Therapeutic vaccination immunomodulation: forming the basis of all cancer immunotherapy

Introduction

The consideration of vaccines being applied therapeutically to treat cancer dates back over 100 years or more and constitutes the first form of immunotherapy.^1–26 Coley’s toxins could produce complete responses, and induced fever from a nonspecific innate immune response which most likely augmented an existing adaptive immune response to the tumour. The mechanism remains unclear but may have provided ‘danger signals’ through costimulatory pathways to remove regulatory adaptive T-cell suppression. Immune stimulation for cancer therapy has a long and chequered history predating knowledge of an ‘immune system’ and possibly alchemy as a primitive medical science, since many early herbal remedies often had assumed ‘tonic’ elements of inflammatory stimulation or suppression by which they modulated diseases, which is only now being more scientifically realized. This ‘folklore’ history has not helped the modern understanding or acceptance of the immune system’s involvement in cancer therapy. Indeed, as recently as less than a decade ago, many medical oncologists and cancer therapists (now unbelievably) did not seriously consider that the immune system was involved ‘at all’ in cancer or cancer treatment, and that vaccines or vaccination had no role. Historically, vaccination was ascribed a ‘passive’, protective role against diseases, usually infective in origin. However, vaccination is almost always a truly ‘active’ process where the immune system is specifically responding to antigenic signals that stimulate B-cell (plasma) or T-cell responsiveness, and often both. As a consequence, either antibody is produced, or T cells are activated, for example, by production of cytokines or cytotoxicity. The nonspecific or innate immune system is not exempt from this process either, occupying a pivotal role in immune stimulation during vaccination events.

When the term ‘immunotherapy’ is considered, it often has the connotation of a more active exogenous form of treatment where the immune system is overtly stimulated and an effect is observable, compared with ‘vaccination’. However, the two are almost indecipherable in real, practical terms, because the immune system is activated in both, and both influence a wide range of diseases or disorders, not exclusively of infective origin.

Only now is vaccination technology and consideration of ‘vaccines’ for clinical treatment of cancer being seriously reconsidered in light of the recent immunotherapy successes and understanding. Indeed, vaccines may be essential for improving checkpoint-immunotherapy clinical response rates. This article revives understanding about vaccines and vaccination for cancer therapy in this new light, placing it within our contemporary understanding of recent immunotherapies and how they appear to be working for the benefit of the cancer patient.

Melanoma, for which most information exists, has been used as a model for immunotherapies, including therapeutic vaccines, and gradually, many principles of melanoma therapy are being demonstrated for other cancer types. In this review, the recent advances in melanoma, where immunotherapies including cancer vaccine therapies, have been explored to demonstrate technical approaches that have advanced cancer immunotherapy more generally. This presents the case for in vivo vaccination as the basis for most, if not all, cancer (immuno) therapies. Recent knowledge suggests that many of these approaches can be extrapolated and applied to other cancers, including lung, bladder, renal, Merkel cell, colon, haematological, head and neck squamous cell carcinoma (SCC), gastric and breast cancers, revealing common underlying immunotherapy mechanisms between different cancer types, and therefore suggesting that in vivo vaccination is a generalized phenomenon which needs unlocking for great benefit.

Cancer vaccine therapy

Cancer vaccines of various types have been devised and utilized for over 100 years with variable success, but notably with numerous reports of complete clinical responses (CR) with regression of all measurable disease in patients with advanced cancers of different types through immunomodulation.^1–38 Such CRs are highly significant events because they often underpin long-term durable survival periods of decades or more, effectively amounting to clinical cancer ‘cure’.^23–26 The problem has been the generally low and unpredictable rate of CR induction, or even partial response (PR) or stable disease (SD) outcomes. However, the very fact that these types of clinical useful responses occur is of great importance because they demonstrate convincing evidence that cancer vaccines offer an effective therapeutic approach which requires more intensive investigation and development.^23,25 The lack of consistency of results across many cancer vaccine platforms has unfortunately biased the view of many influential commentators against the ‘vaccine’ approach, perhaps understandably. Moreover, largely because of the lack of understanding of the mechanisms of action of cancer vaccines, and the paucity of information on the immune-system functions in general over the years, the pharmaceutical world has tended to favour simpler commercial-scale molecular synthetic options for production and marketing with the security of more financial return, such as cytotoxic chemotherapies and more recently, monoclonal antibodies. However, it should be recognized that monoclonal antibodies were recently considered relatively commercially unfavourable, too, until these mechanisms of action and production were better understood and when enough clinical trials demonstrated efficacy.

Many of the vaccine studies have been performed in the adjuvant setting following surgical removal, for example, higher-risk resected stage II and III patients rendered no evidence of disease.^33–38 That assumption undoubtedly arose from the tenet that ‘vaccination’ would be more effective for treating residual low-volume microscopic metastases by ‘preventing’ tumour recurrence, in a similar manner to the way vaccines have been utilized in preventing infection. Interestingly, the highest clinical complete response rates from vaccination have been obtained in surgically unresectable stage IV melanoma patients, indicating that the strength of the active antigenic signal might be of central importance.^23,25

Clinical efficacy has been another issue with immunotherapies, including therapeutic vaccines. The ‘clinical effectiveness’ of a treatment is where a clinical response occurs to prolong survival. That is, induction of a CR, PR or SD associated with that treatment, especially where it is the only therapy administered, or where prolonged survival results. The strength of the association of a drug with a useful clinical response makes further use of that agent more compelling for an observer. However, for the specific patients concerned who are successfully treated with resulting CRs, the treatment has been 100% effective. This aspect has made evaluation and comparison of many agents problematic, as most therapies are only ‘clinically effective’ in a small or moderate cohort of patients (even many newer therapies), and selection of responders at the outset is often difficult. For vaccine immunotherapy (and indeed, all immunotherapy until recently), the relative clinical efficacy of inducing CR, PR, SD or prolonged survival has been low, and this has tempered acceptance of immunotherapy in general terms.

