The history of immunotherapy of cancer dates back to 1890s when New York surgeon William Coley used Streptococcus and Serratia bacterial extracts to treat cancer. Up to the mid-1930s ‘Coley’s mixed toxins,’’ were used to treat various tumors. Better understanding of the human immune system led to the identification of a number of tumor-associated antigens (TAAs) in the 1980s  and development of various immunotherapeutic approaches. Of particular relevance to melanoma immunotherapy was the identification of various antigens expressed specifically in melanocytes and, respectively, in the majority of melanomas. These melanoma-associated antigens include tyrosinase (Tyr), a key enzyme in melanin biosynthesis, tyrosinase-related proteins 1 and 2 (TRP1, TRP2), gp100 (aka pmel17), Melan-a, and MART1. These and several other melanoma-associated antigens formed the basis for the immunologic targeting of the tumor. Up to date, multiple peptide, dendritic cell, adjuvant, lymphocyte, antibody, DNA and virus-based strategies were tested in pre-clinical and clinical studies with varying degrees of success. In recent years, identification of the specific antigenic MHC class I epitopes, advancements in genetic engineering, gene delivery, and cell-based therapeutic approaches allowed development of the novel melanoma-targeting immuno-therapeutics.
2. Genetic engineering of antigen-specific T cells
2.1. Recombinant T cell receptors
Identification of the tumor-reactive T cells among a population of the tumor-infiltrating lymphocytes led to the development of the T cell-based therapies, particularly to the strategy known as adoptive T cells transfer. This strategy is based on the isolation of the tumor-infiltrating lymphocytes following analysis of their ability to target tumor cells and clonal expansion of tumor-reactive T cells via stimulation of cell proliferation with anti-CD3 and antiCD28 antibodies in the presence of IL-2. Upon obtaining a large quantity (> 108 cells), these cells are infused back to a tumor-bearing patient along with the lymphodepleting chemotherapy to temporary knock down circulating immunocytes and repetitive administration of the IL-2 (Fig. 1).
Presently, 87 clinical trials using TIL are completed or on-going. These clinical trials are aimed at treatment of multiple cancers including: Malignant Melanoma, Nasopharyngeal Carcinoma, Hepatocellular Carcinoma, Breast Carcinoma, Leukemia, Lymphoma, Multiple Myeloma, Plasma Cell Neoplasm, Kidney Cancer, Metastatic Colorectal Cancer, Metastatic Gastric Cancer, Metastatic Pancreatic Cancer, Metastatic Hepatocellular Carcinoma, Cervical Cancer, Oropharyngeal Cancer, Vaginal Cancer, Anal Cancer, Penile Cancer, Non-Small Cell Lung Cancer, Brain and Central Nervous System Tumors. Several completed clinical trials on malignant melanoma clearly demonstrated that infusing TILs along with IL-2 and pre-conditioning with reduced-intensity circulating lymphocyte-depleting chemotherapy mediates tumor-targeting immune response in up to 50% of patients . The highest response rate up to 70 % with up to 30% complete remission lasting for up to 3 years was reported when radiation sensitization was used in conjunction with the transfer of the tumor-reactive TILs.
Despite the success of the pioneering studies at the Surgery Branch of the US National Cancer Institute and the consequent clinical trials, this approach, although holding much promise in treating melanoma, is facing several challenges that limit broad application of the TIL-based immunotherapy. As TILs are isolated from resected tumors, the first and foremost requirement is the eligibility for surgery, which should be conducted, preferably, in the facility equipped for the isolation of TILs, identification and expansion of the tumor-reactive T cells.
