Cancer immunotherapy: dawn of the death of cancer?

International Reviews of Immunology ISSN: 0883-0185 (Print) 1563-5244 (Online) Journal homepage: https://www.tandfonline.com/loi/iiri20 Cancer immunotherapy: dawn of the death of cancer? Sidhant Jain & Sahil Kumar To cite this article: Sidhant Jain & Sahil Kumar (2020): Cancer immunotherapy: dawn of the death of cancer?, International Reviews of Immunology, DOI: 10.1080/08830185.2020.1775827 To link to this article: https://doi.org/10.1080/08830185.2020.1775827 Published online: 12 Jun 2020. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=iiri20 INTERNATIONAL REVIEWS OF IMMUNOLOGY https://doi.org/10.1080/08830185.2020.1775827 REVIEW Cancer immunotherapy: dawn of the death of cancer? Sidhant Jaina and Sahil Kumarb a Department of Zoology, University of Delhi, Delhi, India; bDepartment of Pharmacology, Maulana Azad Medical College and Lok Nayak Hospital, New Delhi, India ABSTRACT ARTICLE HISTORY Cancer is one of the proficient evaders of the immune system which claims millions of lives every year. Developing therapeutics against cancer is extremely challenging as cancer involves aberrations in self, most of which are not detected by the immune system. Conventional therapeutics like chemotherapy, radiotherapy are not only toxic but they significantly lower the quality of life. Immunotherapy, which gained momentum in the 20th century, is emerging as one of the alternatives to the conventional therapies and is relatively less harmful but more costly. This review explores the modern advances in an array of such therapies and try to compare them along with a limited analysis of concerns associated with them. Received 29 December 2019 Accepted 16 May 2020 Introduction Cancer is an extensive aberration in the cellular behavior leading to continual unregulated proliferation of cells. Cancer generally arises from ‘gain-offunction’ mutation into proto-oncogenes converting them to oncogenes or ‘loss-of-function’ mutation into tumor-suppressor genes [1,2]. Normal growth promoting cellular genes are called as proto-oncogenes and mutation in these genes can alter their delimited function converting them into cancer inducing cellular oncogenes. Certain viruses like Rous Sarcoma Virus, Epstein-Barr Virus etc. contain oncogene derived from a cellular proto-oncogene in their genome, termed as viral oncogene, which they integrate in the cells they are infecting, inducing cancer [2; 3]. Worldwide, 8.2 million people die of cancer annually. Approximately, 14 million people suffer from it every year, which is expected to increase to 22 million in the next two decades [4]. Chemotherapy, hormone therapy, radiotherapy and surgery are the conventional methods for treating cancer, however since late 20th century, immunotherapy and other targeted therapies got approval for the treatment [5]. The development of immunotherapy began as early as in 1890s which involved injecting bacteria or bacterial products named as Coley’s toxins into inoperable CONTACT: Sidhant Jain 110007, India [email protected] ß 2020 Taylor & Francis Group, LLC KEYWORDS ATCT; cancer; CAR T cell therapy; conventional therapy; immunotherapy; monoclonal antibodies; therapeutic vaccine tumors [6; 3] but its development gained momentum in late 20th century. Immunotherapy involves a set of procedures which help in elimination of a tumor by reviving, instigating or enhancing the in-vivo anti-tumor immune response or by neutralizing immune-inhibitory pathways like immune checkpoints. To put simply, immunotherapy directs the immune system to fight cancer [7; 3]. Immunotherapy encompasses an array of therapies like Therapeutic Vaccines, Adoptive T Cell Therapy, Monoclonal Antibodies, Inhibition of immune checkpoint etc. or a combination of these therapies (Figure 1). The aim of this review is multi-directional. First, it consolidates the information available on the novel methodologies in various fields of cancer immunotherapy, reflecting upon newer trials that are being carried out. Second, it tries to broadly compare different immunotherapies in terms of their advantages and disadvantages. Finally, this review, in brief, talks about concerns related with these immunotherapies. Earlier reviews have covered many similar aims as this review tries to do, however, most of them [4,8–10] concentrate on a specific aspect of cancer immunotherapy. Other reviews (e.g. [6]) which did cover information in various fields of immunotherapy at one place are now somewhat old. New data from multiple clinical trials as well as new approaches have come up, which needs to be discussed. Instead of Department of Zoology, University of Delhi, Gate No.:3, Chaatra Marg, North Campus, Delhi 2 S. JAIN AND S. KUMAR Figure 1. Conventional therapies and immunotherapies for cancer treatment. covering each therapy in detail, we try to cover the important aspects of most of the therapies so that an overall understanding of various immunotherapeutics can be presented. Cancer antigens Majority of antigens associated with tumor cells are self-antigens and hence are subject to tolerance inducing process which prevents auto-immunity. However, many tumors express unique antigens where as others inappropriately express self-antigens (e.g. overexpression of an oncogene product). Both of these types of antigenic expression is recognized by immune system [3]. Peptide antigens can be either Tumor Associated Antigens (TAAs) or Tumor Specific Antigens (TSAs). TAAs are expressed on both normal and tumor cells but they are much more in number in the later. TSAs are specifically expressed by tumors [11]. TAAs can have many sub categories as described in the Table 1. Certain carbohydrates can also be TAAs. Most of these carbohydrate antigens cannot elicit powerful immune response as they are T cell independent. Conjugating these carbohydrate antigens into proper carriers can help eliciting an immune response and is a breakthrough in carbohydrate antigen based therapeutic vaccines [13]. TSAs generally arise as a result of either genetic mutations or viral infections (Table 2). These neoantigens are totally foreign proteins which are not expressed on normal human cells [17]. Theoretically, these antigens