Another issue increasingly being recognized is the fact that immune responses are variable in their speed of onset, intensity and duration. Some clinical responses to vaccine therapy for advanced malignancy can be rapid, occurring within a few weeks, with immediate noticeable regression of cancer deposits, although this speed of onset is not commonplace for most vaccine/immunotherapy regimens, it has been observed. Usually, vaccine responses are slower and can take many months to develop. This has been a real issue arising chiefly from our contemporary understanding of chemotherapy and radiotherapy clinical responses which are usually considered to be more immediate, gauged in terms of 2–3 months to observe an effect. With vaccines, and with multiple other forms of immunotherapy, it is being increasingly appreciated that responses take longer, sometimes up to 6–12 months, where partial responses and stable disease can sometimes gradually convert to a complete clinical response. Pseudo progression can also occur where the tumour enlarges due to inflammatory swelling. Therefore, the classical World Health Organization and RECIST criteria perhaps need to be considered and applied differently, or modified, which has led to the newer immunological response criteria,^39–41 which is gradually gaining acceptance, including by the US Food and Drug Administration (FDA). Future refinements of the currently available methods of measuring successful responses to immunotherapy might include better volumetric assessments, such as utilization of three-dimensional computed tomography scanning, positron emission tomographic volume scanning, heat mapping or magnetic resonance spectroscopic analyses. Part of the remaining issue with all of these methods has been establishing techniques that are time effective in more accurately assessing changes in multiple metastases, because most current evaluation tools for tumour size assessment only provide approximations for the response to therapies but are rapid, practically. It should be noted, however, that CRs are essentially detectable by most methods, although detection of small tumour volume (<4 mm diameter) remains an issue by any method except pathological sampling.

Tumour antigens and mutational load or burden

Tumours are genetically and antigenically heterogeneous.^42,43 A wide range of internal and externally expressed antigens are present within tumour cells, including peptides and glycolipids. Most of the focus has been on peptide antigens and major histocompatibility complex (MHC) antigen presentation; however, glycolipids and CD1 presentation of these has been relatively ignored. ⁴⁴ Moreover, refined selected peptide antigens appear less capable of inducing effective immune responses against tumour than mixed multiple antigens which can provide a stronger signal to the immune system.^22,25,32 There is evidence that intense selection pressure is applied from the tumour microenvironment in early clinical untreated non-small-cell lung cancers (NSCLCs) by the immune system which produces multiple routes to immune evasion, leading to different levels of mutational burden, clinical behaviour and disease outcomes. ⁴⁵ Indeed, neoantigen depletion appears to occur in many tumours that have become ‘unresponsive’ to therapies of different types, including inhibitory-checkpoint blockade. This is likely a mechanism for evasion of antitumour immune responses and resistance to therapies. Moreover, the immune microenvironment is highly variable between tumours, patients, and between metastases, and even within the same tumour deposit within patients with almost 30% of tumours showing diverse levels of immune infiltration. This implies that different metastases within the same individual and even parts of the same tumour mass will demonstrate differential susceptibilities to many therapies, including checkpoint blockade, due to their relative expression of tumour antigens, and thus proportionate T-cell stimulatory capacity, leading either to immune evasion (low mutation/antigen expression) or an effective response (higher mutation/antigen expression). ⁴⁵

Higher tumour mutational burden across a number of tumour types showed an association with improved survival. Patients treated with immune-checkpoint inhibitors showed an improved survival among those with a higher mutational burden, although the degree of mutational load required to confer the improved survival varied with tumour type. ⁴⁶ This infers that the greater the mutational load, the greater the antigenic signal and therefore the stronger the in vivo (vaccine) immune response to the tumour. When high-mutational-burden tumour cells are killed, multiple tumour-associated antigens are released and further (potentially stronger) T-cell-mediated (in vivo vaccine) immune responses can proceed.

In patients with NSCLC treated with first-line nivolumab plus ipilimumab, higher tumour mutational burden was associated with significantly longer progression-free survival than for chemotherapy-treated patients, regardless of the degree of programmed cell-death ligand 1 (PD-L1) expression, ⁴⁷ suggesting tumour mutational burden as a potential independent biomarker for response to checkpoint agents. Transfer of ex vivo-cultured autologous tumour-infiltrating lymphocyte (TIL) has been associated with higher CR rates of around 20%, and Prickett and coworkers have also demonstrated that these autologous TILs recognize a range of distinct mutated gene products so that the TILs represent an enriched population of T cells capable of recognizing autologous tumour antigens but are inhibited, so that responsiveness is reduced; however, in the right circumstances (autologous transfer), can elicit a powerful response inducing a CR. ⁴⁸