Nevertheless, isolation of the individual melanoma-reactive T cell clones allowed the development of another immunotherapeutic approach – generation of the T cells expressing recombinant antigen specific T cell receptors (TCRs). TCRs are members of the immunoglobulin family proteins. Each TCR consists of 2 different membrane-anchored chains that are joined by the disulfide bridges to form heterodimer. About 95% of the T cells are characterized by the expression of the α and β chains, whereas the remaining 5% express γ and δ chains. Respectively, T cells expressing these receptors are often referred to as α/β and γ/δ T cells. Each chain is comprised of the variable and constant regions. The variable domain of both α- and β-chains have three hypervariable regions also known as complementarity determining regions (CDR), however, the β-chain has an additional area of hypervariability that is not involved in antigen binding. TCR α and γ chains are generated within T cells by VJ recombination, whereas β and δ chains by the V(D)J recombination. Currently, the majority of the TILs selected for the ability to target tumors are α/β T cells expressing respective TCR chains that determine T cell specificity to an antigenic peptides presented by the major histocompatability complex (MHC) proteins. Therefore, it was proposed that sequences encoding tumor antigen recognizing TCR chains can be obtained from tumor-reactive T cells and then used for the gene transfer into patient-derived lymphocytes, thereby creating large quantities of tumor-reactive T cells. The first TCRs specific to melanocytic antigens MART-1 and gp-100 were cloned in 1990s. Pioneering clinical studies using human peripheral blood lymphocytes transduced with these TCRs demonstrated melanoma regression in lymphodepleted patients  (Fig. 1). Although these and other initial clinical studies demonstrated a feasibility of the recombinant T cells-based approach, they also revealed multiple challenges. For example, the ability of recombinant TCR chains to interact and pair with the endogenous chains could lead to the generation of 4 different TCRs in a single cell (Fig. 2). Chain misparing decreases the expression of the function, tumor-reactive TCRs and therefore reduces T cell-mediated tumor targeting. To overcome misparing, several strategies were proposed. Recent pre-clinical and clinical studies demonstrated that replacement of the human TCR constant region with murine counterpart reduced misparing and allowed enhanced expression of the functional TCRs and improved T cell functional activity . It was also reported that targeted mutagenesis and generation of the additional cystein residues in recombinant α and β chains permitted stronger pairing of these chains, higher expression of functional TCRs and improved T cell function [6,7]. Recent studies also showed that targeting of the endogenous chains by siRNA allows higher expression of the functional recombinant TCR. Of particular interest is the proposed approach to encode siRNA along with the TCR chains to concurrently express recombinant and inhibit translation of the endogenous chains . Protein engineering was also employed to improve pairing of the recombinant chains. Thus, substitution of specific amino acids within constant regions of the antigen-specific TCRs supported paring and enhanced functional activity of these receptors . It remains to be determined which of these recombinant DNA-based methods will provide better targeting of melanoma (Fig. 2). Nevertheless, recent studies using chimeric murine-human hybrid highly avid tyrosinase-specific TCR demonstrated a favorable clinical outcome .
It is apparent that both α and β chains of the antigen-specific TCR should be expressed in each individual T cell. To date, internal ribosomal entry site (IRES) elements , double promoters , or co-infection with several viral vectors  were used to express several heterologous proteins in cells. However, these methods have their imperfections. For instance, in IRES-mediated co-expression, the upstream protein is usually more strongly transcribed than the downstream protein. Expression of the proteins from two different or biscistronic promoters or the use of multiples viruses also do not provide equal concurrent expression of multiple transgenes. A more promising approach involves the use of the self-processing viral peptide bridges such as 2A or 2A-like peptides described in Picornaviridae . In picornavirus, these sequences share a highly conservative 18 amino acids motif mediating cleavage between C-terminal glycine and N-terminal proline of the 2B peptide. At present 2a and 2A-like sequences are commonly refer to as
Transfer of the recombinant TCR genes into the T cells is another somewhat limiting factor for the broad application of the genetically engineered T cells for melanoma immuno-targeting. Currently, for human applications, a gene transfer platform that can mediate stable transfer of the TCR genes is retroviral system . Lentiviral vectors and transposons were also tested [16, 17]. Use of retroviruses provided several advantages including a rather high infectivity and rapid integration of the transgene into host genome. With multiple vector backbones, virus packaging cell lines, and well-established GMP protocols, a retroviral system offers relative simplicity of viral vector construction and production of viruses. Since retroviruses can infect only dividing cells, stimulation of the T cell proliferation must be done prior to the gene transfer. Also, these viruses have limited capacity for the packaging. For instance, high virus titers cannot be obtained with larger retroviral vectors. Although an average size of a viral vector encoding typical α/β TCR is around 7 kb, this limits possible alternative approaches such as inclusion of various regulatory elements or another transgenes that may enhance T cell activation. Use of the viral system also presents certain safety concerns relevant to the random integration of the transgenes into the host genome that may result in the activation of oncogenes or inactivation of tumor suppressors. This may lead to the various adverse invents including development of a lymphoproliferative disease resembling leukemia due, in part, to the integration of the retroviral gene transfer vehicle near an oncogene [18, 19]. Thus far, the development of lymphoma-like symptoms has not been reported in patients treated with recombinant T cells. It is also essential to note that production of the TCR-encoding cGMP virus substantially increases the cost of the treatment with recombinant T cells. On the contrary to the retrovirus-based gene transfer, lentiviruses can infect non-dividing cells and therefore can be used for the gene transfer into quiescent T cells. Although “safe” lentiviral systems are developed to minimize the chance of producing replication-competent virus (eg. ViaraSafe from Cell Biolabs), transduction of patient-derived T cells for the adoptive transfer will always present some degree of risk.