are perfect targets for developing therapeutics and have shown promising initial results. Neo-antigen prediction became rapid with the use of next-generation sequencing [14]. As normal cells do not express these viral or mutated gene products, these antigens are recognized as non-self by the immune system. Immunotherapeutic measures Therapeutic vaccines Cancer vaccination involves multiple approaches to induce and/or amplify anti-tumor immunity by INTERNATIONAL REVIEWS OF IMMUNOLOGY 3 Table 1. Different types of Tumor associated antigens. Tumor Associated Antigen Description Examples Differentiation Antigens These are expressed on normal tissues and the tumor which originate from these tissues [11]. These are expressed at a relatively small levels in normal cells of a healthy tissue but are amplified manifold at DNA, mRNA or/and protein levels in tumor cells arising from that tissue. [4] In normal conditions, a restricted expression of these antigens is reported in human germ cells in testis and in trophoblast. However, their expression is reported in a variety of human cancers [4,11]. In spite of being expressed in normal germ line cells and trophoblastic cells, cancer-testis antigens can also be put under TSAs. As male germ line cells and trophoblastics cells do not have MHC class I molecules on their surface, they cannot display antigens to T cells and hence appear to be strictly tumor specific [12]. These are normal fetal antigens which are turned off during development but are turned on again in some transformed cells [3,4]. MelanA/MART-1, gp100/pmel17, CEA, gp75/TRP1, TRP-2 Her2/neu, WT1, Human mucin 1, RAGE-1, antigens derived from PRAME gene. Overexpressed Antigens Cancer-Testis Antigens Onco-fetal Antigens NY-ESO-1/LAGE, Melanoma antigen- encoding genes or MAGE (MAGE-A, MAGE-B, MAGE-C family), BAGE, GAGE family of antigens aFetoprotein (AFP) Table 2. Different types of tumor specific antigens or neo-antigens. Tumor Specific Antigen Viral antigens Description These antigens are viral proteins and their expression is extremely essential for the maintenance of tumor malignancy. Any epitope(s) derived from the open reading frames in the viral genome becomes a potential neo-antigen [11,14]. Mutant antigens These antigens arise as a result of genetic mutations hence encoding for a mutant protein which is not expressed on the cells in the normal conditions. An array of genetic alterations leads to the derivation of these mutant neo-antigens which involve point mutations (leading to single-nucleotide variants or causing a frameshift), gene fusions, insertions or deletions and structural variations [12,14]. administrating tumor antigens with or without immune modulators like Antigen Presenting Cells (APCs) and adjuvants [4]. After going through the literature, we divided these approaches into five major methodologies as shown in Table 3. First methodology involves vaccination with peptide antigens. Antigenic peptides expressed by cancer cells can be recognized by CD8þ TC cells (Cytotoxic T lymphocytes or CTL) and CD4þ TH cells (Helper T cells) in association with Class I MHC molecules and Class II MHC molecules respectively [18]. Vaccines which are based upon TAAs, theoretically, should not produce any immune response as TAAs are self-antigens which are overexpressed in cancers. Indeed, it was observed that vaccines against TAAs, based on high affinity epitopes, do not produce an Examples Long peptides from E6 and E7 onco-proteins of HPV-16 were used in a clinical trial for vaccination of women who were HPV-16 positive with vulvar intraepithelial neoplasia (grade 3). A relief in symptoms was reported in about 3 months with adverse effects ranging up to second grade [15] Altered beta-catenin having an alteration in single base pair in cDNA at 37th position and hence coding for phenylalanine residue instead of a serine residue [16]. Other examples involve mutant p21/ras, mutant EGFR VIII and mutant CDK4. immune response as T cells to these high affinity selfepitopes must be deleted during T cell development as a result of positive selection leading to tolerance [9]. The solution to this problem lied in selecting low to medium affinity peptide which can help in overcoming tolerance [19]. Further, if antibodies are generated against such low to medium affinity epitopes (which are overexpressed in cancer but otherwise are expressed in low amounts by normal cells), an autoimmune response can be expected, however, in transgenic mice models or clinical human trials, no such auto immune reactions have been observed [19]. Two examples are here discussed to explain the peptide antigen based vaccines. Gp100 is a non-mutated differentiation antigen expressed only by pigmented retinal cells and normal melanocytes and by melanomas [20,21]. A study [22] 4 S. JAIN AND S. KUMAR Table 3. Different methodologies for therapeutic vaccination. Methodology Antigenic peptides Modified DCs Tumor cell lysates Viral Vectors Naked DNA or RNA Description Vaccination with antigenic peptides expressed by tumor cells like gp100, HER2/neu. Vaccination with autologous DCs which are modified to express tumor antigen. Vaccination with irradiated tumor cells or tumor cell lysates along with APCs. Vaccination with tumor antigen DNA using viral vectors which uses infection potential of virus. DNA or RNA coding for tumor antigen, under a strong viral promoter, is directly administered, usually intra-muscularly or intra-dermally. showed that native peptides can generate melanoma reactive CTLs in humans. However, their efficiency can be increased by modifying these peptides and it was shown that the modified peptides have more potential to produce melanoma reactive CTLs. In another study, gp100 was injected in subcutaneous tissue with interleukin-2 (IL-2) and it was found that the rate of response and progression free survival was longer when these two components were given together [23]. Human Epidermal growth factor Receptor 2 (HER2), the 185-kDa protein receptor, is overexpressed in about 25% of all primary breast carcinomas. It acts as a non-mutated over-expressed antigen [24] and it is overexpressed in other cancers involving ovarian, colorectal, stomach and pancreatic carcinomas