In vivo and in situ vaccination

If the hypothesis is correct that tumour-cell death or injury leads to tumour antigen release, and further, that antigen release and immune stimulation is necessary for an effective antitumour immune response, then it follows that many forms of local (in situ) injury or damage to, or lysis of, the cancer cell ‘in situ’ or ‘in vivo’ (in the patient) can result in multivalent antigen stimulation of the immune system. However, ‘immune symbiosis’ or ‘immune homeostasis’ is what probably prevents elimination of cancers in vivo unless enough immune perturbation occurs and sufficient immunomodulatory stimulation can then reject the cancer (Figure 1). Clearly, cytotoxic therapies such as chemotherapy and radiation are directly aimed at damaging tumour cells. Complete surgical resection of metastases is reported with over 20% of patients surviving 4 years or more, some with durable longer-term survival periods.^42,52 It is currently unclear whether this 20% of survivors is similar to the 20% longer-term survival mentioned above for immunotherapy and vaccine therapies. Surgery itself is associated with local and systemic inflammatory responses and in some cases, tumour-cell lysis. Therefore, many forms of cytotoxicity or cellular injury through the spectrum of available and utilized cancer therapies, including traditional chemotherapy, radiation therapy and surgery, or any of the newer therapies, such as inhibitors, receptor blockers or immunotherapies, can exert this effect of antigen release and immune stimulation. ²⁴ This means that many of the available cancer therapies are in fact capable of exerting a vaccine effect, intentionally or otherwise. The mechanisms of this vaccination effect through repeated waves of release/exposure of tumour antigen in vivo is not widely appreciated, although it has been observed that tumour antigens are released when cells are lysed or when irradiated, intact, whole tumour cells have been used as vaccines. Further, T-cell antigens specific for the respective tumour have been extracted (eluted) from TIL and these antigens have been used either directly or synthesized from their sequences in peptide- or glycolipid-based vaccines, and combination with the recent immunotherapeutic monoclonal antibody agents is being explored in trials. The processes of antigen spreading, immunoediting, tumour antigen identification and sequencing, the presence of mutations within the tumour and the extent of these (mutational burden), antigenic immune selection with neoantigen emergence, tumour–antigen heterogeneity and multiclonality, the role of pluripotential stem cells, host tumour microenvironment, tumour extracellular matrix and vascular properties, proteomic and metabolic properties and pathways in tumours, mitochondrial function, and tumour microenvironmental (and systemic) regulatory immunomodulation through lymphocytic, dendritic cellular and macrophage pathways are being actively explored. All of these facets are pertinent to the discussions and understanding of tumour immunology and therapeutic vaccine design, and although germane to the argument of in vivo vaccination, some are beyond the scope of this review and are discussed elsewhere.

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Figure 1.

Cancer immunomodulation immunotherapy vaccination cycle.

In vivo natural tumour oncolysis

During the growth of cancers, small, early, cancer-deposit expansion of cancer cells initially keeps pace with the vascularity, so that nutrition is adequately provided to the cancer cells in the growing mass of tumour tissue. However, it is apparent that many cancer cells are not entirely immortal and a proportion of the population of cells within the cancer die through the natural mechanisms of apoptosis and necrosis. This process becomes accentuated when the growth of cancer cells outstrips the vascular supply to the mass of tumour tissue, leading to death of cells that do not receive adequate nutrition. ‘Central necrosis’ of the tumour is a commonly observed phenomenon which is most associated, although not always, with larger tumour masses. It has been assumed that the reason necrosis occurs centrally rather than elsewhere is the progressive loss of vascularity in the deepest and most remote parts of the tumour mass away from the outer vascular shell, leading to effective devascularization of deeper parts. This assumption arises largely because the main vascular supply derives from outside the tumour mass, often with the peritumoural blood supply being observably relatively rich at the tumour surface, with communicating vessels into and out of the tumour, typically becoming progressively less prolific deeper into the tumour mass. However, it is also well recognized, that for some tumours, the cut in vivo tumour surface can bleed freely, demonstrative of high internal vascularity, so there are other mechanisms that produce the necrosis, such as tumour necrosis factor alpha (TNF-α) and other cytokines. Importantly though, the natural cell death typically results in a combination of apoptosis and necrosis, and these processes serve to release tumour antigens and cause in vivo vaccination. Repeated cycles of therapy can produce waves of repeating in vivo vaccination.