Besides viral approaches, non-viral gene transfer may also be used for the expression of the TCRs in T cells. Recently, a Sleeping Beauty Transposon System was tested for the transduction of the T cells . Sleeping Beauty Transposon System consists of two components - the transposon, composed of inverted terminal repeat sequences (IRs) with desired genetic material in between, and a SB transposase enzyme. Most recently, a number of IRs and hyperactive transposases with increasing enzymatic activities were developed to mediate transposition of transposon-encoding proteins into the genomic DNA . Although transposition of SB transposons appears to be unregulated, it has certain advantages over viral based approaches. For instance, expression of transgenes, TCRs in particular, could be regulated by specific promoters that provide either T cell specific expression (eg. CD3 promoter; [21, 22]), or high level of expression (eg. elongation factor 1 promoter; [23,24]. Promoters may also be selected for further specific applications (discussed below). On the contrary to the viral gene transfer, non-viral systems also permit significantly simpler production of the cGMP-grade material (plasmid DNA) and lesser safety testing. Up to date, the Sleeping Beauty transposon-mediated approach was shown to mediate a long-term stable integration of the T-cell receptor genes targeting melanoma-derived antigen, MART-1, in laboratory settings (Fig. 3b). This system provided 50% efficiency of the TCR integration into the genome of the T cells and sustained functional reactivity of lymphocytes to the antigen-positive melanoma .
Other non-viral strategies could be useful in genetic engineering of the T cells. For example, integrase-mediated insertion of the genetic material may provide stable, site-directed integration of the transgenes (TCRs) into T cell genome. This strategy involves integrase from the
Collectively, viral and non-viral strategies for the genetic engineering of the T cells expressing melanoma-specific TCRs are suitable for the
At present time, about 20 clinical studies involving melanoma-specific T cells expressing recombinant TCR were conducted in US alone (some of them reviewed in ). The result of some of the completed trials opened new perspectives for the improvement of the TCR-based strategies. For instance, adoptive transfer of the T cells genetically engineered to express highly avid MART-1-specific TCR has achieved objective clinical responses in a 13% of treated patients . Analysis of CTL-resistant tumor cell revealed that these resistant clones exhibited hyperactivation of the NF-κB survival pathway and overexpression of the antiapoptotic Bcl-2, Bcl-x, Bcl-xL, and Mcl-1 genes . These studies suggest that sensitivity of melanoma to the recombinant T cells could be increased by the pharmacological inhibition of the NF-κB pathway and/or Bcl-2 family members. Multiple investigative studies are on-going to further improve recombinant TCR-based approach.
