, whereas Gardasil® (Merck), a tetravalent vaccine, contains additional VLPs of HPV6 and HPV11 for protection against benign genital warts. These vaccines precipitate the VLPs in aluminum salt, a commonly used adjuvant. Cervarix is administered in the adjuvant system AS04 additionally containing monophosphoryl lipid A.9
Despite the current prophylactic vaccines, cervical cancer cases are still on the rise with over 80% occurring in developing countries. Standard treatment consists of surgery, radio- and/or chemotherapy. All three procedures contribute to a 90% efficacy rate, but they unfavorably affect healthy tissue and do not always clear
4
oncoproteins are favorable targets for therapeutic immunization strategies due to their sustained expression in cancer tissue and, as previously mentioned, their involvement in malignant transformation. Different strategies consist of HPV vaccine platforms in the form of peptide- or protein-based vaccines, nucleic acid-based vaccines, whole cell-based vaccines, and bacterial or recombinant viral vectors. Recombinant viral vectors possess the advantage of high infection efficiency and expression of encoded antigens with the disadvantage of possible anti-vector responses, limiting the use of these vaccine platforms in humans. In contrast, peptide- and nucleic acid-based vaccines, although lacking the ability to amplify in vivo, are safer and inexpensive due to the low manufacturing costs.11 Protein vaccination has the advantage over peptide-based platforms in delivering all epitopes to antigen presenting cells of the immune system. Despite these advantages, immunotherapeutic strategies are still fraught with the challenge of inducing a sufficient immune response in the clinical setting. This is attributed to the immuno- compromised state of cancer patients and to the various immune escape mechanisms employed by viral-infected cells or tumors.12
It is favorable to assume that immunotherapeutic strategies may require the addition of immunomodulators as a combination therapy for enhancing HPV-specific immunity and providing substantial immunological memory. This refers to, but is not limited to, the more widely used peptide- and protein-based therapeutic immunizations that do not consist of inherent immunomodulatory activity. This is additionally the case for DNA-based strategies (for review, see [13]). On the one hand, the use of immunomodulators in immunotherapy has been described by Berzofsky et
al. to provide a ‘push’ by increasing immunogenicity quantitatively and qualitatively
against an antigen; i.e. providing adjuvant activity.14 Adjuvants can either be classified as immunostimulatory agents, passive depots or vehicles.15 Regarding therapeutic immunization, cellular immunity is paramount with the careful selection of an adjuvant in favor of a type 1 response. Examples of such adjuvants include T helper 1 (Th1) cytokines, costimulatory molecules and certain microbial products (e.g. toll-like receptor ligands). Most of commonly used adjuvants, particularly those already used in human vaccines, such as aluminum salts and oil-in- water emulsions, are associated with an antibody-mediated humoral response and therefore ineffective concerning active immunization against malignancies.16 This is in contrast to passive immuno- therapy using monoclonal antibodies as targeted therapy. On the other hand, immunomodulators may serve to overcome immuno- suppressive mechanisms in the tumor microenvironment and provide a ‘pull’ approach by targeting various cells or molecules involved in immune inhibition.14 The success of cancer vaccines in preclinical trials is often impeded by the moderate responses observed in the clinic. Besides one possible reason being the application of these vaccines in advanced stage cancer patients, negative regulatory mechanisms, such as T cell exhaustion in
4
the tumor microenvironment, may also contribute to a poor success rate.17 Targetingthese mechanisms would enhance the potency of vaccine-induced, antigen-specific immune responses.
In this review, we provide an extensive update of mainly preclinical studies (since 2008) relating to immunotherapeutic strategies against HPV that include immunomodulators. These are divided into the following groups: TLR adjuvants, adjuvants targeting intrinsic adaptive immune activation and those targeting immunosuppression (see Table 4.1-4.3). Other strategies to improve immune responses include the enhancement of antigen immuno- genicity with linkage to carrier proteins (e.g. heat shock proteins), explored mainly within the context of DNA vaccination (for review, see [11]). These strategies are not involved in direct enhancement of immune activation and therefore will not be discussed in this review.
Guiding the Innate and Adaptive Therapeutic Response Towards HPV
Recent immunotherapeutic strategies against HPV-associated malignancy have employed a number of different adjuvants in vaccine formulations for enhancing cellular immunity. Immunological adjuvants improve the quantity and quality of the immune response by targeting both innate and adaptive immunity.17 Innate immunity is characterized by the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). This interaction triggers innate immune responses with recruitment, activation and maturation of antigen presenting cells (APCs). Through cytokine production, these cells play a crucial role in initiating and guiding adaptive immune responses. Adaptive immunity includes humoral (B cell) and cellular (T cell) responses. It is imperative that therapeutic cancer vaccines ultimately target the CD4+ and CD8+ cytotoxic T cell adaptive immune components for an effective tumor-specific response with efficiency in spontaneously clearing HPV infection in the individuals with impaired T cell immunity. For this purpose, a strong, T helper (Th)- 1-driven, type 1 response is required, characterized by the cytokine profile (mainly interferon (IFN)-y and interleukin (IL-12)). In the case of HPV-specific immunotherapy, immunological adjuvants eliciting potent E6- and/or E7-specific cytotoxic CD8+ T cell responses are appropriate for antitumor therapeutic efficacy. Most preclinical studies testing vaccine formulations make use of the TC- 1 tumor model, comprising of epithelial cells of C57BL/6 mice cotransformed with oncogenes HPV16 E6 and E7 and c-Ha-ras.18
4
Table 4.1. Overview of HPV immunotherapeutic studies with inclusion of TLR ligands.
TLR Vaccine Formulation Vaccine Type Immunomodulator[s]Additional Study Type Key Result[s] of Study Ref.
TLR 3
HPV16 E7 protein or long HPV16 E7 peptide + poly[I:C] Peptide/ protein - Preclinical Rapid and extensive increase in antigen-specific CD8+ T cells with cluster immunization in mice [38] TriVax [includes poly[I:C] + aCD40 mAb] Peptide aCD40 mAb Preclinical Durable E7-specific responses elicited eradicating established tumors in mice [39] HPV16 E7 peptide + poly[I:C] Peptide PADRE Preclinical Improved E7-specific CD8+ T cell responses and antitumor effects in mice than either PADRE or poly[I:C]
alone
[41] HPV E7 peptide + poly[I:C] Peptide Cyclophosphamide Preclinical E7-specific [1/10th] CD8+ T cells induced with effective antitumor responses in the advanced disease
setting and significantly longer survival of mice with addition of cyclophosphamide
[42] Pentarix + poly[I:C] Protein - Preclinical E7-specific CD8+ T cells increased drastically with cluster vaccination with complete regression of large,
established TC-1 tumors in mice
[43] HPV16 L1 capsomeres + PolyICLC Protein - Preclinical HPV16-specific Th1 immune responses induced with strong anti-HPV16 L1 antibody responses compared
to injected of capsomeres alone in rhesus macaques
[45] HPV16 E7 protein + poly[I:C] Protein CAF01 Preclinical Induced higher cytotoxic CD8+ T cells and significantly reduced TC-1 tumor growth in mice [46]
TLR 4
HPV16 E7 peptide + CGN Peptide - Preclinical Significant enhancement of E7-specific responses with prophylactic and therapeutic antitumor responses elicited in mice
[49]
EDA-HPVE7 Protein Poly[I:C] or
cyclophosphamide + CpG-DOTAP
Preclinical Treatment of EDA-HPVE7 with pIC achieved complete eradication of large established tumors with a CD8+ T cell response; EDA-HPVE7 and CpG-DOTAP with low dose cyclophosphamide rejected large tumors in 50% of mice; EDA-HPVE7 eradicated tumors only with intratumor administration
[50]
TLR7/8
HPV fusion protein + imiquimod Protein - Clinical Significant infiltration of CD4+ and CD8+ T cells; Complete lesion regression in 63% of patients [59] CRT/E7 + imiquimod DNA Calreticulin Preclinical Increased E7-specific CD8+ T cell responses and decreased MDSCs in the tumor microenvironment
concomitant with antitumor effects and prolonged survival in mice
[64]
TLR 9
pBSC/E7GGG.LAMP + ODN1826 DNA - Preclinical Systemic delivery of high doses of CpG ODN reduced activation of CD8+ T cells in mouse spleen, but enhanced antitumor effects
[71] HPV16 E7SH[contains optimized CpG cassette] DNA - Preclinical Highly optimized CpG motifs enhanced IFN-y and granzyme B responses with more potent tumor
regression; Antitumor effects were synergistically increased by 10-fold with electroporation
[72] HPV16 E7 peptides + ODN 1826 Peptide - Preclinical Enhanced humoral and celluar responses close to or comparable to those elicited with tattoo injection
of E744-62; Administration of E749-57 + CpG decreased CTL responses and protection against TC-1 tumor challenge
[73]
HPV16 E7 long peptide + CpG 1826 Peptide - Preclinical CpG1826 required for generation of E7-specific CD8+ T cell responses in mice further enhanced with electroporation
[74] E7 + CpG ODN Protein - Preclinical Approx. 5-fold increase in IFN-y-producing CD8+ T cells in the genital mucosa of mice with intravaginal
instillation of CpG ODN after s.c. injection of E7; CpG ODN increased the percentage of mice with complete regression of large tumors from 20% to 75%
[75]
4
Table 4.1. Overview of HPV immunotherapeutic studies with inclusion of TLR ligands.