Oncolytic viruses

Another wave of intense interest has developed relatively recently concerning viruses, and modifications of them, for treatment of cancer. Virus therapy for cancer, also termed virotherapy, falls into two basic groups: (a) viruses that infect cancer cells and induce expression of viral antigens on the surface of cancers cells, making them more visible to the immune system through improved antigen recognition, resulting in better stimulation of the immune response, thereby eliciting a more effective anticancer response, together with enhanced stimulation of the production of interferons and other cytokines by adjacent immunological and stromal cells (innate immunity activation); and (b) oncolytic viruses, which are termed ‘oncolytic’ because they are capable of lysing tumour cells (more) selectively over normal cells, again with direct in situ stimulation of the immune response and cytokine cascades. The term ‘oncolytic’ has been used less precisely than desirable for viral therapies that induce immunogenic apoptosis rather than true ‘oncolysis’ (bursting) of cancer cells via the necrotic death pathways. Indeed, there is becoming much imprecision in the terminology that is used. This ‘looseness of terminology’ has, and continues to, hamper the proper definitions in the immunological therapeutic virotherapy field. Immunogenic apoptotic-cell death probably should properly be termed ‘oncoptosis’ rather than oncolysis, because no bursting lysis occurs; rather, the cells involute and shrivel. The use of oncological virotherapy has a long history, much greater than many people may think, dating back to the early 1930s, with the first larger-scale research trials being performed and reported over the 1960s and into the 1980s.^33–38 Many types of animal and human viruses have been tested, including adenovirus, vaccinia, Newcastle disease virus, reovirus, measles, herpes simplex and zoster, parvo, and ECHO-7, principally as a consequence of their ability to infect and cause selective lysis of tumour cells over normal cells, and thus act as a vaccine. The action of oncolytic viruses can be harnessed either through systemic infection of the patient and in situ oncolysis in vivo, or through ex vivo means first lysing the cultured tumour cells in vitro external to the patient, and then administering the lysate in in vivo doses. The principal effect of oncolytic viruses is typically natural oncolysis of abnormal cell membranes, usually on malignant cells, but oncolytic actions on normal cells can occur as well, for example the vesicular eruptions of normal tissues seen with the cutaneous manifestations of measles or pox-virus infections, as in herpes simplex (coldsores or shingles) or small pox.

Most routes of administration have been used, intravenous, intramuscular, inhaled, oral, subcutaneous and intratumoural, but perhaps the most clinically effective route has been intradermal, probably due to the abundance of dendritic and other antigen-presenting cells in the skin with relatively rapid drainage via lymphatic channels to regional lymph nodes. The most recent addition to the entourage of oncolytic viral agent therapies is T-Vec (talimogene laherparepvec; Imlygic®, Amgen, Thousand Oaks, California, USA) which is a genetically engineered herpes virus with two genes removed and a gene for GM-CSF being added. ⁵³ Injection of T-Vec into tumour deposits has been associated with necrosis/apoptosis of the tumour cells with observable clinical responses in the injected tumour deposits, with reported bystander responses in adjacent lesions, and also some systemic responses indicative of a systemic immune response.^44,54,55 T-vec has been combined recently with anti-PD-1 therapy with some improvement in the response rates, but the full results are currently not available yet,^56–59 and combined with ipilimumab, showing an objective response rate (ORR) of 39% versus 18% with ipilimumab alone. ⁵⁸ Interestingly, there was a doubling of the visceral response rate in the combined versus single-agent ipilimumab arm, a phenomenon (systemic; abscopal) also discussed later, and CRs with combined radiotherapy.^58–60 A Coxsackie-virus-based vaccine therapy has been reported and is being commercialized with similar application, injected into cutaneous and subcutaneous melanoma deposits with observable responses in the injected and adjacent non-injected masses.^56,61,62

Vaccination from cytotoxic chemotherapy and chemical oncolysis

Most current cytotoxic chemotherapy kills a percentage of cells in the tumour which releases tumour antigens. Cytotoxic chemotherapy primarily interferes with cell division through interrupting nucleic acid replication and processing. The interaction of cytotoxic agents with the deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins or cell cytoskeletal functions (mitotic spindle) is generally well understood, but the effects leading to cell death and consequently on cancer growth is perhaps less clearly determined. Further, the effects of cytotoxic chemotherapy on the cell membrane and antigens is currently quite poorly understood. Classical cytotoxic chemotherapy induced cell death is chiefly via an apoptotic pathway by acceleration of senescent cell death pathways (apoptosis). This itself can have immunogenic consequences arising from macrophage and dendritic-cell engulfment and presentation of tumour antigens to the immune system in vivo. However, the resulting onco-apoptosis can follow the usual line of normal senescent cell death with low or little stimulation of host immune mechanisms (even immune-response regulation or suppression), the so-called ‘nonimmunogenic’ apoptotic pathways. This constitutes a type of involutional cell death. The distinction between induction of nonimmunogenic versus an immunogenic route of apoptotic-cell handling is not clearly understood at this time, but the presence of danger signals appears to be important in determining whether the immune system responds and the strength of that response to apoptotic end products of cell death. Consequently, inflammation and immune responses can arise from vaccination by apoptotic cancer-cell bodies as a result of cytotoxic chemotherapy cell death. The critical problem, as far as cancer destruction is concerned, is that apoptosis does not usually act as sufficiently strong a stimulus for inflammation, nor engender an active immune response, in most cases.

However, it is less well appreciated that cytotoxic chemotherapy can damage the cancer-cell membranes and that this can give rise to a different type of immune response. ⁶³ When the cancer cell membrane is damaged, and this can occur through a variety of mechanisms related to the membrane structure or function, the cell can no longer maintain its integrity to the degree that the cell lyses or bursts. This process is chemical oncolysis, and necrotic cell death occurs (rather than apoptosis). Lysis of cancer cells produces release of membrane fragments and antigenic molecules associated with the cancer-cell membrane. Not only are a variety of cancer antigens released, but danger signals are typically present as well. Tumours can become erythematous, hot and inflamed, sometimes associated with fever.

Chemical damage to the cancer-cell membrane from cytotoxins can be sufficient to release antigens from the cell surface but without killing the cancer cell, thus liberating antigens that effectively act as an in vivo vaccine to stimulate the immune response.