2.2. Chimeric antigen receptors
Independently, an alternative approach involving recombinant DNA technology was developed to generate tumor-targeting T cells. It utilizes fusion of the variable chain of the tumor-antigen-specific antibody, TCR constant region, and intracellular signaling domains. Initially, these structures were called T-bodies . They are comprise of the single-chain antibody (sFv), TCR transmembrane domain and the intracellular signaling domain of the TCR-ζ. One of the first tumor associated antigens targeted by T cells expressing T-bodies was erbB2 (HER2/neu) receptor that is over-expressed in multiple cancers . Later, a more general term – chimeric antigen receptors or CARs emerged. As compared to the TCRs, CARs allow overcoming dependency on HLA type, antigen presentation, and restricted intracellular signaling of the recombinant α/β TCRs. Initial studies with T-bodies (and recombinant TCRs) demonstrated a rather short lifespan of the engineered T cells and the inability of the recombinant receptors to fully support persistence of the T cell. To address this issue, several studies were conducted to identify the most potent CAR structures by testing several signaling molecules involved in T cell activation (Fig. 5). It was demonstrated that fusion of TCR-ζ with the intracellular domain of CD28 can augment cytokine production by CAR-expressing T cells upon encountering antigen and enhance antitumor efficacy . Inclusion of CD134 (OX-40) into CAR structure also led to the elevated tumoricidal activity of the recombinant T cells . Comparative analysis of the different CARs comprised of TCR-ζ signal transduction domain, CD28 and/or CD137 (4-1BB) intracellular domains demonstrated that addition of the CD137 supports T cell function to a greater extent as compared to other constructs . Collectively, addition of these signaling domains to the CAR structure allowed overcoming (to certain extent) inefficient effector function and anergic status of the T cells.
As recognition of target cells by CAR depends on the antibody, CARs can recognize not only polypeptides but also non-protein molecules such as tumor-associated glycolipids and carbohydrates. However, antibody-mediated binding require surface expression of an antigen and strict selection of TAAs to avoid autoimmune side effects. Also, use of the mouse monoclonal antibody sequences in CAR design may lead to the unwanted immune recognition of the CAR-expressing T cells and limit long-term clinical use [36, 37]. Nevertheless, existence of a large number of the tumor antigen-specific antibodies and robust anti-tumor response by CAR-expressing T cells suggest great clinical utility of these recombinant molecules. Currently, in US alone there are 18 clinical trials aimed at treatment of various malignancies with CAR-engineered leukocytes, with 16 trials in the recruitment phase. Eight of them are aimed at targeting different B cell malignancies with anti-CD19-CAR. Three trials are intended to test HER2-specific CAR-modified T cells for the treatment of sarcoma, glioblastoma, and advanced Her2-positive lung malignancy.
With regard to melanoma, several CAR designs were tested for the ability to target this malignancy. Thus, recent studies demonstrated that treatment of melanoma xenografts in nude mice using engineered T cells expressing tandem CAR (CD28/TCRζ) specific to ganglioside GD3 with IL2 supplementation led to complete remissions of the established tumors in 50 % of treated animals . As GD3 is often over-expressed in melanoma, this approach could be potent in eliminating melanoma in human patients.
Another attractive target for the CAR-mediated T cell therapy for melanoma is a high molecular weight melanoma-associated antigen (HMW-MAA) encoded by CSPG4 gene. This is a cell-surface proteoglycan expressed on more than 90% of the tumors. Recent studies on targeting of this antigen using CAR that is comprised of the anti- HMW-MAA antibody chain and intracellular signaling domains of the CD28, CD137, and CD3ζ demonstrated that T cell genetically modified to express this CAR were cytolytic to the HMW-MAA–positive melanoma cells, produced cytokine and proliferate
As progression of most tumors including melanoma depends on the microenvironment, T-cell mediated targeting of the microenvironmental components could also be a viable strategy for melanoma immunotherapy. Particularly, tumor survival was shown to be dependent on the
Presently, there is an accumulating body of evidence suggesting clinical utility of the T cell genetically engineered to express melanoma antigen-specific CARs. It is likely that in the near future CAR-mediated targeting of different melanoma antigens will evolve into general practice of cancer immunotherapy.
3. DNA vaccination
Another immunotherapeutic approach directly relevant to recombinant DNA is genetic or DNA vaccination. The original idea of DNA vaccination emanated from the observations that intramuscular injection of DNA encoding influenza A virus protein resulted in the robust activation of the immune responses that protected the host from viral challenge . Generally, DNA-mediated activation of immune response involves multiple processes. First, plasmid DNA should be delivered intracellularly and expressed in the host cells. Next, in most of cases the antigen has to be secreted from the cells and picked up by the dendritic cells (DC), processed and presented in the context of the MHC class II to the CD4+ T helper (Th) cells. Alternatively, if the antigen is expressed directly in the DCs, it could be processed intracellularly and presented via MHC class I molecules, leading to the activation of the CD8+ T cells and induction of the cytotoxic immune responses. Initial studies on DNA vaccination were carried out using an intramuscular route of vaccine administration (Fig. 6). This allowed high levels of antigen expression and secretion from the elongated muscle cells into perimysium, the resident site of the intramuscular DCs. Later, DNA vaccination through the skin was suggested to be superior over the intramuscular route. Skin has evolved as a barrier to prevent the entry of pathogens, with efficient immune surveillance complex including Langerhans cells, dendritic cells, lymphocytes, and other leukocytes. Skin is also rich in lymphatic vasculature network that provides an efficient route for DC and T cell trafficking. Depending on the physical methods of into-skin DNA delivery, DNA-based vaccines can be targeted to specific locations in the skin .