TLR Vaccine Formulation Vaccine Type Immunomodulator[s]Additional Study Type Key Result[s] of Study Ref.
TLR 3
HPV16 E7 protein or long HPV16 E7 peptide + poly[I:C] Peptide/ protein - Preclinical Rapid and extensive increase in antigen-specific CD8+ T cells with cluster immunization in mice [38] TriVax [includes poly[I:C] + aCD40 mAb] Peptide aCD40 mAb Preclinical Durable E7-specific responses elicited eradicating established tumors in mice [39] HPV16 E7 peptide + poly[I:C] Peptide PADRE Preclinical Improved E7-specific CD8+ T cell responses and antitumor effects in mice than either PADRE or poly[I:C]
alone
[41] HPV E7 peptide + poly[I:C] Peptide Cyclophosphamide Preclinical E7-specific [1/10th] CD8+ T cells induced with effective antitumor responses in the advanced disease
setting and significantly longer survival of mice with addition of cyclophosphamide
[42] Pentarix + poly[I:C] Protein - Preclinical E7-specific CD8+ T cells increased drastically with cluster vaccination with complete regression of large,
established TC-1 tumors in mice
[43] HPV16 L1 capsomeres + PolyICLC Protein - Preclinical HPV16-specific Th1 immune responses induced with strong anti-HPV16 L1 antibody responses compared
to injected of capsomeres alone in rhesus macaques
[45] HPV16 E7 protein + poly[I:C] Protein CAF01 Preclinical Induced higher cytotoxic CD8+ T cells and significantly reduced TC-1 tumor growth in mice [46]
TLR 4
HPV16 E7 peptide + CGN Peptide - Preclinical Significant enhancement of E7-specific responses with prophylactic and therapeutic antitumor responses elicited in mice
[49]
EDA-HPVE7 Protein Poly[I:C] or
cyclophosphamide + CpG-DOTAP
Preclinical Treatment of EDA-HPVE7 with pIC achieved complete eradication of large established tumors with a CD8+ T cell response; EDA-HPVE7 and CpG-DOTAP with low dose cyclophosphamide rejected large tumors in 50% of mice; EDA-HPVE7 eradicated tumors only with intratumor administration
[50]
TLR7/8
HPV fusion protein + imiquimod Protein - Clinical Significant infiltration of CD4+ and CD8+ T cells; Complete lesion regression in 63% of patients [59] CRT/E7 + imiquimod DNA Calreticulin Preclinical Increased E7-specific CD8+ T cell responses and decreased MDSCs in the tumor microenvironment
concomitant with antitumor effects and prolonged survival in mice
[64]
TLR 9
pBSC/E7GGG.LAMP + ODN1826 DNA - Preclinical Systemic delivery of high doses of CpG ODN reduced activation of CD8+ T cells in mouse spleen, but enhanced antitumor effects
[71] HPV16 E7SH[contains optimized CpG cassette] DNA - Preclinical Highly optimized CpG motifs enhanced IFN-y and granzyme B responses with more potent tumor
regression; Antitumor effects were synergistically increased by 10-fold with electroporation
[72] HPV16 E7 peptides + ODN 1826 Peptide - Preclinical Enhanced humoral and celluar responses close to or comparable to those elicited with tattoo injection
of E744-62; Administration of E749-57 + CpG decreased CTL responses and protection against TC-1 tumor challenge
[73]
HPV16 E7 long peptide + CpG 1826 Peptide - Preclinical CpG1826 required for generation of E7-specific CD8+ T cell responses in mice further enhanced with electroporation
[74] E7 + CpG ODN Protein - Preclinical Approx. 5-fold increase in IFN-y-producing CD8+ T cells in the genital mucosa of mice with intravaginal
instillation of CpG ODN after s.c. injection of E7; CpG ODN increased the percentage of mice with complete regression of large tumors from 20% to 75%
[75]
4
Table 4.2. Overview of HPV immunotherapeutic studies with inclusion of adaptive immunomodulators Adaptive
Immune Modulators
Vaccine Formulation Vaccine Type
Additional
Immunomodulator[s]Study Type Key Result[s] of Study Ref.
Cytokines TG4001 Virus - Clinical 48% clinical efficacy in patients with CIN 2/3 at 6 months; Correlated with absence of HPV16 transcripts; No enhanced antibody responses to E6 and E7
[79] Nanoparticles Tat- E7/pGM-CSF Peptide Tat from HIV-1 Preclinical Prophylactically and therapeutically protected mice with increase in long-term survival and decrease in tumor
growth; Enhanced CD8+ memory T cell responses
[83] B9 cells [expressing E6, E7 and GM-CSF] Whole cell - Preclinical Protected mice against challenge with MK16 cells or TC-1 cells; MHC class I-restricted and non-restricted
mechanisms were postulated to be involved
[84] P13-H5-FL- SigE7LAMP Virus Sig from LAMP1 Preclinical P13-H5-FL-SigE7LAMP induced prophylactic and therapeutic antitumor efficacy with Flt3L coexpression [85] pE7 + pIL-12 DNA - Preclinical Suppressed E7-specific CTL, antibody and Th cell responses leading to a loss of protection from tumor
challenge
[86] rSFV-eE6,7 + SFV- IL12 Virus - Preclinical Enhanced antigen-specific effector memory CD8+ T cell responses and antitumor responses in 28% of mice
with addition of low doses of SFV-IL12; Higher doses reduced these effects
[87] TC-1-IL-12 Whole cell Gemcitabine Preclinical Combined treatment of gemcitabine and tumor cells led to a 29% reduction of mice with lung metastasis with
MK16 residual disease
[88] E7SH/IFN-y WT; E7SH/IL-2 WT;
E7SH/GM-CSF WT; E7SH/IL-12 WT
DNA Chemokine MIP1-a Preclinical Best CD8+ T cell responses with IFN-y or IL-12 administered 3 days after and MIP1-a administered 5 days prior to E7SH immunization
[89] Chemokines pcDNA-[E7-NT-gp96] + pcDNA-IP-10 DNA Glycoprotein 96 Preclinical Significant suppression in tumor growth with increased numbers of IFN-y and IL-2 in the lymph nodes of mice [90] pcDNA3-CHI-E7 DNA IL-2 Preclinical HLA-A2 mice were protected from TC-1/A2 tumor formation [93] Costimulatory
molecules
DC- and B-cells transduced with CFm40L-complexed adenovirus with E6 and E7[AdE6E7-CFm40L]
Whole cell Gemcitabine Preclinical Enhanced activation of APCs and CTL responses with greater inhibition of tumor growth in mice [94]
B1 cells [expressing E6, E7 and B7-1] Whole cell - Preclinical Protected mice against challenge with MK16 cells [84] E7 + SA-4-1BBL Protein - Preclinical E7 protein-specific CD4+ and CD8+ T cell primary and memory responses induced as well as NK cell killing
responses; A single vaccination eradicated TC-1 tumors in 70% of mice
[95] E7 peptide + SA-4-1BBL Peptide - Preclinical Effective for reversal of anergy, induction of CD8+ T cell effector and memory responses and in eradication of
TC-1 tumors in mice; More effective immune modulator than TLR agonists LPS, MPL and CpG
[96] Natural adjuvants LacE7 + Japanese herbal medicines Bacterium - Preclinical Higher mucosal E7-specific type I T cell responses induced with secretion of IFN-y and IL-2 into the intestinal
lumen
[97] Bryo-I/E7 Peptide - Preclinical Produced IFN-y from lymphocytes and induced CTL responses in mice; Mice remained tumor free after 15 days [99] pcDNA3-CRT/E7 + a-GalCer [prime]
+ E7- pulsed DC-1 [boost]
DNA Calreticulin Preclinical E7-specific CD8+ T cell responses generated with prime immunization of pcDNA; Booster with E7-pulsed DC-1 had the best antitumor effects
[101] HPV-16 E7 + Fve Protein - Preclinical Enhanced E7-specific antibodies and IFN-y-producing CD4+ and CD8+ T cells; Tumour protection in 60% of
mice
[102] TA-CIN [HPV16 L2E6E7] + GPI-0100 Protein - Preclinical Enhanced HPV16 neutralizing antibody responses and E7-specific IFN-y-producing CD8+ T cells by 20-fold in
mice; Induced complete protection against tumor growth
4
Table 4.2. Overview of HPV immunotherapeutic studies with inclusion of adaptive immunomodulators Adaptive
Immune Modulators
Vaccine Formulation Vaccine Type
Additional
Immunomodulator[s]Study Type Key Result[s] of Study Ref.