as well [25]. A study [26] showed that intradermal injection of vaccines based on Her-2/neu peptides/protein mixed with granulocyte-macrophage colony stimulating factor (GM-CSF) in patients overexpressing these antigens developed immunity to both Her-2/neu peptides and proteins. Phase I/II clinical trials were conducted for another Her-2/neu based vaccine, E75 (Nelipepimut-S), which is also injected with GM-CSF and produced safe and clinically efficient results and is under phase III trials [27]. Second methodology for cancer vaccination involves vaccinating patients with modified APCs like Peripheral Blood Mononuclear Cells (PBMCs), Dendritic cells (DCs) and activated B-lymphocytes. It was suggested that by using DCs, improved strategies can be designed which can boost immunity against metastatic cancers [28]. The immunological basis of cancer vaccines which utilizes the knowledge of DC system has also been described [29]. DC system based vaccines involve harvesting of DCs from cancer patients which are modified to express tumor antigen. Like peptide based vaccines, DC system based vaccines are being injected with adjuvants, Interleukins etc. to produce a heightened response in the patients. Viruses have been used to transduce peptide antigens in autologous DCs. Initial gene targeting techniques using viruses for DNA modification in dendritic cells have been summarized in a review [30]. These techniques are being modified to generate more effective vaccines. In a study, phase I/II trials were conducted for a vaccine given to patients suffering from metastatic melanoma. This vaccine contains autologous DCs transduced with a replication lacking adenovirus encoding for complete melanoma antigen MART-1. The patients showed an immunogenic positive and safe response towards this vaccine [31]. In another study, the effect of vaccination with autologous dendritic cells was studied in ten patients who were suffering from Acute Myeloid Leukemia (AML) by targeting Wilm’s Tumor 1 (WT-1) antigen. These dendritic cells were loaded with full length WT-1 mRNA via electroporation and injected intradermally in partial/complete remission state. These patients showed improved clinical outcomes along with enhanced immune activation [32]. Majority of the trials which were carried out in the past decade, involved patients at advanced stages of cancer. It had been hypothesized that DC cell vaccination can yield better and more promising results in patients who are at early stages of cancer with less tumor burden and before immunological impairment occurs as a result of chemotherapy [33]. Some reviews summarize the clinical trials based on DC system of vaccination which are in progress [34,35]. Third methodology for cancer vaccination is based upon using the killed tumor cells or tumor cell lysates supplied along with APCs and/or co-stimulatory cytokines like GM-CSF. Studies were conducted using the aforesaid methodology and a potent anti-tumor immunity was seen in patients in phase 1 trials suffering from metastatic melanoma when irradiated autologous melanoma cells, which were manipulated to secrete GM-CSF, were injected in them [36]. In another phase I/II study, vaccination of 28 patients suffering from stage IV metastatic melanoma, with similar irradiated autologous tumor cells which were retro-virally transduced with GM-CSF gene showed heightened T-cell activation and limited degree of INTERNATIONAL REVIEWS OF IMMUNOLOGY toxicity against melanocyte antigens but led to induction of vitiligo in a few patients [37]. Certain studies combined the use of APCs like dendritic cells with tumor cell lysates to produce new combinations of vaccines. Promising results came from another study involving 17 patients, many of whom are suffering from active metastatic melanoma, who were immunized intra-dermally with autologous APCs which were cultured with GM-CSF and mixed with tumor lysate 48 hours prior to immunization proving feasibility of such vaccines two decades back [38]. In another study, dendritic cells obtained from PBMC were treated with tumor lysates and immunogenic protein Keyhole Limpet Hemocyanin (KLH) invitro along with GM-CSF and Interleukin-4 and given intra-dermally for treatment of relapsed cancer in children with solid tumors. It was observed that the treatment was feasible and apparently nontoxic and was able to regress tumor in a child with metastatic R -L, which tarfibro-sarcoma [39]. A vaccine, DCVaxV gets Glioblastoma Multiforme (GBM) contains mature dendritic cells along with tumor lysate or peptides is in stage III trials [40]. Fourth methodology is based upon the use of viral vectors. Viruses have been used to transduce antigens in autologous APCs ex-vivo in the case of modified DC based vaccines discussed above. Further, irradiated autologous tumor cells can be retro-virally transduced with genes of various factors like GM-CSF, etc. to produce heightened immune response against cancer antigens as described above [37]. With respect to vaccination, viral vectors carrying antigen DNA can be designed to directly inject tumor antigens into tissue which can be easily delivered to tissue cells owing to infective potential of virus. In a study, two patients suffering from hepatocellular cancer having an AFPþ tumor, received vaccination with plasmid DNA injections. Plasmid construct had full length AFP followed by replication deficient but AFP expressing adenovirus and it was observed that the vaccine was largely nontoxic and well tolerated with immunogenic nature. Larger clinical trial for this methodology is proposed [41]. Currently, Adeno-associated virus (AAV) vectors are being studied extensively for delivery of transgenes. AAV vectors are showing increased clinical success. The current status, applications and future prospects for AAV vectors has been strikingly summarized in a recent review [42]. Proposed and ongoing clinical trials based on DNA vaccines utilizing viral vectors majorly have been clubbed in another review [43]. 