Use of adjuvants for immunotherapy

ABSTRACT

Cancer vaccines are designed to stimulate the body's immune system to kill tumor cells. To improve their immunogenicity, vaccine antigens must be combined with adjuvants which are able to stimulate the innate immunity and potentiate the adaptive immune response. In the last years a new generation of adjuvants mimicking the natural microbial ligands have been developed. In particular, several TLR ligands have been extensively explored as vaccine adjuvants and many preclinical and clinical studies have been conducted. However, the road to approval of such adjuvants for clinical use is still to go.

KEYWORDS:

• adjuvant
• cancer immunotherapy
• cancer vaccine
• Toll-like receptors

Introduction

Immunotherapy is the most promising therapeutic strategy for cancer. The concept of cancer immunotherapy is based on using the immune system to attack the tumor cells targeting tumor-specific antigens.^1,2 This can be achieved using passive and/or active strategies. The optimal active immunotherapy strategy is represented by therapeutic cancer vaccines which are designed to elicit a T cell response specific to tumor-associated antigens (TAAs).

Provenge is the first and only FDA-approved therapeutic cancer vaccine specific for prostate cancer. It is based on dendritic cells (DCs) obtained from the patient and stimulated ex vivo with the prostatic acid phosphatase (PAP), an antigen that is highly expressed in most prostate cancer cells. The activated DCs are then re-infused in the patient to stimulate T cells for attacking and eliminating the prostate cancer cell.³ On the other hand, the only approved preventive cancer vaccines are the hepatitis B virus (HBV) and human papilloma virus (HPV) preventive vaccines. Indeed, given that chronic infection with the 2 virus possibly leads to liver cancer and cervical cancer, respectively, such vaccines are de facto preventing development of cancer.^4,5

In the last years, several TAAs have been identified and tested in early stage clinical trials as different formulations (e.g. peptides, DCs loaded ex vivo, oncolytic viruses, genomic vectors).^6,7 Overall, most of the vaccines in clinical trials suffer from weak immunogenicity due to limited capacity of stimulating the innate immunity, which is the prerequisite for eliciting a strong adaptive immune response.^8,9 This has required the need for developing adjuvants to improve the immunogenicity of cancer vaccines.

Adjuvants in cancer vaccination

The term adjuvant comes from the Latin word ‘adjuvare’, meaning help. Adjuvants are a crucial component of cancer vaccines, as they help in eliciting a potent immune response against the vaccine antigens.

Adjuvants have a long history as potentiators of immunity against preventive vaccines to pathogens' infection.¹⁰ In particular, the role of adjuvants has become more relevant when the traditional vaccines based on attenuated or killed pathogens have been substituted with subunit vaccines. Indeed, the latter lack the adjuvanting molecules naturally present in the pathogens (namely nucleic acids) and their immunogenicity is definitely weak.^11,12 Consequently, addition of adjuvants in the formulation is required to potentiate immunogenicity of subunit vaccines. The aluminum salt has been for decades the only approved adjuvant for human vaccination. Its effect is the induction of humoral response due to i) increment of the biological or immunologic half-life of vaccine antigens (depot effect); ii) improvement of the antigen delivery to antigen-presenting cells (APCs), as well as antigen processing and presentation by the APCs and iii) induction of immunomodulatory cytokines production.¹³ The strong limitation of Alum is the inefficient elicitation of Th1-dependent cellular immunity, which is the one effective in the anticancer activity.¹⁴

In the last years the key role of Dendritic Cells (DCs) as trait d'union between the innate and adaptive immune response has been defined in great details and they have been identified as the optimal cell target for adjuvants.¹⁵ In particular, DCs express many pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), Nod-like Receptors (NLRs), Retinoic acid-Inducible Gene 1-Like Receptor (RIG-I) or Stimulator of interferon genes (STING) that act as sensors whose physiological role is to recognize conserved pathogen-associated molecular patterns (PAMPs) and trigger activation and maturation of DCs.¹⁶

The maturation process induces the migration of DCs to draining lymphoid organs, upregulation of MHC antigens and co-stimulatory molecules as well as cytokine secretion to drive the priming and polarization of naīve CD4+ T helper cells. In particular, CD4+ Th1 cells are able to initiate and sustain an anti-tumor and anti-viral cellular immunity, CD4+ Th2 cells are able to initiate and sustain an anti-bacterial humoral immunity. The detailed understanding of activation mechanisms of DCs through the PRRs has promoted an explosion in the discovery of novel adjuvants which are able to mimic the PAMPs inducing activation and maturation of DCs for an optimal presentation of the vaccine antigen to T cells.¹⁶

TLR agonists as adjuvants

Over the past 2 decades, high-throughput compound screening approaches have contributed to the discovery of many novel adjuvants able to bind and activate specific PRRs, leading to DC maturation and inducing patterns of inflammatory responses.¹⁷ Among different PRRs, the potential of TLR ligands as vaccine adjuvants has been extensively explored.^18,19,20

TLRs have been identified in several 10 in humans (TLR1–TLR10) and 12 in mice (TLR1–9, TLR11–13).²¹ They are expressed by a variety of cell types and are classified according to their microbial ligand specificity, signal transduction and cellular localization.²²