Cytokines TG4001 Virus - Clinical 48% clinical efficacy in patients with CIN 2/3 at 6 months; Correlated with absence of HPV16 transcripts; No enhanced antibody responses to E6 and E7
[79] Nanoparticles Tat- E7/pGM-CSF Peptide Tat from HIV-1 Preclinical Prophylactically and therapeutically protected mice with increase in long-term survival and decrease in tumor
growth; Enhanced CD8+ memory T cell responses
[83] B9 cells [expressing E6, E7 and GM-CSF] Whole cell - Preclinical Protected mice against challenge with MK16 cells or TC-1 cells; MHC class I-restricted and non-restricted
mechanisms were postulated to be involved
[84] P13-H5-FL- SigE7LAMP Virus Sig from LAMP1 Preclinical P13-H5-FL-SigE7LAMP induced prophylactic and therapeutic antitumor efficacy with Flt3L coexpression [85] pE7 + pIL-12 DNA - Preclinical Suppressed E7-specific CTL, antibody and Th cell responses leading to a loss of protection from tumor
challenge
[86] rSFV-eE6,7 + SFV- IL12 Virus - Preclinical Enhanced antigen-specific effector memory CD8+ T cell responses and antitumor responses in 28% of mice
with addition of low doses of SFV-IL12; Higher doses reduced these effects
[87] TC-1-IL-12 Whole cell Gemcitabine Preclinical Combined treatment of gemcitabine and tumor cells led to a 29% reduction of mice with lung metastasis with
MK16 residual disease
[88] E7SH/IFN-y WT; E7SH/IL-2 WT;
E7SH/GM-CSF WT; E7SH/IL-12 WT
DNA Chemokine MIP1-a Preclinical Best CD8+ T cell responses with IFN-y or IL-12 administered 3 days after and MIP1-a administered 5 days prior to E7SH immunization
[89] Chemokines pcDNA-[E7-NT-gp96] + pcDNA-IP-10 DNA Glycoprotein 96 Preclinical Significant suppression in tumor growth with increased numbers of IFN-y and IL-2 in the lymph nodes of mice [90] pcDNA3-CHI-E7 DNA IL-2 Preclinical HLA-A2 mice were protected from TC-1/A2 tumor formation [93] Costimulatory
molecules
DC- and B-cells transduced with CFm40L-complexed adenovirus with E6 and E7[AdE6E7-CFm40L]
Whole cell Gemcitabine Preclinical Enhanced activation of APCs and CTL responses with greater inhibition of tumor growth in mice [94]
B1 cells [expressing E6, E7 and B7-1] Whole cell - Preclinical Protected mice against challenge with MK16 cells [84] E7 + SA-4-1BBL Protein - Preclinical E7 protein-specific CD4+ and CD8+ T cell primary and memory responses induced as well as NK cell killing
responses; A single vaccination eradicated TC-1 tumors in 70% of mice
[95] E7 peptide + SA-4-1BBL Peptide - Preclinical Effective for reversal of anergy, induction of CD8+ T cell effector and memory responses and in eradication of
TC-1 tumors in mice; More effective immune modulator than TLR agonists LPS, MPL and CpG
[96] Natural adjuvants LacE7 + Japanese herbal medicines Bacterium - Preclinical Higher mucosal E7-specific type I T cell responses induced with secretion of IFN-y and IL-2 into the intestinal
lumen
[97] Bryo-I/E7 Peptide - Preclinical Produced IFN-y from lymphocytes and induced CTL responses in mice; Mice remained tumor free after 15 days [99] pcDNA3-CRT/E7 + a-GalCer [prime]
+ E7- pulsed DC-1 [boost]
DNA Calreticulin Preclinical E7-specific CD8+ T cell responses generated with prime immunization of pcDNA; Booster with E7-pulsed DC-1 had the best antitumor effects
[101] HPV-16 E7 + Fve Protein - Preclinical Enhanced E7-specific antibodies and IFN-y-producing CD4+ and CD8+ T cells; Tumour protection in 60% of
mice
[102] TA-CIN [HPV16 L2E6E7] + GPI-0100 Protein - Preclinical Enhanced HPV16 neutralizing antibody responses and E7-specific IFN-y-producing CD8+ T cells by 20-fold in
mice; Induced complete protection against tumor growth
4
Table 4.3. Overview of HPV immunotherapeutic studies with inclusion of immunomodulators targeting immunosuppression.
Targets Vaccine Formulation Vaccine Type
Additional
Immunomodulator[s]Study Type Key Results of Study Ref.
Tregs Ad-p14 + anti-GITR T cell - Preclinical Achieved complete and permanent eradication of TC-1 tumors in mice [107] pcDNA3-E7/Hsp70 + anti- CD25 [PC61] DNA Hsp70 Preclinical Long-term protection against tumor growth; Generated higher levels of E7-specific CD8+ T cell
responses compared to administration with isotype Ab
[108] rSFV-eE6,7 + a-FR4 Virus - Preclinical Prior depletion with a-FR4 did not enhance immune or antitumor responses to rSFVeE6,7 [109] IL-6 E7 peptide-pulsed BMDC transfected with IL-6Ra DC - Preclinical Decreased tumor growth and prolonged survival in TC-1 bearing mice; Increased tumor-specific
CD8+ T cells
[111] IL-10R DC2c4-Sig/E7/LAMP-1 modified with siRNA
targeting IL-10R
DC - Preclinical Generated higher frequency of HPV E7-specific CD8+ T cells and stronger antitumour effects in mice expressing TC-1 tumors
[113] TGF-B R Ad-E7 + TGF-B receptor kinase inhibitor [SM16] Virus - Preclinical Significantly increased in TC-1 tumor regression in mice [114] TGF-B VACV co-expressing SigE7LAMP & sTBRII Virus - Preclinical Increased E7-specific CD8+ T cell responses in mice [115] CCL2 Ad.E7 + a-CCL2 mAb Virus - Preclinical Increased in E7-reactive T cells; Significantly reduced the percentage of Tregs in the spleen; Tumor
cure rate of 50% in mice
[117] CTLA-4 HPVm16E7-eCTLA4 Protein - Preclinical Low dose induced higher antibody titres and strong CTL responses towards E7 with higher levels of
IFN-y and IL-12; Slowed growth of established tumors
[121] PD-1 E7 DNA + sPD-1 DNA - Preclinical Increased antigen-specific T cells and potent antitumor responses against TC-1 tumor cells [124] BTLA pgD-E7E6E5 DNA pGM-CSF/pIL-12 Preclinical 70% of mice challenged with TC-1 were protected from tumor growth with three vaccine doses;
Cytotoxic E7-specific CD8+ T cell responses were induced; Antitumor response increased to 100% with addition of pGM-CSF or pIL-12
[127, 128] Ad-LIGHT Virus - Preclinical Increased E7-specific TILs as well as IFN-y and chemokines IL-1a, MIG and MIP-2; Regressed tumors and
increased survival of mice
[129] DR5 4a-CRT/E7[detox] + MD5-1 DNA Calreticulun Preclinical Generation of higher susceptible TC-1 cells to cytotoxic E7-specific T cells with potent antitumor effects [130]
4
Table 4.3. Overview of HPV immunotherapeutic studies with inclusion of immunomodulators targeting immunosuppression.