5 Viruses can be utilized as an effective measure against cancer in an altogether different way. Extensive research is being done on oncolytic viruses to develop them as an effective therapeutic measure against cancer. Oncolytic viruses specifically infect and propagate via tumor cells, hence damaging them and this process is minimally toxic to the normal cells [44,45]. Adenoviruses are being majorly researched to develop them as oncolytic viruses. One strategy involves mutating the E1 genes of the Adenovirus whose gene products are responsible for preventing the apoptosis of the host cell. This makes the virus, tumor selective as products of E1 genes are essential for propagation of virus [46]. Therefore, propagation in normal cells is aborted and host cells undergo p53 mediated apoptosis. However, in majority of tumors, p53 is itself nonfunctional and hence E1 mutated/ deleted viruses can still spread through them which ultimately causes their lysis [47]. These oncolytic Adeno viruses have been modified to express immune-stimulatory cytokines. In one study, oncolytic Adenovirus was modified to encode TNFa and IL-2 which induced danger- and pathogenassociated signaling (DAMP/PAMP) which activated cytosolic protein sensors like AIM2 leading to modulation of tumor microenvironment [48]. Other studies, used oncolytic Adenovirus or Helper dependent Adenovirus (HDAd) expressing IL-12 and these treatments inhibited cancer progression. It was also observed that for IL-12 delivery, HDAd are safer as compared oncolytic Adenovirus [49,50]. Similar to the expression of cytokines, oncolytic viruses have been engineered to express PD-L1 inhibitor and GM-CSF [51]. This PD-L1 inhibitor, inhibited PD-L1 on immune cells as well as on tumor cells which overcame the PD-L1 mediated immunosuppression and this led to provocation of systemic tumor neoantigen-specific T cell response. This therapy can be extremely useful to patients resistant to PD-1/PDL1 immune checkpoint blockade (discussed ahead). An array of clinical trials are in progress with respect to oncolytic viruses but it would be beyond the scope of this work to collate all of them. A recent review can be referred [52] which summarizes many of these clinical trials. An interesting candidate for virus based therapies is Zika virus. Although it causes microcephaly by killing brain cells but studies have shown its oncolytic potential against aggressive glioblastoma stem cells providing us with a possible brain tumor therapy [53,54]. 6 S. JAIN AND S. KUMAR Another strategy for cancer vaccination is based on physically delivering DNA or RNA. Naked DNA vaccines for immunotherapy are made using bacterial plasmid coding for a targeted tumor antigen under a strong viral promoter for continuous and maximal expression and are usually administered intra-muscularly or intra-dermally. Naked DNA vaccines are usually injected with adjuvants like polysaccharides, liposomes, aluminum salts, nano-particles etc. which act as immune stimulators and/or delivery system [55,56]. Intra-muscular electroporation is also used to deliver plasmid DNA [57]. Plasmid DNA vaccines are nearly nontoxic and rarely integrates into host genome. The chances of insertional mutagenesis are even less than that of spontaneous mutation [56]. In a study, plasmid DNA vaccine was made which encoded for truncated Carcino-embryonic Antigen (CEA) overexpressed in many cancers like colorectal, breast, lung, ovarian cancer etc. and was fused with T-helper epitope DNA. 10 patients suffering from colorectal cancer were immunized intra-dermally with this vaccine three times via jet injection. Prior to first vaccination, cyclophosphamide was injected intravenously whereas GM-CSF was administered subcutaneously with every vaccination. Except for grade I and II adverse effects (AEs), the vaccine showed positive results with minimal toxicity and no signs of autoimmunity [58]. The use of mRNAs/siRNAs is also increasing in development of therapeutics. Conventionally, RNA vaccines are made by transcription of a template DNA, which encodes for a particular peptide antigen that has to be targeted, into mRNA using RNA polymerase derived from bacteriophage in-vitro. After administration and internalization by host cells, translation directly takes place in host cells’ cytoplasm and the translated peptide antigens are presented to APCs and an immune response is hence generated. On the other hand, autologous DCs can be transfected with TAAs’ mRNA and delivered to respective patient to generate an immune response [55,59]. In a study, DCs were harvested from patients suffering from metastatic prostate cancer and transfected with mRNA encoding for Prostate Specific Antigen (PSA). PSA specific Tcell responses were recorded in these patients confirming the bioactivity of this vaccine [60]. On-going clinical trials based on such vaccines has been summarized in a recent review [61]. In-spite of decades of research in cancer vaccination, only two cancer vaccines got US Food and Drug Administration (FDA) approval till May, 2018. One of them is Sipuleucel-T used for treating Prostate cancer [62]. Sipuleucel-T uses a recombinant fusion protein, PAP-GM-CSF (PA2024), to activate PBMCs. PAP-GM-CSF is made up of Prostatic Acid Phosphatase (PAP), an antigen expressed in prostate cancer which is joined to the N-terminus of GM-CSF via its C-terminus. Each dose of this vaccine has about 50 million activated CD54þ cells [63]. Second vaccine to be approved by FDA and first one to be based on oncolytic virus is talimogene laherparepvec, also known as T-VEC, or OncoVEXGM-CSF for use in melanoma patients [64]. Adoptive T cell therapy Adoptive T cell therapy (ATCT) involves infusion of autologous tumor specific CTLs derived from cancer patients for the recognition and destruction of the cancerous cells. These T cells can be harvested and expanded from the tumors themselves (Tumor Infiltrating Lymphocytes or TILs) or can be harvested from peripheral blood and tumor specificity can be induced either via antigen specific expansion or genetic engineering [9]. The first trial with ATCT was done in a 1988 study, when TILs, extracted from melanomas were given to 20 patients along with IL-2 after an intravenous dose of cyclophosphamide. Regression was observed in 11 out of 20 patients which lasted for approximately 2–13 months along with reversible toxic effects of IL-2 [65]. Many modifications and advancements have been