The DNA vaccination approach has several advantages over other types of vaccinations: (i) multiple expression vectors coding for different antigen and co-stimulatory molecules can be concurrently delivered into the skin (or the muscle); (ii) the use of cell-type-specific promoters can provide specificity of protein expression; (iii) protein expression from designed plasmids can be controlled by inducible promoters, the use of ubiquitous chromatin opening elements (UCOE), or chemically (e.g. sodium butyrate). Also of note is the relative simplicity and inexpensiveness of the cGMP grade DNA vaccine production and pre-clinical testing. These attractive characteristics of DNA vaccines have prompted extensive research within the past 10 years.
Multiple studies on pre-clinical animal models of melanoma and other cancers have been conducted. Studies on the canine model of aggressive and metastatic melanoma (stages II-IV) demonstrated that xenogeneic vaccination of dogs with DNA vaccine coding for human tyrosinase led to an excellent clinical response in the majority of vaccinated dogs. A long-term survival of dogs with advanced stage IV disease with bulky lung metastases (on average 400 days) was observed . Vaccinated dogs with stage II/III disease also had long-term survivals (on average 500 days) with no evidence of melanoma on necropsy. Overall, median survival time for all treated dogs was 389 days. Another canine model study  showed that xenogeneic DNA vaccination induces melanoma-specific antibody response, which coincides with observed clinical responses. As a result, in 2010 Merial, an animal health company has gained full-licensure from the U.S. Department of Agriculture (USDA) for ONCEPT™ Canine Melanoma DNA Vaccine. Up to date, ONCEPT is the first and only USDA-approved therapeutic vaccine for the treatment of cancer in either animals or humans. (The first DNA vaccine was licensed by the USDA in 2005 for prevention of West Nile virus infection in horses).
However, presently only a few human clinical trials on DNA vaccination were conducted. One of such study, aimed at the evaluation of the immune response in patients with hormone-refractory prostate cancer showed that DNA vaccination with a prostate-specific antigen (PSA) encoding plasmid given with GM-CSF and IL-2 is safe in doses of up to 900 μg, and that the vaccination can induce cellular and humoral immune responses .
Similar to the reference above canine studies, DNA vaccines were shown to be effective in mouse melanoma models when mice were vaccinated with heterologous DNA encoding human melanoma-associated antigen gp-100 . This vaccination regimen was augmented by the GM-CSF and was most effective in the prophylactic setting. It was also effective in suppressing pre-established melanoma. However, vaccinations with autologous mouse melanoma antigens were less successful. Nevertheless, the relative simplicity of modifying recombinant DNA allowed testing of various genetic alterations aimed at breaking the immunologic tolerance and enhancing immune responses to DNA vaccines. For example, concurrent vaccination with DNA encoding several melanoma-specific epitopes can be used. This approach was tested in several studies with different degree of success. As a result, vaccination of mice with gp10025–33 and TRP-2181–188 encoding minigene was effective in preventing melanoma development . As many of the melanoma MHC class I epitopes were characterized for melanoma including those derived from tyrosinase, TRP1, TRP2, gp-100, MART-1, and MC-1R (some of them shared between mouse and human MHC molecules , one can envision generation of an ultimate genetic immunogen capable of targeting several melanoma-associated antigens.