Targets Vaccine Formulation Vaccine Type
Additional
Immunomodulator[s]Study Type Key Results of Study Ref.
Tregs Ad-p14 + anti-GITR T cell - Preclinical Achieved complete and permanent eradication of TC-1 tumors in mice [107] pcDNA3-E7/Hsp70 + anti- CD25 [PC61] DNA Hsp70 Preclinical Long-term protection against tumor growth; Generated higher levels of E7-specific CD8+ T cell
responses compared to administration with isotype Ab
[108] rSFV-eE6,7 + a-FR4 Virus - Preclinical Prior depletion with a-FR4 did not enhance immune or antitumor responses to rSFVeE6,7 [109] IL-6 E7 peptide-pulsed BMDC transfected with IL-6Ra DC - Preclinical Decreased tumor growth and prolonged survival in TC-1 bearing mice; Increased tumor-specific
CD8+ T cells
[111] IL-10R DC2c4-Sig/E7/LAMP-1 modified with siRNA
targeting IL-10R
DC - Preclinical Generated higher frequency of HPV E7-specific CD8+ T cells and stronger antitumour effects in mice expressing TC-1 tumors
[113] TGF-B R Ad-E7 + TGF-B receptor kinase inhibitor [SM16] Virus - Preclinical Significantly increased in TC-1 tumor regression in mice [114] TGF-B VACV co-expressing SigE7LAMP & sTBRII Virus - Preclinical Increased E7-specific CD8+ T cell responses in mice [115] CCL2 Ad.E7 + a-CCL2 mAb Virus - Preclinical Increased in E7-reactive T cells; Significantly reduced the percentage of Tregs in the spleen; Tumor
cure rate of 50% in mice
[117] CTLA-4 HPVm16E7-eCTLA4 Protein - Preclinical Low dose induced higher antibody titres and strong CTL responses towards E7 with higher levels of
IFN-y and IL-12; Slowed growth of established tumors
[121] PD-1 E7 DNA + sPD-1 DNA - Preclinical Increased antigen-specific T cells and potent antitumor responses against TC-1 tumor cells [124] BTLA pgD-E7E6E5 DNA pGM-CSF/pIL-12 Preclinical 70% of mice challenged with TC-1 were protected from tumor growth with three vaccine doses;
Cytotoxic E7-specific CD8+ T cell responses were induced; Antitumor response increased to 100% with addition of pGM-CSF or pIL-12
[127, 128] Ad-LIGHT Virus - Preclinical Increased E7-specific TILs as well as IFN-y and chemokines IL-1a, MIG and MIP-2; Regressed tumors and
increased survival of mice
[129] DR5 4a-CRT/E7[detox] + MD5-1 DNA Calreticulun Preclinical Generation of higher susceptible TC-1 cells to cytotoxic E7-specific T cells with potent antitumor effects [130]
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Innate Immunostimulatory Adjuvants in HPV Immuno- therapy: TLR Ligands In the past 20 years, characterizing adjuvants, within the category of immunostimulatory agents, with their associated cellular receptor(s) has provided a better understanding into their mechanisms of action. Most of the immunostimulatory adjuvants are ligands of PRRs with one of the main families being toll-like receptors (TLRs). TLRs are transmembrane proteins expressed on the surface or in endosomal compartments of immune and nonimmune cells. TLR ligands, types of PAMPs, comprise of conserved motifs on pathogens recognized by the leucine-rich repeats of TLRs. Upon ligation, TLRs initiate a signaling cascade leading to the activation of nuclear factor-KB (NF-KB) with subsequent induction of costimulatory molecules and cytokine expression necessary for activation of adaptive immunity. The resultant adaptive immune response elicited is dependent on the type of TLR ligand and its corresponding TLR.
Regarding Th1-polarized cellular responses, TLR3, 4, 7/8 and 9 ligands were applied in numerous preclinical studies of cervical cancer and are discussed as separate immunological adjuvants below. These ligands are known to induce APC maturation and promote cross-presentation of antigen to CD8+ T cells.19-22 For instance, TLR3 and 4 have been shown to aid in the maturation of E7-loaded myeloid-derived dendritic cells (DCs) favoring Th1 responses.23 An association between a decrease in TLR expression in the cervical mucosa and HPV persistence or progression of cervical neoplasia has been demonstrated.24,25 In order to elicit significant CD8+ T cell responses of peptide vaccines, TLR ligands may require conjugation to the antigen itself.26 TLR ligands have also shown therapeutic benefit in the clinical setting. For instance, the TLR7 agonist 852A has demonstrated promising immune responses in both phase I and II clinical trials. Subcutanous (s.c.) administration of the agonist in patients with recurrent ovarian, cervix and breast cancers leads to significant increases in the levels of interferon y-induced protein 10 (IP-10), indicative of immune activation and Th1 responses.27 Such immune activation was also observed in patients with metastatic melanoma with some cases resulting in overall stable disease.28 Moreover, TLR9 ligands have provided superior immunogenicity in the clinic when combined in vaccine formulations. TLR9 ligands in the form of bacterial DNA or synthetic oligonucleotides (i.e. immuno- stimulatory sequences (ISS)) included in a hepatitis B vaccine (HBV-ISS) have shown significantly higher antibody responses with long-lasting seroprotection compared to the currently licensed alum-adjuvanted vaccine.29,30 To note, combinations of TLR ligands have also been suggested to provide a potent adjuvant effect. In a recent study, a detoxified, nonhemolytic form of listeriolysin O (dtLLO) from Listeria monocytogenes was postulated to act as a PAMP with direct immunomodulatory activity by targeting multiple PRR pathways and contributing to antitumor, E7- specific responses with significant reduction in tumor burden when fused or combined in a mixture with E7 recombinant protein.31
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TLR3 Ligands
TLR3 is found in endosomal compartments of conventional DCs (cDCs) and macrophages as well as on the surface of various nonimmune cells. The receptor is activated by the binding of double-stranded viral RNA triggering DC activation. This leads to expression of major histocompatibility class (MHC) class II as well as cross-presentation with production of IL-12 and type I IFN.32 In addition to TLR3, TLR3 ligands can interact with other receptors, such as retinoic acid-inducible gene I (RIG-1).33 Synthetic analogs of dsRNA, a prime example being Poly(I:C)(polyriboinosinic:pol yribocytidylic acid), have been used as adjuvants for the development of potent cellular immune responses in vaccination.34 Their mechanisms of action include increasing cross- presentation in DCs, promoting Th1 cytokine production and suppressing Th2 cytokines both in vitro and in vivo.19,35-37
Poly(I:C) had been used in several preclinical studies concerning peptide and protein HPV immunotherapeutic strategies. For example, one study administered a soluble E7 protein or a 19mer, long synthetic E7 peptide admixed with poly(I:C) daily in mice over a four day period (a cluster immunization strategy). With this strategy extensive antigen-specific CD8+ T cell responses were elicited, which are akin to those seen during acute viral infections. This was only observed with involvement of poly(I:C) with continuous exposure to minimal amounts of antigen.38 Another peptide- based vaccine induced long-term immunological memory and prevented tumor recurrences when admixed with poly(I:C) and costimulatory anti-CD40 antibodies (referred to as TriVax). It was demonstrated that poly(I:C) was mainly responsible for this adjuvant effect.39 The same peptide (E7