introduced since this trial including treatment with T cells after depleting the immune elements which increases anti-tumor efficacy [66]. Promising results have been obtained from multiple studies which have been done utilizing TILs for treatment, where in some of them, the other immunotherapies have failed [67,68]. A recent review highlights the opportunities which can be harnessed in the field of ATCT along with the challenges being faced and possible solution to those challenges [69]. The evolution of Chimeric Antigen Receptor T cells (CAR T cells) can be thought as most promising development in ATCT. This therapy uses high numbers of tumor targeting T cells, produced by genetic manipulation of autologous T cells using plasmid or retroviral vectors which aims at introducing tumor specific chimeric T cell receptors (TCRs) [10]. TCRs require peptide antigen presentation derived from proteins in association with MHC whereas CARs exhibit MHC independent recognition of these antigens which, theoretically, can be any surface antigen INTERNATIONAL REVIEWS OF IMMUNOLOGY 7 Figure 2. Production of CAR T cells and Generations of CARs. i.e. a peptide, protein or phospholipid [70; reviewed in 10]. Manufacturing CAR T cells involves a patient or a T cell donor, from whose blood T cells are harvested via leukapheresis. T cells are activated in presence of magnetic beads covered with CD3 and CD28 antibodies followed by genetic engineering (via viral transduction to express CAR) and T cell expansion. This is followed by bead removal and adoptive transfer or freezing for storage (Figure 2). A conditional chemotherapy is preferred before the adoptive transfer [71,72]. The recognition of an antigen by TCRs and T cell activation is an intricate process. It involves an antigen presenting cell/target cell to present an antigen with MHC (class II/I) molecule to CD4þ/CD8þ T cell. The TCR, itself, is a disulfide heterodimer made up of two chains, a and b, with some CD3 invariant chains (f, e, d, and c). Co-stimulation of T cell by interactions between CD 80/86 on APC/target cell and CD 28 on T cell is also required for its activation. Adhesion Molecules’ interactions like LFA-1 to ICAM-1 and LFA-3 to CD2 further strengthens interaction between the cells [3]. CARs are recombinant receptors made up with an extracellular single-chain variable fragment (scFv) obtained from an antibody linked to a trans-membrane domain, which is again joined to intracellular T-cell signaling domain(s) [73]. A ground breaking study led to the development of first generation CARs where cytoplasmic domain of TCR signaling complex is fused to variable fragment of tumor targeting antibody [74]. A first generation CAR involves fusion of CD3f or Fc receptor c chain, which are cytoplasmic signaling domains, to a scFv of a tumor targeting antibody via multiple hinge and trans-membrane domains [73]. After the development of CAR T cell therapy, some groups started targeting ovarian cancer ex-vivo and in-vivo using monoclonal antibodies against ovarian cancer peptides to generate CARs [75,76]. A trial study was then conducted which involved generation of autologous T cells against a folate receptor (FR) antigen associated with ovarian cancer. The CARs (first generation) were expressed by a chimeric gene which incorporated anti-FR single chain antibody linked to c chain of TCR/CD3 signaling complex. The 8 S. JAIN AND S. KUMAR patients were segregated in two groups where patients in first group received autologous T cells against FR along with high dose IL-2 and patients in second group received dual-specific T cells (against FR and allogenic cells) followed by injecting with allogenic PBMC. Though the treatment was safe, except for the IL-2 associated toxicity, but didn’t reduce tumor burden and it was observed that such tumor reactive cells perished soon [77]. The first generation CAR T cells undergo limited proliferation because of lack of co-stimulatory signals leading to anergy or activation induced cell death [73]. To overcome this issue, second generation CARs were developed (Figure 2) by incorporating signaling domains of T cell co-stimulatory receptors like CD28, OX-40 or 4-1BB because of which the signaling strength enhanced and anti-tumor activity was manifold increased [78,79]. A further improvement to these second generation CAR T cells, involving cytoplasmic domains from two co-stimulatory receptors like CD28-4-1BB or CD28-OX-40, led to production of its third generation [73]. The fourth generation has also been developed, termed as TRUCKs (T Cell Redirected Universal Cytokine Killing) which, along with the primary signal of TCR/CD3 and a second signal from the co-stimulatory signaling domain like CD28, have a CAR inducible transgene which usually codes for a cytokine [80]. CAR T cell therapy has shown positive results in anti-leukemic regime in mice models and human trials [79,81]. FDA in 2017 has approved two CAR T cell therapies. First, Kymriah or Tisagenlecleucel, is approved for the treatment of certain children and young adults suffering from Acute Lymphoblastic Leukemia. Second, Yescarta or axicabtagene ciloleucel, is approved for the treatment of adults suffering from large B-cell Lymphoma. After showing its potential in hematologic malignancies, currently, CAR T cell therapy is also being researched to analyze if it can be used to treat solid tumors. The barriers imposed by the solid tumors against this therapy like penetrating the stroma of tumor, persistence and expansion within the tumor etc. and the possible strategies to overcome such obstacles have been abridged in a recent review [82]. Antibody based therapy Over the last two decades, antibody based cancer therapy has established itself as one of the most effective strategies for treating humans suffering from haematological malignancies as well as solid tumors [8]. The revolutionary process of generation of monoclonal antibodies (mABs) was documented in 1975 and involves the use of cancerous cells to produce hybridomas [83], the very same technology is now being used to generate mABs to target cancerous cells. Although most mABs used in these therapies are immunoglobulin G (IgG) but various modifications like protein-Fc conjugates and drug conjugates are also used [8]. mABs target cancer cells by binding to specific antigens expressed on cell surface like CEA, Vascular Endothelial Growth Factor (VEGF), HER2, Cluster of Differentiation (e.g. CD20, CD33), VEGF Receptor etc [84]. After attachment to a particular tumor antigen there are multiple pathways by which a cancer cell can be destroyed:  Direct tumor cell death.  