TLR2 is a membrane surface receptor and it functions by dimerizing with TLR1 or TLR6. TLR1–TLR2 heterodimer is responsible for sensing triacylated lipopeptides of mycoplasma or Gram-negative bacteria origin, whereas TLR2–TLR6 heterodimers sense diacylated lipopeptides of mycoplasma and Gram-positive bacteria origin.²³

Synthetic triacylated and diacylated lipoproteins for TLR2 activation include Pam3Cys and Pam2Cys, respectively. These TLR2 ligands have been reported to induce long-lived antigen-specific CTL and humoral responses in preclinical tests.^24,25,26

The TLR2 agonist SMP105 was shown to activate NFκB in a TLR2- and MyD88-dependent manner. Administration of SMP105 enhanced levels of CTLs and IFN-producing cells and reduced tumor growth in mice.²⁷

TLR3 is an endosomal receptor that recognizes double-stranded RNA (dsRNA), a molecular pattern associated with viral infection.²³ The Polyinosine-polycytidylic acid (Poly(I:C)), a synthetic analog of dsRNA, is the ligand of choice for TLR3. Poly I:C has been reported to function as a potent type 1 adjuvant capable of activating Th1 type immune responses with a balanced induction of antigen-specific antibodies and CD8+ CTL. However, it was found to activate intracellular RNA sensors, RIG-I and MDA5, which are associated with toxic effects, such as systemic cytokine storm induction.²⁸ To overcome such toxic effects, modified synthetic dsRNA: Poly ICLC polyinosinic–polycytidylic acid stabilized with polylysine and carboxymethyl cellulose (Hiltonol) and phosphorothioate ODN-guided dsRNA (sODN-dsRNA) were recently developed to bind only the TLR3 but not RIG-I or MDA5, which showed similar adjuvant potency without toxic effects in a mouse model.^29,30,31

TLR4 recognizes the lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria.²³ The native form of LPS is toxic and cannot be used as it is. Therefore, 2 nontoxic derivatives of LPS, such as monophosphoryl lipid A (MPL) and glucopyranosyl lipidA (GLA), have been developed and evaluated in vivo.^32,33 However, the only approved TLR4 agonist for human applications is MPL, tested in many clinical trials as a cancer vaccine adjuvant with a good safety and immunogenic profile.^34,35

TLR7 and TLR8 are endosomal receptors that recognize single- stranded (ss) RNA molecules, such as polyU strands or imidazoquinoline derivatives, including resiquimod R-848 (TLR7 and 8) and imiquimod R-837 (TLR7 only).^36,37,38 Imiquimod, originally used as an anti-viral medication for the treatment of genital warts, is the only TLR7 ligand approved by the FDA for the treatment of precancerous skin lesions (actinic keratosis).³⁹

It has already been shown that synthetic RNA molecules protected against RNase-mediated degradation are able to activate dendritic cells and macrophages via murine TLR-7 and human TLR-8, in a MyD88 - dependent manner.^40,41,42 Recentely, Curevac have developed a novel RNA-based adjuvant, RNAdjuvant®, containing a synthetic RNA and a polymeric carrier. The RNAdjuvant® efficiently activates the innate immune system and has been shown to elicit effective anti-tumor immune responses in mouse.⁴³ The immunomodulatory efficacy of RNAdjuvant® on immune cells from cancer patients has been recently assessed in an ex vivo multiparametric platform showing comparable effects on PBMCs from both HCC and healthy subjects.⁴⁴

Finally, TLR9 is capable of recognizing unmethylated CpG dinucleotides of bacterial DNA.²³ CpG ODN is a synthetic TLR9 ligand capable of activating the TLR9–MyD88–IRF7 signaling pathway to induce type I interferons as well as the TLR9–MyD88–NF-κB signaling pathway to induce pro-inflammatory cytokines. Clinical trials using CpG ODN as immunotherapeutic agents in cancer patients, such as melanoma and non-small cell lung cancer (NSCLC) demonstrated that CpG ODNs can induce Th1 immune response and that the combination with chemotherapy can induce potent anti-tumor immune effects that correlate with clinical benefit^45,46,47

Conclusions

Cancer vaccines are weakly immunogenic due to low immunogenicity of subunit antigens. Several adjuvants have been and are currently developed to stimulate the innate immunity and induce a Th1/Th2 balanced immune response. The appropriated targets for such adjuvants are the PRRs, such as TLRs, which recognize pathogens molecules to activate maturation of DCs. Several ligands mimicking the pathogen molecules are evaluated in pre-clinical and clinical settings but only one is FDA-approved for human use in cancer immunotherapy. Heavy investigation is needed for evaluating additional adjuvants which are a key component of vaccine formulation for cancer immunotherapy, together with appropriate target antigens.

Disclosure of potential conflicts of interest

All authors declare that they have no competing interests.

Funding

The study was funded by the European Union´s 7th Framework Programme for Research, Project “Cancer Vaccine development for Hepatocellular Carcinoma – HEPAVAC” (Grant Nr. 602893) and the Italian Ministry of Health through Institutional “Ricerca Corrente.” Luisa Circelli, is HEPAVAC fellow.