49-57), complexed to oxygen-regulated protein 150
(ORP150), was also recently shown to induce more potent antitumor effects in vitro and in vivo when combined with poly(I:C).40
Additionally, one strategy employed a dual adjuvant approach using the Pan HLA-DR epitope peptide (PAHLA-DRE) and poly(I:C). PAHLA-DRE supports the generation of CD4+ T helper cell responses, necessary for CD8+ T cell response development, by binding with high affinity to MHC class II molecules. Combining these two agents in an E7 peptide-based vaccine strategy, with s.c. administration in mice, generated significantly higher E7-specific CD8+ T cells and anti-TC-1 tumor activity compared to PADRE or poly(I:C) alone. This response was even higher after intratumoral administration.41 In another combination strategy, treatment including cyclophosphamide-driven depletion of regulatory T cells (Tregs), with intra-lymph node immunization of E7 peptide and poly(I:C), was associated with higher antigen-specific responses and regression of more advanced tumors (C3.43 cell line expressing HPV16 E7).42
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as adjuvants. These proteins were recombined as a fusion protein (referred to as Pentarix). Mice receiving a single dose of Pentarix admixed with polyI:C had higher IFN-y responses. Stronger E7-specific T cell responses and complete regression of large, established TC-1 tumors were demonstrated when administered by cluster immunization.43
Additionally, in a preclinical study using rhesus macaques, the effect of polyICLC, an analog of poly(I:C), was assessed. This TLR3 ligand is more suitable for antiviral immune responses in primates. Also, derivatives of poly(I:C) are more preferably used in the clinic due to the safety issues associated with high doses of poly(I:C).44 PolyICLC injected s.c. in rhesus macaques with HPV16 capsid protein L1 (capsomeres) enhanced HPV16-specific Th1 responses by 5-fold compared to vaccine alone. Even though this study did not include HPV16 E6 and/or E7, polyICLC was nevertheless proven to aid in the induction of HPV-specific responses with an increase in the number of cells producing CXCL9 (MIG) and CXCL10 (IP-10), chemokines that attract naïve T cells and DCs, in the lymph nodes.45
Lastly, a study assessed a cationic adjuvant formulation (CAF01) with poly(I:C) administered with E7 protein. Combination of poly(I:C) with another immunomodulator, mycobacterial cord factor, incorporated into CAF01, was able to lyse target cells and significantly regress TC-1 tumors.46
TLR4 Ligands
Like TLR3, TLR4 is expressed on cDCs, macrophages and various nonimmune cells.32 All clinical trials that include TLR4 adjuvants use lipopolysaccharide (LPS) derivatives. A less toxic component of LPS is monophosphoryl lipid A (MPL), a natural glycolipid from Salmonella and the only TLR ligand licensed in humans thus far. Cervarix® is adjuvanted with MPL absorbed onto aluminum hydroxide or aluminum phosphate (AS04). Despite the use of Cervarix® for prophylactic vaccination, the mechanism of AS04-induced immune response upon intramuscular (i.m.) injection involves the transient production of cytokines through NF-kB activity with rapid antigen migration of DCs in the draining lymph node, stimulating B cells through APC-driven activation of Th1 cells.47
There are a scant number of therapeutic preclinical studies assessing the effect of TLR4 ligands in antitumor immune efficacy due to its dubious stimulation of CD8+ T cell responses. However, in a recent study, a sulfated polysaccharide compound extracted from red algae, carrageenan (CGN), was demonstrated to activate the TLR4 pathway. For chemical structure (see Figure 4.1A).48 CGN significantly enhanced E7-specific responses via prolonged antigen enhancement in the lymph nodes in conjunction with an HPV16 E7 peptide-based vaccine. The agent was able to elicit both prophylactic and therapeutic antitumor responses in mice, comparable to those induced by MPL.49 In a protein-based vaccine strategy, the extra domain A (EDA) from fibronectin,
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another TLR4 ligand, fused to HPV E7 (EDA-HPVE7), induced CD8+ T cell responsesby targeting antigen to DCs. This formulation was not sufficient to eradicate tumors and caused only a slight delay in tumor growth. On the other hand, the combination with Poly(I:C), resulted in complete eradication of established tumors. The paper also explores a more extensive combination approach in which the use of TLR9 ligand CpG mixed with cationic lipids (DOTAP), and a low dose chemotherapeutic agent, was able to reject even larger, established tumors. This suggests that combination strategies in this regard are essential for an effective antitumor response, unless the vaccines were to be repeatedly administered intratumorally (where the proinflammatory effects of EDA are deemed to be favorable).50
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TLR7/8 Ligands
TLR7 and 8, expressed in endosomal compartments of human DCs, monocytes, macrophages, lymphocytes, Langerhans cells (LCs) and natural killer (NK) cells, recognize single stranded (ss) RNA sequences rich in polyU or GU. Cells containing these receptors can be activated by synthetic imidazoquinolines such as imiquimod (Figure 4.1B) and resiquimod (Figure 4.1C) which secrete IFN- a, IL-6, tumor necrosis factor (TNF)-a and IL-12, cytokines that guide Th1 responses.51-54 A study confirming the expression of TLR7 and 8 on human LCs showed that resiquimod (TLR7/8 agonist) and 3M-002 (TLR8 agonist) activated LCs, and upon in vitro immunization with HPV16 VLPs, initiated a specific CD8+ T cell response. To note, this was not observed with imiquimod.55
Imiquimod is the only TLR7/8 ligand used in the clinic thus far. The agonist was licensed for topical application of lesions developed with HPV infection.56-58 A recent clinical study using a fusion protein of HPV with imiquimod led to regression of vulval intraepithelial neoplasia (VIN) lesions in 63% patients with a significant increase in local infiltration of CD8+ and CD4+ T cells.59 A DNA vaccine prime, viral vector boost regimen in combination with imiquimod is currently underway in a phase I clinical trial for patients with CIN III lesions.60 Regarding other cancers, imiquimod has been used for the treatment of malignant melanoma as a topically applied cream following intradermal administration of recombinant NY-ESO-1 protein. In a phase I trial, this combined treatment induced antibody titres and CD4+ T cell responses in a significant amount of patients.61 In a phase I/II trial for prostate cancer, the comparison of topical imiquimod was made to adjuvants granulocyte macrophage colony-stimulating factor (GM-CSF), hyperthermia and mucin-I-mRNA/protamine complex combined with prostate-specific antigen vaccine consisting of peptides that bind to MHC class II. Imiquimod was determined to be most beneficial compared to the others with more stable or slower progression of PSA.62 Although topical administration of imiquimod results in no severe local reactions or systemic events, its effect on disease progression of various cancers has yet to be proven.