Immune mediated tumor cell killing.  Vascular ablation. Direct cell killings involve three different mechanisms: Firstly, there can be delivery of cytotoxic drugs/radioisotopes using conjugate antibodies leading to cell lysis [85]; secondly, apoptosis can be induced [86–88]; lastly, signaling pathways necessary for cell survival can be targeted and inhibited [87]. Immune mediated cell killing incorporates measures like activation of cellular phagocytosis, engagement of Antibody Dependent Cell-mediated Cytotoxicity (ADCC) or complement mediated cell lysis [89–92]. Further, mABs’ SCFvs can be used in generation of CARs [75,76,93]. Finally, few mABs can inhibit the inhibitory receptors on T lymphocytes leading to activation of T lymphocytes [94, reviewed in 95] and have been discussed in next section. Angiogenesis is a necessary component for growth and metastasis of cancer. Vascular ablation restricts blood supply to the tumor decreasing tumor perfusion [96]. The total number of mABs approved US FDA for different types of cancers reached 64 in 2018 [97] indicating its importance as a milestone therapy in cancer therapeutics. However, in spite of the promising results one can achieve with these monoclonal antibodies, there exists a major lacunae as resistance can be generated among cancer cells to such targeted therapy due to down regulation of pathways which are being targeted and inhibited by mABs and up regulation of the alternative pathways. For example, Cetuximab, a mAB against Epidermal Growth Factor Receptor (EGFR) is an effective strategy to treat patients with colorectal INTERNATIONAL REVIEWS OF IMMUNOLOGY cancer but resistance to the same has been reported due to uncharacteristic ERBB2 signaling (ERBB2 gene amplification or hergulin production) leading to resistance [98]. Resistance in HER-2 targeted therapy [99,100] has also been reported. To limit this issue of resistance and increase the efficacy of targeted therapy, bi-specific antibodies (bsABs) have been developed. A bsAB can simultaneously bind to two antigen like a tumor antigen and effector cell or drug [101]. bsABs can help in multiple ways:  More than one tumor cell antigen can be simultaneously engaged due to presence of different antigen binding sites.  Non-conventional immune cells like T cells, which are not activated by mABs due to lack of FC receptor, can be activated [102].  bsABs can be directed against receptor tyrosine kinase (can be used in HER-2 targeted therapy), tumor angiogenesis and even intracellular antigens [103].  Further, all the targeting methods discussed above like delivery of cytotoxic drugs, activation of apoptosis etc. can be used in combination to target cancerous cells using bsABs [104]. Given the overabundance of combinatorial options, bsABs can be used in multiple combinations to counter this lacunae of resistance. Inhibition of immune checkpoint inhibitors Immune response inhibitory interactions are common in natural conditions like expression of CTLA-4 on T cell after T cell activation, which like CD28 binds to B7 (CD80/CD86) expressed on an APC but binds with higher affinity. This interaction, instead of providing a co-stimulatory signal, provides an inhibitory signal. CTLA-4 limits CD28-B7 interaction to reduce hyper T cell activation which can lead to splenomegaly, lymphadenopathy, autoimmunity and even death but it helps the unchecked growth of tumors as CTLA-4 is constitutively expressed on cancer cells [105,106]. Another such molecule is PD-1 which is present on the surface of T cells. It binds to PD-L1 or PD-L2 expressed on professional APCs and this PD-1 to PDL1/PD-L2 interaction down regulate the T cell response [107,108]. PD-L1 is overly expressed on cancer cells [108] and this combined with overexpression of PD-1 on TILs can lead to poor surveillance by 9 immune system helping in unchecked growth of tumor cells [109]. Clinical trials using mABs which block PD-1/PDL1 interaction, by binding to any one of them, gave excellent responses especially for melanoma, bladder cancer and renal cell carcinoma [110]. Pembrolizumab (Keytruda) and Nivolumab (Opdivo) are two FDA approved mABs for certain types of cancers which block PD-1. Phase II/III trials are undergoing to check the efficacy of certain mABs blocking PD-L1, which have shown significant results in pre-clinical and Phase-I trials [111]. Similarly, Ipilimumab (Yervoy) is a mAB that binds to CTLA-4 and blocks its activity [112]. In the absence of these inhibitory signals, an array of T cells get activated and mount an immune response against the tumor cells. These immune checkpoint receptors are essential to keep in check the body’s immune system to prevent it from causing damage and harm but in the presence of active malignancies, inhibitory signals may be dominant and needs to be curbed to generate effective immunity against tumor cells [112]. However, as these mABs are blocking all these inhibitory molecules, they can pave way for a serious and even life threatening side-effects in some people. Common side effects of these drugs can include nausea, fatigue, itching, skin rash, dirrahea and less often one can have serious problems in the lungs, intestines, liver, kidneys and endocrine glands [113]. Many other molecules are under study which are co-inhibitory molecules which can be inhibited to mount an effective immune response against tumor cells. Lah-3, CD40, CD137 have already reached Phase I or II trials whereas B7x, BTLA, and Tim-3 are under pre-clinical study [114] clearly indicating that this therapy is still in the very initial stages and many more therapeutics, developed on similar lines, are expected in near future. Combinatorial therapies Some cancer patients may respond to a single therapy but for a majority of them, it is ineffective [115]. It is a necessity to combine various cancer therapies to achieve complete remission [116]. In this review, we describe some of these combinatorial therapies.  