Recombinant DNA technology has also allowed introduction of immuno-augmentation sequences into the DNA vaccine composition. Identified universal pan HLA DR helper binding
Besides introducing immuno-enhancing alteration to the DNA vaccine, other strategies could be employed to enhance DNA vaccination efficacy including addition of the immuno-enhancing molecules to vaccine composition, alteration of the microenvironment at vaccine administration site, and use of the prime-boost immunization regimens. Recent studies demonstrated that antibody-mediated inhibition of the cytotoxic T lymphocyte antigen 4 (CTLA-4) enhances melanoma-specific immune response. This strategy was recently tested in treatment of stage III-IV melanoma and the drug (Ipilimumam) was approved by the FDA as first anti-melanoma immunotherapeutic [59, 60]. CTLA-4 presents its immuno-inhibitory function during activation of the T cells by the antigen-presenting cells. It also inhibits TCR-mediated intracellular signaling in activated T cells and down-modulating T cell mediated immunity. Therefore, it is possible that inhibition of CTLA-4 in conjunction with DNA vaccination may provide significant enhancement of the vaccine-mediated immune response induction. Although providing CTLA-4 inhibiting antibodies like Ipilimumab along with DNA vaccination is not feasible, other options could be explored. For example, recently characterized genetically engineered lipocalin (LCN2) exhibits a strong cross-species antagonistic activity to CTLA-4 . It is likely that this molecule could be included into DNA vaccine composition to enhance DC-mediated activation of the T cells. Other immuno-modulatory strategies may include addition of CD40 ligand, which was shown to stimulate expression of maturation markers CD80, CD86 and IL-12 in APC [62, 63] and its ability to activate CD8+ T cells and increase cell-mediated immunity [64, 65]. Addition of different cytokines and growth factors including GM-CSF, IL-2, IL12 for stimulation/support of the T cells was also tested in several studies (as exemplified in preceding paragraphs) and could be further explored. Alteration of microenvironment via application chemokines to recruit specific sets of the leukocytes to the vaccine administration site may also provide a favorable milieu for the launch of the effective DNA-vaccine induced immune response [66, 67]. These and many other strategies can be proposed; however, the clinical utility of the DNA vaccination combination with other approaches remains to be determined. Nevertheless, presently in the US alone, 10 clinical studies utilizing xenogenic (mouse) or human DNA vaccines coding for melanoma associated antigens have been completed. In these trials, tyrosinase, gp75, gp100, and TRP2 were used as antigens. Although most of these studies are already completed, currently no study results are posted nor are follow-up reports available on patient survival and characterization of immune response. Nevertheless, DNA vaccination remains to be a promising modality that could provide cost-effective and generic immunotherapy for patients with melanoma and other cancers.
4. Other strategies involving recombinant DNA technology
At the present time, almost every immuno-therapeutic approach utilizes recombinant DNA in one way or another. Understanding of the immuno-regulatory functions of DCs and the molecular mechanisms involved in the capture, processing and presentation of antigens by DCs allowed the development of the DC-based vaccines. Initially, in the mid 1990’s, several pre-clinical and clinical studies were conducted using autologous DCs pulsed with melanoma-associated antigens. These studies demonstrated that antigen-loaded DC can trigger active melanoma specific immune responses [68, 69]. However, it became apparent that enforced expression of the antigens in DC rather than loading of these cells with peptides allows for presentation of the tumor-derived antigens via MHC class I complex and priming of the CD8+ T cells to elicit cytotoxic immune response. Moreover, to provide DC specific expression of the antigens, long and short CD11c promoters were characterized and used in several studies [70, 71]. These promoters allow effective and cell type specific expression of the antigens in DCs, as well as more efficient priming and activation of the T cells
During last decade, various melanoma-specific immunotherapeutics that utilize recombinant DNA have been developed and tested in pre-clinical and clinical studies with varying degrees of clinical success. Many of these approaches, including recombinant TCRs and CARs, have already demonstrated promising clinical results, thus providing us with the hope that in the near future melanoma immunotherapy will become curable for melanoma patients.
AcknowledgmentsPresented here data regarding clinical trials was obtained through registry and results database of publicly and privately supported clinical studies (clinicaltrials.gov). Experimental data presented on Fig. 4 was obtained in Dr. Alexeev’s laboratory. Plasmid DNA encoding tyrosinase-specific TCR was kindly provided by Dr. S.A. Rosenberg (NCI, NIH). ΦC31 integrase-encoding plasmid was obtained from Dr. M.P. Calos (Stanford University, CA).
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