TLR7/8 ligands were used in preclinical studies for cancer vaccines to enhance antigen-specific T cell and antibody responses in both quality and magnitude.63 Yet, only one recent study researched its effect in combination with HPV immunotherapy. This study utilized a DNA-based vaccine linking calreticulin to HPV16 E7 (CRT/E7) and combined it with the topical administration of imiquimod at the tumor site of mice. Treatment with this formulation enhanced E7-specific CD8+ T cell responses with a concomitant increase in natural killer T cells (NK1.1+ cells) and macrophages. In addition, a decrease in myeloid-derived suppressor cells (MDSCs) was observed in the tumor microenvironment which led to a lower tumor size and increased survival in tumor-bearing mice.64
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TLR9 Ligands
TLR9, localized in endosomal compartments expressed in human plasmacytoid DCs and B cells, recognizes unmethylated CpG motifs on bacterial DNA.65 Upon recognition, the activation leads to type I IFN production. CpG motif-expressing oligonucleotide (CpG ODN) is a synthetic TLR9 ligand used to mimic bacterial DNA and induces the production of tumor-specific cytotoxic CD8+ T cells in various clinical studies. In a pilot trial consisting of patients with stage III/IV NY-ESO-1-expressing melanoma and other cancer types, CpG was immunized in combination with the NY-ESO-1 peptide and Montanide, inducing peptide-specific CD8+ T cell responses.66-67 Other responses due to the cross-priming effect of CpG, including the antibody and Th1-type, were increased in patients with different cancers, e.g. prostate cancer, that were given the same vaccine but as a protein formulation.68-69
HPV was proven to abolish the expression and function of TLR9 which may contribute to the cancer-inducing effect of the virus. Upregulation of TLR9 expression may therefore be crucial for immunotherapeutic efficacy.70 Co-administration of CpG ODN with DNA-, peptide- or protein-based vaccines has been assessed in a number of preclinical studies against HPV. Systemic administration of CpG ODN with a DNA vaccine encoding mutated HPV16 E7 and lysosome-associated membrane protein (LAMP) (pBSC/E7GGG.LAMP), delivered by gene gun, demonstrated that despite the suppression of cytotoxic lymphocytes (CTLs) in the spleen, high doses of CpG ODN coincided with enhanced reduction of TC-1 tumors in mice compared to the administration of DNA vaccine alone.71 In addition, DNA vaccination may offer the advantage of inclusion of CpG motifs in the plasmid itself. For instance, one study introduced a CpG cassette with four optimized mouse and four human CpG motifs into the plasmid backbone containing the HPV16 E7 gene that lacked transforming abilities (HPV16 E7SH). This plasmid triggered Th1 responses that were concomitant with higher C3 tumor regression in mice. The usage of electroporation technology further enhanced these responses with a 10-fold decrease in tumor size.72
In the context of peptide vaccination, a study assessed responses against different CTL epitopes of HPV16 E7. T cell and antibody responses towards CTL epitope
E749-57 were similar between peptides administered s.c. with CpG ODN versus those
administered with tattoo injection. The cellular responses were also comparable to gene-gun immunization with the DNA vaccine addressed above, pBSC/E7GGG.LAMP. This effect was very much dependent on the peptide but with tattoo administration being beneficial for all formulations.73 Another study making use of a vaccine based on E7 long peptide administered with CpG ODN by i.m. injection followed by
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hence, the E7-specific antitumor response was not addressed.74 Furthermore, a recent report on the intravaginal instillation of CpG ODN after s.c. vaccination with E7 protein completely regressed HPV tumors in 75% of mice compared to the 20% vaccinated without prior exposure to the TLR9 ligand.75 Lastly, a study assessing heat-resistant and chemically-stable, soluble oligomers of E7, appearing most abundantly in tumor tissue, combined with ODNs with optimized human CpG motifs, showed prolonged antitumor activity. The cellular immune responses involved were not classified.76 Skewing the T Cell Response Against HPV with Adaptive Immune Adjuvants Adjuvants that manipulate the complex networks that govern adaptive immunity with the aim of augmenting T cell responses may employ endogenous immunomodulators. These immuno- stimulants are largely cytokines, chemokines and costimulatory molecules which bypass the need for the initial PRR interaction.15
Systemic administration of cytokines is associated with serious systemic toxicity, as cytokines need to be administered at high doses to elicit antitumor effects.77 Therefore, these endogenous adjuvants are used in combination with vaccine strategies as recombinant proteins or are incorporated within the vaccine itself.78 Besides the cytokines that are involved, the activation of T cells is governed by two signals. One is derived from the interaction with the MHC class I or II complexes on APCs and the other from the costimulatory signal governed by the B7-1 (CD80) or B7-2 (CD86) ligands that bind to the CD28 receptor on T cells. B7-1/B7- 2 ligand-CD28 interaction is crucial for the development of Th1 responses. Additionally, the interaction of CD40 on B cells with CD40L is important for adaptive immune signaling. Various adaptive immune adjuvants may exploit these interactions. In addition, compounds derived from natural sources have been used as adjuvants in HPV immunotherapeutic strategies for direct activation of adaptive immune responses.
Cytokines as Endogenous Adjuvants
Th1-specific pro-inflammatory cytokines such as IL-2, IL-12, IFN-y, GM-CSF and Flt3L are preferentially used to elicit cell- mediated immune responses and are therefore most favorable for therapeutic vaccination against cancer as a single or a multiple modality treatment.
There are a plethora of clinical studies regarding the use of cytokines in immunotherapy against cancer. For example, in a phase 2 clinical trial, 48% of patients with CIN 2/3 lesions receiving s.c. injections of vaccinia virus encoding HPV16 E6 and E7 as well as IL-2 (TG4001) had regressions of their lesions.79 In the last decade, GM-CSF has been used in a number of cancer vaccine clinical trials based on the observation that the cytokine showed protection from tumor rechallenge in mice readministered with irradiated tumor cell lines transduced with the cytokine.80 The most prominent example is sipuleucel-T, the first FDA- approved therapeutic cancer
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vaccine. Sipuleucel-T, or Provenge®, effective against metastatic prostate cancer,consists of patient- derived peripheral blood mononuclear cells that are activated by ex vivo incubation with a fusion protein of recombinant human PAP (prostatic acid phosphatase) and GM-CSF.81 Surprisingly, GM- CSF has also been negatively implicated in both preclinical and clinical responses, likely due to its ability to increase the population of suppressor cells. It was suggested that the fine balance between immunostimulatory versus immunosuppressive effects is due to the dosage of GM-CSF administered. Other clinical studies of interest include the adjuvantation with bolus IL-12 of gp100 peptide which significantly improved progression-free survival of melanoma patients compared to using IL-12 alone.82
The adjuvant effect of cytokines has been shown in a number of recent preclinical studies against cervical cancer. For instance, a nanoparticle encoding a fusion peptide of HPV16 E7 (amino acids 49-57) and Tat (amino acids 49-57), a cell-penetrating peptide from HIV-1, was demonstrated to drive a higher memory CD8+ T cell response and prolong survival in mice, particularly when combined with a plasmid encoding GM-CSF (Tat-E7/pGM-GM-CSF). Tat is considered an immunomodulatory agent by delivering peptides across the plasma membrane into the cytoplasm of APCs.83
Another study, using a live cell vaccination strategy, tested the immunogenicity of a genetically-modified cell line producing GM- CSF, B9. B9, expressing E6 and E7, was able to protect mice against both MK16 (a metastasizing murine cell line) and TC-1 challenge. The study additionally assessed the B1 cell line, producing B7-1 costimulatory molecule, as discussed later on.84 Apart from GM-CSF, another important cytokine in the development of DCs and studied within the context of HPV-induced disease, is Fms-like tyrosine kinase 3 ligand (Flt3L). Enhanced anti-tumor activity,
using the TC-1 model, was observed when recombinant vaccinia virus (rVACV) coexpressing soluble Flt3L under a strong VACV promoter. This was concomitant with an increase in splenic myeloid dendritic cells and a decrease in MDSCs.85
Treatment with IL-12 has been correlated with antitumor and antimetastatic activity. On the contrary, the unexpected effects of this cytokine on vaccine-induced immune responses were demonstrated in one study in the context of DNA vaccination. IL- 12 complementary DNA (cDNA) was coinjected with E7 DNA which led to a 100% loss in tumor suppression concomitant with a decrease in the CTL, antibody and T helper cell responses through nitric oxide production.86 The effects of IL-12 were further evaluated with s.c. immunization of IL-12 expressed by a vector based on the alphavirus Semliki Forest virus (SFV) (SFV-IL12). In combination with recombinant SFV encoding a fusion protein of HPV16 E6 and E7 (rSFV-eE6,7), the antitumor responses
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positive effect of IL-12 was observed in one study using recombinant IL-12 expressed by TC-1 cells at the site of local tumor growth decreasing the tumor burden, but only after administration of the chemotherapeutic drug gemcitabine. This is likely due to the role of IL-12 in inhibiting tumor neovascularization with the production of high levels of IFN-y by splenocytes.88
Lastly, an approach comparing the effects of a range of different cytokines (IL-2, IL-1(IL-2, GM-CSF, IFN-y), immunized with a nontransforming HPV16 E7-based DNA vaccine, was given in different combinations and at different time points in mice. The authors also included in the study a plasmid encoding the chemokine, macrophage inflammatory protein ((MIP)1-a). It was shown that enhanced tumor responses were associated with secretion of granzyme B and an increase in the cytotoxic response with the best responses elicited upon administration of plasmids encoding IFN-y or IL-12 given 3 days after immunization with E7SH and MIP-1a given 5 days prior to E7SH. Significant responses were also obtained with IL-2 administered 5 days after E7SH immunization.89
Chemokines and Costimulatory Approaches
Apart from cytokines, chemokines and costimulatory molecules are currently being extensively explored as immunomodulators in preclinical studies.