Checkpoint inhibitors with conventional therapies Linking antibodies against checkpoint inhibitors with conventional chemotherapy may take an advantage of the fall in tumor burden caused by this 10 S. JAIN AND S. KUMAR Table 4. Comparison of different immunotherapies. Immunotherapy (Examples) Vaccines (Sipuleucel-T, OncoVEXGM-CSF) Advantages     ATCT and CAR T cell therapy (Kymriah, Yescarta)      mABs (Rituximab, Cetuximab) Immune checkpoint blockade (Pembrolizumab, Nivolumab, Ipilimumab)       different References/ Reviewed into    Lack of universal antigens Relatively poor efficacy Not Cost effective.    Expensive Relatively more time consuming Certain CAR T regimes are detrimentally toxic Neurotoxicity with CAR T regimes Approved CAR T cell therapies are extremely costly Potent anti-tumor activity Prolonged survival Can deal with broad range of cancers  treatment curbing cancer. A chemotherapeutic agent, Decarbazine was given with ipilumumab (anti-CTLA4) to patients suffering from metastatic melanoma which was not treated previously. The survival rate with this regime, relative to mono-treatment with decarbazine plus placebo, was longer though the grade 3 and 4 AEs were higher [117]. In a phase III trial involving males with ‘minimum one bone metastasis’ from castration-resistant prostate cancer which had progressed after docetaxel treatment, 8 Gy radiotherapy, directed to bone, was provided followed by ipilimumab or placebo. Though, the survival rates in both the groups were not much different, certain amount of anti-tumor activity was observed in the radiation treated group warranting further research [118].  Combination of point inhibitors Disadvantages Specificity Engagement of host immune system Relatively low toxicity Potential for durable treatment effect owing to memory. Specificity High response rate Efficacy can be increased using lymphodepletion. CARs exhibit MHC independent tumor antigen identification CARs can theoretically identify any surface antigen (phospholipid peptide, protein) Specificity Relatively low toxicity Engagement of host immune system. immune check- The inhibition of both the checkpoint inhibitors i.e. CTLA-4 or PD-1 leads to prevention of a heightened T cell response but both of them use different signaling pathway to reach the outcome. CTLA-4 diminishes the initial T-cell activation whereas PD-1 obstructs effector T-cell responses in tissues [119]. Due to this, synergy is expected from a rational combination of these two antibodies. Combination of PD1 and CTLA-4 blocking (in combination with Fvax) is more than two times as effective as anyone alone in supporting the elimination of B16 melanomas in mice [120].       Development of resistance. mAB specific Hepatitis B virus reactivation Development of severe infections in some cases. Relatively few patients obtain clinical benefit. Serious side effects in vital organs in a few patients.  [126–128]      [70] [129] [130] [131] [132]  [98,100,133–135]    [113] [116] [115] In humans, combination therapy blocking CTLA4 and PD1 causes distinct immunologic changes as compared to changes that occur by blocking one of them [121]. In a phase I trial, concurrent treatment, with nivolumab (anti PD-1) and ipilimumab (antiCTLA-4), of patients suffering from melanoma achieved higher response rate than any of them alone along with manageable safety profile [122]. The frequency, magnitude and onset of immune related toxicities, however, also increased in this combinatorial approach relative to treatment with a single checkpoint blocking agent [123].  Therapeutic vaccines point inhibitors with immune check- In a study involving mice, treatment with GVAX (GM-CSF-expressing irradiated tumor cells) along with CD-1 and CTLA-4 blockage induced significant rejection of tumors and in one case 100% mice rejected tumor [124]. In a Phase III study, patients with metastatic melanoma, were given one of the three treatments i.e. either gp100 peptide vaccine alone, ipilimumab alone, or both gp100 vaccine and ipilimumab to study the overall survival. The median overall survival with gp100 peptide vaccine alone was 6.4 months and the same in combination with both was 10 months, showing a high overall survival [125]. Interestingly, in the same study, treatment with ipilimumab alone gave a median overall survival of 10.1 months. Further research is required in INTERNATIONAL REVIEWS OF IMMUNOLOGY this combinatorial therapy to access multiple other factors and their efficacy. Immunotherapies at a glance All the therapeutics described in this review have been broadly compared in Table 4. Concerns with immunotherapy  Cost Cancer immunotherapies are relatively less toxic and generate efficient responses but the primary concern lies with their cost. Majority of these therapies are extremely expensive [136] and hence are out of the reach of poor individuals or the developing nations. Taking into consideration CAR T cell therapies, Tisagenlecleucel has a price tag of $4,75,000 whereas Axicabtagene ciloleucel costs around $3,73,000 [132]. Owing to such high prices, CAR T cell therapy stays out the reach of poor people. Similarly, immune checkpoint inhibitors have shown promising results but many of them are not economical. A study found that use of nivolumab for metastatic head/neck cancers was not cost-effective over chemotherapy and use of nivolumab and pembrolizumab for renal cell cancer or bladder cancer didn’t show adequate cost-effectiveness [137]. However, the same study found few other inhibitors economically reasonable for other types of cancer and have noted that the patient selection criteria is an important parameter for determination of cost-effectiveness which can alter their conclusions [137]. The first FDA approved therapeutic vaccine, Sipuleucel-T, used for prostate cancer, is relatively minimally toxic but costs around $93,000–$100,000 for a full therapy course given for one month [138,139]. Abiraterone, an anti-androgen medication, is a conventional alternative to Sipuleucel-T and it costs almost the same though, it may cost more as the cost depends upon the