Classical chemical cell death occurs when damage to the cell membrane, nuclear material or cytosol is sufficiently great to lead to rupture of the cancer cells (oncolysis or necrosis). Cytotoxic chemotherapeutic agents create chemical injury that may damage the nuclear material (e.g. mustard compounds), cytosolic elements (e.g. antibiotic agents like dactinomycin), or the cell membrane structurally or functionally. The intensity of the damage and the capacity of repair mechanisms to correct the injuries determines the ability of the cell to survive or if sufficiently deleterious, causes the death of the cell. For example, if the membrane is damaged such that the sodium/potassium adenosine triphosphate (ATP) pump is affected then the cell swells and bursts, which effectively constitutes lysis and an in vivo vaccine event.

A multitude of mechanisms exist which result in cellular damage to the cancer cell, altering, for example, functional capacity and survival, with the outcomes not always being predictable due to the inherent cellular heterogeneity that exists within most tumours.

Other chemical agents have been reported to act directly on cancer cells to induce cell killing, such as the pink dye Rose Bengal (also known as PV-10; Provectus, Tennesee, USA).^64,65 This agent selectively penetrates the cancer-cell membrane, but not the cell membranes of normal cells, to act internally at the lysosomal level, leading to cell death. The cell death appears to be apoptotic, leading to autophagy. PV-10 is injected directly into cutaneous and subcutaneous melanoma masses causing lysis and necrosis of tumour, inducing local inflammatory responses in the injected lesions. Interestingly, ‘bystander’ responses are reported in non-injected lesions and some systemic responses were also noted, indicating generation of a systemic immune response. Although the cellular response to PV-10 is reported to be involutional and apoptotic, clinically, the injected tumour masses undergo overt ulceration more indicative of necrosis. It is therefore likely that the response to direct injection of PV-10 into tumours is a mixed necrotic and apoptotic process. This would explain the observed clinical responses of bystander and systemic responses, which come from induction of local, regional and systemic immune responses, most likely as a result of immunogenic danger signals arising from necrosis. Indeed, this model probably serves as a generalizable model for many agents that locally damage the tumour, causing an element of tumour necrosis, for instance T-Vec, Cavatak® Merck, New Jersey, USA (Coxsackie virus) and radiation therapy.^53–66

Vaccination from physical oncolysis

Radiation therapy causes both apoptosis and necrosis in tumour cells exposed to sufficient levels of radiation to damage DNA. The resulting cell-membrane damage and lysis releases tumour antigens from the malignant cells, producing in vivo vaccination events as each cell is damaged or dies. Radiation-induced antitumour immunity has been previously described and can act as an immune adjuvant by exerting systemic immunological effects beyond the local radiation field of delivery.^66–71 In another study following radiation exposure in murine and human melanoma, DNA damage-response-marker expression was upregulated days prior to immune-marker expression of death receptors and T-cell costimulatory/co-inhibitory ligand changes indicating a time-dependent effect. ⁶⁹ Complete regression of established murine tumours in most animals using combined local radiation and intratumourally injected IL-2-linked tumour-specific antibody immunocytokine and regression of large tumours was reported with the addition of systemic anti-CTLA-4 T-cell-checkpoint blockade. ⁶⁹ The ‘abscopal effect’ of radiation therapy where tumour regresses outside of the field of radiation has been well recognized both clinically and in animal models, where locally administered radiation can cause systemic immune response against remote tumours not receiving radiation exposure. ⁷¹ Indeed, MacManus and colleagues showed that 1.1% of 2337 patients with NSCLC receiving localized palliative radiation therapy survived 5 years or more after therapy, indicating that radiation could induce systemic responses with improved survival. ⁷²

Radiation and other forms of physical cellular damage induces damage (or danger)-associated molecular patterns (DAMPs), intrinsic danger signals (also known as alarmin) that are capable of eliciting an inflammatory response in a non-infectious situation. These are a diverse variety of predominantly intracellular molecules that are not usually revealed at the external aspect of the cell membrane but can become expressed on the cell surface of damaged cells or are released when cells are lysed when the contents are released into the surrounding tissues and vessels. DAMPs are released in response to trauma or injury to tissues, for example, and when cancer cells are injured from a variety of mechanisms, the DAMP molecules are released into the tissues and are capable of inducing an inflammatory response.

DAMPs are tissue dependent and include intracellular proteins, for example, heat-shock proteins (HSPs), or the chromatin-associated lysosomal protein high-mobility group box 1 (HMGB1), and also include extracellular-matrix-associated stromal proteins, such as hyaluronan fragments. DAMPs also include nonprotein molecules like DNA, adenosine, ATP, uric acid, and heparan sulfate. Calcium regulatory proteins such as S-100 are also able to act as DAMPs in some situations, and even calcium itself, depending on the concentration and location. DAMPs appear to be concentration and location dependent in terms of how great their stimulatory capacity is on inciting danger signals in the immune system in a particular situation.