In a combination approach, a DNA vaccine encoding E7 protein fused to the N-terminal of glycoprotein (gp)-96 (E7-NT-gp86), delivered via a non-viral gene delivery system (PEI600-Tat), was coadministered with IP-10. IP-10, or interferon inducible protein, belongs to the CXC chemokine family (CXCL10). Higher IFN-y and IL-2 production was observed with IP-10 administration at the TC-1 inoculation site rather than with s.c. administration of E7-NT- gp96 and PEI600-Tat at footpad concomitant with higher tumor reduction.90 Others observed the same immunostimulatory properties of IP-10 in DC and DNA vaccination studies.91-92 In another combination approach, a chimeric protein encoded on a plasmid consisting of the chemokine CC ligand 19/macrophage inflammatory protein-3B (CCL19/MIP-3B) fused at the N-terminus of hepatitis B small surface antigen (HBsAg(S)) and IL-2 and HPV16 E7 flanked at the C-terminus (pcDNA3-CHI-E7) prevented the development of TC-1/ A2 tumors expressing E6 and E7 in HLA-A2 mice. This approach circumvented the requirement of coinjection with an adjuvant.93
The activation of B cells and DCs requires the interaction of CD40 with its CD40 ligand (CD40L). One recent study exploiting this requisite is one that delivers the E6 and E7 genes to DCs in vitro using an adenoviral vector. This was made possible with the expression of the CD40L fused with the ectodomain of coxsackievirus-adenovirus receptor (CAR), as CAR is absent on APCs. This improvement in an APC-based vaccine approach was proven by the high levels of E7 expression in both DCs and B cells. In addition, high levels of cell surface markers CD80, CD86 and MHC class II molecules
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were induced for subsequent antigen- specific stimulation of effector T cells andsignificant suppression of tumor growth in mice.94
Another study exploiting costimulatory interactions investigated the properties of different HPV16- transformed tumor cells that were genetically modified to express B7-1 co-stimulatory molecule. The B7-1-producing cells (B1) that were given prior to challenge of MK16 cells (less oncogenic than TC-1 cells) were able to protect mice from tumor outgrowth. Growth of TC-1 tumors, however, was much higher in mice immunized with B7-1-producing cells.84
Lastly, a study evaluated a potent tumor necrosis family (TNF) costimulatory ligand, a soluble form of 4-1BBL chimeric with streptavidin (SA-4-BBL), as a vaccine adjuvant. This adjuvant served as a vehicle for increasing delivery of antigens to DCs. The signaling pathway ensued upon ligation allows effector T cells to become resistant to suppression by T regulatory cells. SA-4-1BBL conjugated with HPV16 E7, and administered as a single injection in mice, was able to eradicate established TC-1 tumors in about 70% of mice, either at the primary site of development or in metastatic tissue.95 This agent was additionally shown to be effective in inducing robust antitumor responses in combination with HPV16 E7 peptide vaccination.96
Nature-Derived Immunostimulatory Agents
There are also immunomodulatory agents derived from nature that serve as natural adaptive immune adjuvants. For instance, a novel study using recombinant
Lactobacillus casei expressing HPV16 E7 (LacE7) orally administrated in mice with a
Japanese herbal medicine induced 2-2.5-fold higher mucosal E7-specific type I T cell responses as compared to administration of LacE7 alone, with enhanced secretion of IFN-y and IL-2 into the intestinal lumen.97 An additional study using a peptide-based strategy tested the adjuvant effects of Bryostatin-I (Bryo-I), a macrocyclic lactone isolated from the marine bryozoan Bugula neritina.98 Mice s.c. immunized with Bryo-I fused to an E7 peptide (Bryo-I/E7) prevented outgrowth of TC-1 tumors through IFN-y expression in DCs and triggering CCL2 and CCL3 production from bone- marrow-derived DCs (BMDCs). Counterintuitively, CCL2 has been implicated as a protumorigenic and immunoregulatory chemokine, but its effect may be tumor-type dependent.99 Furthermore, a study assessing the NKT ligand a-galactosylceramide (a-GalCer), a glycolipid isolated from marine sponges, vaccinated with DNA (pcDNA3-CRT/E7) or tumor-antigen loaded DCs (pulsed DC-1) induced high numbers of E7-specific CD8+ T cells for protection in mice against the TC-1 tumors.100,101 Furthermore, a fungal immunomodulatory protein (FIP)-fve, isolated from the golden needle mushroom, Flammulina velutipes, a major fruiting body protein, enhanced production
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a semi-synthetic quillaja saponin analog, derived from naturally occurring saponins, was shown to enhance the generation of E7-specific, IFN-y-producing CD8+ T cells by 20-fold with vaccination of HPV16 L2E6E7 as a single tandem fusion protein (TA-CIN).103
Targeting Immunosuppression in the Tumor Microenvironment
The introduction of HPV-specific therapeutic vaccinations into the clinic has met with moderate clinical success. This is in part due to insufficient immune surveillance as tumors are consistently under selective pressure by immune recognition to evolve escape mechanisms and become resistant against immune attack.17
Circumventing different forms of negative regulation through specific targeting of immunosuppressive components may be imperative for effective immunotherapeutic regimens against HPV. Non-specific suppression of regulatory cells has been demonstrated in combination of vaccination with chemotherapy, e.g. low dose cyclophosphamide. However, these studies are non-specific and therefore will not be addressed in the following section.
Regulatory Cells
One mechanism of immune suppression includes the recruitment of suppressive cells such as CD25hiFoxp3+CD4+ Tregs, regulatory type II natural killer T cells (NKT cells) or MDSCs by tumor cells. A successful clinical strategy utilizing synthetic long peptides derived from HPV16 E6 and E7 given to patients with vulvar intraepithelial neoplasia (VIN) lesions resulted in complete regression in half of the patients with HPV-induced lesions with a high effector T cell to Treg ratio being predictive of clinical success.104,105 Tregs were shown to increase in the peripheral blood of patients with high-grade CIN lesions and correlated with disease progression.106
So far, preclinical immunotherapeutic strategies against HPV have primarily focused on specific depletion of Tregs with monoclonal antibodies (mAbs) in combination with vaccination. For example, depletion using anti-GITR mAb in combination with T cell vaccination based on adenovirus (Ad-p14) was shown to provide complete protection against tumor growth in mice.107 Another strategy using a DNA vaccine encoding E7 linked to heat shock protein 70 (Hsp70) (pcDNA3-E7/ Hsp70), with prior depletion of Tregs using anti-CD25 Ab (PC61), resulted in significant long- term protection from tumor growth compared to administration of the isotype control.108 Lastly, it is of value to point out that a study utilizing a vaccine based on SFV demonstrated that Treg cell depletion with anti-folate receptor 4 (FR4) mAb was not required for enhancing antitumor immune responses.109 Therefore, depending on the vaccine approach, it is evident that removal of Tregs from patients may have beneficial effects in the clinical setting.