DAMPs perform a similar function to pathogen-associated molecular patterns (PAMPs) which are a variety of molecules associated with (extrinsic) pathogens usually recognized by the innate immune system through TLRs and other pattern-recognition receptors in plants and animals. Although there are many PAMPs, examples include glycans, bacterial lipopolysaccharides, glycoconjugates, endotoxins, flagellin, lipoteichoic acid, peptidoglycan, viral nucleic acids [e.g. double-stranded ribonucleic acid (dsRNA)] and CpG. Many microbes contain natural danger signals capable of inciting an immune response upon exposure to the animal and human immune system, as an evolutionary adaptation. Such responses have been selected for over millions of years. ⁷³ Cancer cells, at least within established tumours, are not only of ‘self’ origin, but are usually lacking the necessary danger signals for eliciting an effective immune response, which forms part of the observed ‘nonresponsiveness’, or natural ‘tolerance’ of tumours in the host. When danger signals are present together with tumour antigens then an immune response can proceed, and this may explain some of the ‘spontaneous regressions’ of cancer observed following an infective or traumatic episode. Many of the current cancer immunotherapy trials are now utilizing adjuvants or danger signals with vaccination, or checkpoint-agent therapy on this basis. Examples are heat-killed vaccines and adjuvants, such as Coley vaccines, site-specific immunomodulators, and systemic immune modulators. Imodulon (IMM-101, Immodulon Therapeutics, Uxbridge, UK) contains heat-killed Mycobacterium obuense and is capable of modulation of the innate and adaptive immune systems in response to cancer, through interaction with a number of receptors (PAMPs) and γδ T cells, granulocytes, and antigen-presenting cells, to downregulate a type 2 (Th2) response bias, in favour of a type 1 (Th1) response.^74–79 Radioprotective effects can also occur in tissues through TLRs; either arising from DAMPs or through secondary infection (PAMPs) or when TLR agonists are administered with radiotherapy. ⁸⁰ In essence, radiation therapy can act to cause tumour cell death with in vivo vaccination events through release of antigen in waves with each successive treatment cycle stimulating the immune system; or produce radioprotective responses in target tumour cells, the balance of which can determine the clinical response.

Topical and systemic sensitizing agents

Topical sensitizing agents have been used for many decades as mechanisms for adjuvant boosting for the immune system and have been shown to induce regression of cancer due to stimulation of a pre-existing in vivo immune response. These agents are usually administered percutaneously by painting them onto the skin overlying the tumour deposit such that local absorption of the agent influences the local tumour microenvironment; however, bystander responses have been observed where nontreated metastases also can regress, indicative of enhancement or development of a systemic immune response.^81–83

Such immunomodulating agents can act as effective therapies, even after standard therapies have failed, although the response is unpredictable. The agent 2, 4-dinitrochlorobenzene (DNCB) was one of the early agents demonstrating effective clinical responses. Using intralesional DNCB for treating in-transit metastases, CRs occurred in about 60% of patients. ⁸¹ DNCB has also been used topically.^82–84

Diphencyprone (DPCP) is another topically administered agent that has been used more recently to induce immune responses in patients with surgically nonoperable deposits, especially in difficult areas, and has shown remarkable results in some patients with high rates of complete (46%) and partial (38%) regressions being recorded.^85–87 DPCP immunotherapy has been used to treat cutaneous warts and alopecia areata, and more recently, has been used as a single agent to successfully treat radiotherapy-resistant extensive, multiple scalp-melanoma metastatic confluent nodules not amenable to surgery.^85,86 The fact that complete responses have been reported in locally advanced melanoma, with bystander effects in nontreated lesions and regression of visceral metastases, are highly significant observations. ⁸⁷ Treatment was well tolerated with daily topical applications, was non-invasive and very cost effective.

Both DNCB and DPCP have been utilized combined with other therapies, notably radiation therapy and chemotherapy, and appear to augment these in some situations. ⁸⁸

Dinitrophenyl (DNP) is another agent, used as a hapten, to bind to tumour-cell surface ex vivo (in vitro culture) and are then injected back into the patient to form a melanoma vaccine.^89–93 Multiple intradermal injections of DNP-modified autologous tumour cells mixed with Bacillus Calmette–Guérin (BCG) acting as an immunological adjuvant/stimulant were used to induce and enhance local inflammation in the metastatic melanoma deposits to incite a systemic immune response against the patient’s tumour. ⁹⁰ Delayed-type hypersensitivity (DTH) developed in almost all patients against autologous DNP-modified melanoma cells and in about 50% of patients exposed to autologous, unmodified tumour cells. Development of DTH was associated with significantly longer survival. The 5-year overall survival (OS) rate in 214 stage III patients was 44%, compared with a survival of 20–25% after stage IV surgical metastasectomy. ⁹² DNP autologous vaccine treatment following low-dose cyclophosphamide was also used for 97 (83 evaluable) unresectable stage IV melanoma patients with 11 (13%) clinical responses, 2 (2.5%) CRs, 4 (4.5%) PRs and 5 (6%) mixed responses. Survival was prolonged for the responders, and those with DTH to unmodified autologous tumour cells. Regression of tumour required at least 4 months, with some cases not achieving maximum regression until up to 1 year after commencing treatment. Long survivals beyond 2 years and 5 years occurred in some patients developing CR and PR.^90–92

Interestingly, antibody responses to DNP have been shown to directly correlate to better outcome. One primary mechanism of action of these agents appears to be haptenation of cell surface and cytoplasmic proteins, which can induce CD8(+) T-lymphocyte-mediated allergic-contact-hypersensitivity responses.

Haptens are small molecules acting as incomplete antigens, often without their own intrinsic activity, but when bound to another, often larger protein, can act with this carrier to generate stronger immune responses. The response strength is determined by several host factors that can determine immunological responses, such as nutrition, tumour burden, the pre-existing immune status and immunosuppression. Haptens can also produce autoimmunity, which is of considerable interest, since the clinical efficacy of checkpoint-inhibitory agents are directly associated with the generation of autoimmune side effects. Haptens can augment or create an effective or partially effective antitumour immune response when bound to a carrier protein that is a suitable tumour antigen ⁹³ Another immune stimulant, imiquimod, stimulates TLR 7/8 and has demonstrable antitumour activity, especially against skin cancers [basal cell carcinoma (BCC), SCC and melanomas] which is addressed later ⁹⁴ (Figure 2).

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Figure 2.

Mechanism of in vivo cancer vaccination from multiple divergent therapies.

Systemic and local intratumoural therapies

Small-molecule inhibitors

BRAF therapies, often combined with MEK inhibitors, have been used in the approximately 40–50% of melanoma patients who have BRAF-mutation-positive tumours. CR rates are variably reported between 0.9 and 6.25%. ²⁶ The median OS was 26.1 months for combined dabrafenib plus trametinib therapy, while in the group treated with the combination of vemurafenib and cobimetinib, the median OS was 22.3 months, while vemurafenib alone was 17.8 months (HR = 0.68). While 3-year OS was 45% and 31%, respectively, for patients treated with vemurafenib and cobimetinib, or vemurafenib alone.

Vemurafenib-alone therapy versus dacarbazine was trialled in 675 patients in the BRIM-3 study and showed a median OS of 13.3 months versus 10 months for V600E-mutant advanced melanoma, respectively.^124,125 In the final results, 84 dacarbazine-group patients crossed over to vemurafenib, and the OS rates for vemurafenib versus dacarbazine at 4 years were 17% versus 16%, and at 1, 2, 3 years were 56% versus 46%, 30% versus 24%, 21% versus 19%, respectively, by Kaplan–Meier estimates. ¹²⁶

The action of BRAF inhibitory agents is to block the BRAF pathway, reducing cancer-cell proliferation and inducing cell death. Spectacular reductions in active metabolizing melanoma metastases have been demonstrated, although gradual development of resistance to the agents is commonplace over time with a median duration of effect of about 11 months before resistance to the therapy develops. ¹²⁵

The induction of cancer-cell death releases tumour antigens that act also as repeated vaccine events, promoting an immune response. Recent reports indicate that clinical efficacy is associated with infiltration of lymphocytes around metastatic deposits, and removal of lymphocytes close to the tumour results in loss of effectiveness, indicating that these agents are operating through immune mechanisms as well.^127–129

Interestingly, breaks in BRAF treatment with later reinstitution, and combination with other immunotherapies, have been associated with durable CRs in patients with BRAF-mutated metastatic melanoma after initial failure of sequential immunotherapies (high-dose IL-2 followed by ipilimumab with or without concurrent radiation therapy). ¹²⁹ The presence of non-Treg, CD4-positive effector-phenotype T cells in these patients was associated with the durable responses. Synergism between conventional or targeted cytotoxic therapy and immunotherapy in cancer treatment is suggested. ¹²⁹ Rechallenge after a break in failed BRAF inhibitor therapy has been demonstrated in several studies to induce clinically useful responses, including PRs and CRs.^130–132

The above local and systemic therapies are capable of killing tumour cells, causing release of tumour antigens in vivo, thus creating local vaccination events, with systemic manifestations. Most therapies rely on repeated cycles of treatment for their clinical effects, thus repeated waves of tumour cell death and repeated vaccine events. The immunotherapies are next addressed with similar consideration due to their ability to induce T-cell-mediated tumour-cell killing and in vivo antigen release.

Recent immunotherapies

Anti-CTLA-4 therapy: ipilimumab

Hodi and colleagues reported a median OS of 10 months for ipilimumab plus gp100-treated advanced-melanoma patients, compared with 6.4 months for gp100 alone (HR for death, 0.68; p < 0.001) and 10.1 months for ipilimumab alone. No difference in median OS was detected between the ipilimumab groups with or without gp100.

Serious immune-related adverse events (grade 3 or 4) were reported in 10–15% of patients with ipilimumab treatment, but 3% treated with gp100 alone, with 14 deaths related to the study drugs (2.2%), most being autoimmunity related. ¹³⁹ The OS at 3 years for ipilimumab alone was 34% in the Wolchok and coworkers ¹⁴⁰ study; the ORR was 10.8% (4/37) in the ipilimumab-alone group (p < 0.001), with a CR rate of 0 in the ipilimumab-alone group. ¹⁴¹

Anti-PD-1 therapies

Checkpoints combined with other agents

Following the response rate of 84% using topical DPCP for cutaneous melanoma metastases in a 50-patient case series with nodal or visceral metastasis regression in four of the patients, ⁸⁷ DPCP was combined with the PD-1 inhibitor nivolumab treatment which showed internal metastasis regression.^153,154

In a small phase Ib clinical trial of 21 stage III/IV patients, 6 (43%) of the 14 patients with visceral metastatic melanoma had complete regression reported following combined intralesional T-Vec and anti-PD-1 therapy. ⁶⁰ The ability of combined intralesional T-Vec and anti-PD-1 therapies to induce complete regression of distant metastases was also demonstrated in two patients with lung metastases in a 10-patient stage III/IV study. ⁵⁷ These are further examples of a local therapy combined with a systemic therapy to induce ‘off-target’ systemic responses. The overall response in injected lesions was reported as 90% with 6 (60%) of the 10 patients achieving lesional CRs.