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Regulatory Cytokines/ChemokinesAnother mechanism of immune suppression includes the production of soluble factors; inhibitory molecules that bind to corresponding receptors on T cells. Cytokines and chemokines, produced by tumor infiltrating lymphocytes (TILs), tumor cells and stromal cells in the tumor microenvironment, have been suggested to act locally and systemically. Immune cells in the form of immature or semimature myeloid dendritic cells, for example, produce immunosuppressive cytokines such as IL-10 and TGF-B. Additionally, immature DCs produce indoleamine 2,3-diogenase (IDO), another strong candidate for suppressing T cell responses, contributing to the cervical cancer progression. It was observed that samples from cervical cancer patients had significant increases in the numbers of IDO and IL-10-producing cells compared with samples from a normal cervix and a high-grade CIN lesion group.110 Within the context of recent HPV-specific vaccination, pre- clinical strategies that target IL-6, IL-10, TGF-B and CCL2 will be discussed.
IL-6, a cytokine produced by tumors, was implicated in the inhibition of DC maturation. In one study, researchers immunized mice, that were previously injected with TC-1 cells, with E7 peptide-pulsed BMDCs that were transfected with IL-6Ra siRNA (siIL-6Ra DC) or treated with an IL-6 receptor signal competitor (IL-6RC DC). Both BMDC types contributed to sustained survival of mice by decreasing tumor growth with generation of effector and memory CD8+ T cells.111
There have been studies on various cancer types reporting increased levels of the cytokine IL-10. IL-10, produced by TILs, tumor cells as well as tumor-associated macrophages (TAMs), was shown to inhibit expression of CD40 on APCs.112 In a recent study, more potent antitumor CD8+ T cells were generated upon administration of DCs transduced with Sig/E7/LAMP-1 (DC2·4- Sig/E7/LAMP-1) and small interfering RNA targeting IL-10R. IL- 10R, expressed on DCs and Treg cells, suppresses through STAT proteins. DCs that were modified by IL-10-targeting with siRNA resulted in upregulation of MHC class II, CD40 and IL-12 upon stimulation with LPS.113
TGF-B is another potent immunosuppressive cytokine. It was shown to induce Treg development from CD4+ T cells as well as recruit MDSCs into the tumor microenvironment. Two recent approaches targeting TGF-B were used. One strategy inhibited TGF-B-mediated suppression with an inhibitor of TGF-B receptor kinase, SM16, administered with an adenoviral vector expressing HPV16 E7. Vaccine efficacy was increased with significant TC-1 tumor regression.114 The other strategy enhanced E7-specific CD8+ T cell responses by utilizing double recombinants of vaccinia virus (VACV) expressing both soluble TGF-B receptor II and SigE7LAMP (sTGFBII).115
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Overcoming Immune Exhaustion
An additional immunosuppressive mechanism in the tumor microenvironment involves tumor-driven inhibition or attack on immune cells by the expression of low levels of costimulatory molecules contributing to T cell tolerance or anergy (i.e. immune exhaustion). Tumor cells are able to downregulate costimulatory molecules on professional APCs.118 Different strategies to shift the balance from tolerogenic to immunogenic immune responses include targeting CTLA-4, PD-1, BTLA and DR5.
Cytotoxic T lymphocyte antigen-4 (CTLA-4), expressed on Tregs and activated T cells, interacts strongly with B7 molecules on APCs or tumor cells; even more so than CD28 by 20- to 50- fold. This interaction leads to T cell anergy or cell death (apoptosis).119 Blocking this interaction has been studied in mice using different forms of anti-CTLA4 antibody treatment. In the TC-1 tumor model, a significant delay in tumor growth was observed upon systemic treatment with anti-CTLA4 antibody or expression of the antibody from tumor cells. This effect, however, was not seen with a stem cell based delivery approach which targeted also non-tumoral tissue.120 With regard to vaccine approaches, a study which modified the E7 gene of HPV16 responsible for cellular transformation (HPV16mE7) was fused to the extracellular domain of CTLA-4, creating the fusion protein HPVm16E7- eCTLA4. Having the ability to bind to DCs via B7 molecules, and thereby blocking CTLA-4 ligation, the fusion protein was able to stimulate potent CTL responses and protect mice from tumor challenge. At a low dose, this was associated with increased survival and growth inhibition of established tumors.121 Furthermore, adenovirus-mediated, intratumoral expression of recombinant CTLA-4 significantly delayed tumor growth through NKT and CD8+ T cell involvement with systemic depletion of Tregs using a-CD25 Abs.122
Programmed death receptor ligand 1 (PD-L1), expressed by tumor cells and in both hematopoietic and nonhematopoietic tissue induces apoptosis upon interaction with T cells expressing the PD-1 receptor (CD279). PD-L1 is able to bind to B7-1 and is expressed by cervical cancers lesion.123 Blocking PD-1 restores the function of exhausted T cells with soluble PD-1 (sPD-1) evaluated in the context of HPV. sPD-1 DNA coadministered with an HPV16 E7 DNA vaccine resulted in strong antitumor responses. This was not found to be vaccine-dependent as the same adjuvant effect of sPD-1 was observed with an adenovirus-based vaccine. The mechanism behind this phenomenon is increased functional activity, proliferation and reduced apoptosis of T cells, thereby not only affecting T cell exhaustion but also primary T cell activation, coupled with enhanced DC maturation.124 A Phase I study that determined the safety and tolerability of blocking PD-1 with a humanized antibody (MDX-1106) in 39 patients with advanced metastatic melanoma, colorectal cancer (CRC), castrate-resistance prostate cancer, non-small-cell lung cancer (NSCLC) or renal cell carcinoma (RCC), gave encouraging results in terms of safety, tumor regression and durability of the antitumor activity.125,126
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Compared to CTLA-4 and PD-1, B- and T-lymphocyte attenuator (BTLA) is anotherinhibitory protein expressed on T cells and interacts with the B7 homolog. This protein can also bind to tumor necrosis family receptors (TNF-R); an example being the herpes virus entry mediator (HVEM). Upon interaction, T cells receive inhibitory signals and hence are negatively regulated. Glycoprotein D (gD) of herpes simplex virus (HSV) binds to HVEM but competes for the same binding site as BTLA, thereby blocking inhibitory signals induced by the HVEM-BTLA interaction. A DNA vaccine carrying the E5, E6 and E7 genes of HPV (pE7E6E5) fused to gD (pgD-E7E6E5) elicited CD8+ T cell responses, independent of help from CD4+ T cells, towards established TC-1 tumors. This anticancer response can be further enhanced from 50 to 100% with coadministration of GM-CSF or IL-12-encoding plasmids. An increase in therapeutic protection was further shown via i.m. administration of HPV16 E7 fused to gD (pgDE7) with pIL-2.127,128 Another ligand for HVEM is lymphotoxin-B receptor ligand (LIGHT). Administration of recombinant adenoviruses encoding LIGHT (Ad-LIGHT) in a C3.43 tumor challenge model increased the frequency of E7- specific TILs as well as IFN-y and chemokines IL-1a, MIG and MIP-2 with resultant tumor regression. This was concomitant with a decrease in TGFb1, another immunosuppressive cytokine, as well as IL-10, albeit the latter showed no statistical significant difference with control adenoviruses.129
Lastly, a study involving a mAb directed against death receptor 5(DR5) (MD5-1), leading to apoptotic cell death in tumors, was administered via gene gun prior to immunization with a DNA vaccine encoding calreticulin linked to HPV16 E7 antigen mutated for the Rb binding site (CRT/E7(detox)). DR5 is a receptor for the tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) which initiates apoptosis via the extrinsic apoptosis pathway and is expressed on the surface of different cancer cells including TC-1 tumors. The combination of CRT/E7 (detox) DNA vaccination and MD5-1 mAb resulted in the higher percentages of E7-specific CTLs with TC-1 tumors more susceptible to lysis compared to treatment with either strategy alone.130
Concluding Remarks
It is of profound importance that durable cellular immune responses are elicited by HPV immunotherapeutic vaccines. Enhanced HPV-specific immunogenicity could be provided by the use of effective immunomodulators with diverse mechanisms of action. For instance, from this review it can be postulated that peptide- and