Other immune-checkpoint protein-modulating agents

A number of clinical trials are currently investigating drugs that target other checkpoint-control proteins such as OX40, B7-H3, and LAG3. ¹⁵⁵

Cellular therapies

These include treatments under the categories lymphokine-activated killer cell, TIL, adoptive cell therapy, T-cell receptor TIL therapy, autologous circulating T cells targeting either unspecified tumour-associated antigen or a tumour-specific antigen, chimeric antigen-receptor T-cell (CAR T) therapies, other experimental T-cell therapies based on pluripotent stem cells, CRISPR or γδ-T cells, or cell therapies based on DCs, NK or NK T cells, macrophages or other cell types. Some of these therapies have been associated with a variety of lymphodepletive and other preconditioning regimens such as marrow ablation, using cytarabine and intensive whole-body radiation. The fact that lymphocytes can be cultured or engineered and reinfused to produce homing and specific tumour killing demonstrates the ability of the immune system to treat cancer, and reinforces the in vivo actions of anticancer vaccines to produce effective lymphocyte clones. Combined therapies, including cellular therapies as a component are being trialled clinically. The field is broad and emerging, even though some aspects have been decades in development, and many of the therapies are currently heavily individualized and costly. Toxicity, including death, has been a problem in some studies leading to holds placed on some trials, but the technique looks promising with future refinement and generalization potentially making it more cost effective on a larger scale, especially if allogeneic methods are developed. Cellular therapies have been reviewed in a number of articles and are not detailed further here.^165–175

Concluding remarks: vaccination as the basis of immunotherapy

What is so remarkable is that many vastly different treatment modalities for cancer can all induce a definite, but often small, rate of complete clinical regression responses. This fact is highly indicative that a common underlying mechanism is likely responsible and is operating to cause this effect and observation. Moreover, it is rapidly emerging that for almost all cancer therapies, the immune system is either directly or indirectly implicated. For example, small-molecule BRAF inhibitory therapies appear dependent on an immune infiltrate into the tumour occurring and are severely reduced or abrogated when the immune system is depleted. Clearly, the checkpoint inhibitors and IL-2, IFNs, T-Vec and vaccines, all being immune stimulants, function through the immune system for their activity. It is rapidly emerging that radiation therapy, chemotherapy, electrochemotherapy, oncolytics, cell-pathway inhibitors and receptor-blocking agents have an immunomodulatory role too, and that due to the ability of these agents to damage or lyse and kill cancer cells, those apparently cytotoxic therapies can provide release of tumour antigens which constitute active waves of in situ, in vivo vaccine events; in essence, they are performing as vaccines to cause their effects (Figure 3).

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Figure 3.

All cancer therapies can lead to in vivo vaccination events.

The tumour microenvironment (TME) comprises tumour cells, cancer-associated fibroblasts, endothelial cells, myelomonocytic cells, MDSCs, TILs, extracellular matrix, and vessels (arterial, venous and lymphatic), nerves, and a variety of peritumoural stromal vascular and leukocytic cells (some normal and some tumour influenced). This level of complexity has made understanding of the interaction between all of these elements in successful and unsuccessful regression of cancer enigmatic.

The phenomenon of peritumoural cuffing of lymphocytic infiltrates has been observed in a number of therapeutic approaches and has been related to successful outcomes. Indeed, the relationship between lymphocytic infiltration and survival has been historically noted for a long time in breast^{176, 177} and many other malignancies. A possible future direction would be more detailed studies of the TME across many different modalities of therapy (and tumours) in order to investigate for possible common features or differences that might more clearly indicate similar underlying mechanisms associated with response or failure. For example, the state of the TME before therapy, such as the activation status of the T cells prior to any therapy, has emerged as pivotal. In general terms, tumours that are ‘hot’ and have higher levels of inflammation with activated T cells and low regulatory-cell densities appear to be better positioned to respond to immunotherapies, while tumours that are ‘cold’ and are in a suppressed state with a low infiltration of T cells respond less well to immunotherapies. One of the key approaches is therefore to modulate ‘cold’ tumours into ‘hot’ ones to prepare the TME for more successful immunotherapeutic intervention.

As we understand more about the immunological mechanisms associated with the therapies above capable of causing tumour regression, that is, antigen release, neoantigen unmasking/exposure, the control of antigen recognition, immune responsiveness, immunosuppressive T-reg responses to inhibit TIL activation, suppressor macrophages, augmentation of initially weak antitumour responses into effective tumour killing, how innate immunity interacts with adaptive responses and how these relate to natural vaccination responses that continually occur to a vast range of exogenous and endogenous antigens throughout life, the prospect of in vivo vaccination and immunomodulation will become clearer. To generate an effective antitumour immune response, antigen must be released/supplied, immune recognition must occur and immune responsiveness proceed.

In essence, whenever tumour cells are killed, antigen release can provide repeated in vivo vaccination events. Most of the treatments used for cancer outlined above require repetitive cycles of therapy to achieve cancer regression when it occurs. This aspect is now emerging as possibly the most important mechanism by which perhaps the entire range of standard and newer cancer therapies are exerting their anticancer effects, by causing waves of antigen release and repetitive in vivo vaccination events.