months of treatment received. However, a study showed that with Abiraterone, the gains in number of total life years and quality adjusted life years are somewhat higher [140]. Hence, a conflict between the cost, efficiency and toxicity of treatment definitely exists. However, with the emergence of biosimilars, the cost is expected to come down for various immunotheraputics. Biosimilars have a comparable biological activity to their respective reference drugs but are generally cost-effective and have the ability to increase treatment accessibility, especially to the poor. Further, 11 they bring competition in the market making the original drug developers to bring down the cost. With the expiry due for the patents of various immunotheraputics in coming years, biosimilars are expected to emerge which can potentially address the cost issue. One such assessment of the impact of biosimilars on the cost and their safety and efficacy has been done in a recent review [141].  Autoimmunity and infections Vaccines, ATCT and mABs also give rise to autoimmunity against normal tissues. It can arise as a result of therapies acting against TAAs expressed on normal tissues, expansion of self-reactive T cell due to inhibition of CTLA-4 or by other less understood mechanisms [142]. mABs have been reported to increase the risk of severe infections as well as reactivation of hepatitis B virus (HBV). In particular, mABs for anti-epidermal growth factor receptor, used in many malignancies, has been reported to significantly increase the chance of developing non-fatal but severe infections [133]. Rituximab, which is an anti-CD20 mAB, is used for the treatment of CD20þ B-cell non-Hodgkin Lymphoma. There exists a well-defined association between Rituximab treatment and reactivation of HBV and hence it is critical to precisely identify patients at risk and therefore require prophylactic antiviral regimes [134].  Side effects The frequent AEs linked with Sipuleucel-T involved pyrexia, malaise, chills, fatigue, nausea and culture positives [143]. Severe Cytokine release syndrome (sCRS) and neurological disorders are major toxicities associated with CAR T cell therapy [81]. The manifestations of sCRS involve hypoxia, end organ dysfunction and hemophagocytic lymphohistiocytosis among others whereas neurological disorders involve cognitive defects, encephalopathy, seizures and cerebral edema [144]. Immune checkpoint blockade involves effects like fatigue, hepatitis, vitiligo, colitis, diarrhea, hypothyroidism, myasthenia gravis [113,145]. Patients administered with PD-1/PD-L1 inhibitors reported 30–40% of dermatological toxicities of all grades which was 50% with anti-CTLA-4 inhibitor, ipilimumab. Further, organ specific inflammation like pneumonitis, hepatitis, autoimmune encephalitis, inflammatory arthritis, hypophysitis, lupus nephritis and posterior reversible 12 S. JAIN AND S. KUMAR encephalopathy syndrome, sarcoidosis, anemia are some of the many other AEs which have been observed with immune checkpoint inhibitors [146]. Combining mABs against immune checkpoints further increases these toxicities [123]. Discussion Immunotherapy is emerging as one of the leading therapeutics to deal with certain types of cancer. Although conventional therapies like surgery, chemotherapy and radiotherapy remain the most widely used mechanisms and generally are the first line of treatment in many cancers, however, immunotherapy is slowly gaining speed and is becoming relevant for first treatment line in certain cancers like non-small cell lung cancer (NSCLC) [147]. Other than that, immunotherapies are used as adjuvant therapies in the line of cancer management in certain cases like in advanced melanoma [148] or when first line of treatment fails. Most immunotherapeutics are highly specific, relatively less toxic and offer better survival compared to conventional therapies. In our view, every immunotherapy offers different niche of advantages but also has certain challenges, most common among them being their high cost. In the development process of therapeutic vaccination, new antigens are being tested regularly for a potential vaccination candidate. Multiple ways have been developed for therapeutic vaccination involving infusion of antigens, irradiated tumor cells, mutated cells which express specific antigen, or even the antigen DNA with different types of vector systems. Relative to other immunotherapies, vaccines are less toxic but also are less efficient. The development of oncolytic viruses can be considered as a major breakthrough in therapeutic vaccination for their very ability to selectively infect and propagate via tumor cells and causing minimal harm to the normal cells. However, lack of universal tumor antigens hinder this development. In spite of these obstacles, vaccines are being developed targeting both TAAs and TSAs. ATCT and specifically CAR T cell therapy are evolving at a rapid rate. Since its inception in 1988, ATCT has demonstrated positive results all along. The rapid evolution of CAR T cell therapy can only be judged from a simple fact that there now exists four generations of CAR T cell. CAR T cell therapy is highly efficient, specific and has a high response rate. However, research and development in CAR T cell therapy regime seems to benefit only a few as of now owing to its extremely high cost of administration. Further, toxicity related to CAR T cell therapy also possesses great challenges. sCRS, measured in terms of cytokine maximum fold change, fever, hypoxia, neurological disorders etc., is related with CAR T cell treatment. This sCRS requires immediate medical intervention and steroid treatment in the conventional strategy [81]. Davila and his group studied efficacy of using tocilizumab, a monoclonal antibody blocking IL-6 receptor as a measure to control sCRS against steroids which lead to five-fold decline in CAR T cells and got positive results [81]. More of such novel alternatives are required while dealing with such severe toxicities. Monoclonal antibodies are one of the most rapidly rising regime in the field of immunotherapy. Owing to the fact that it is extremely specific and relatively less toxic, it can soon become most widely accepted treatment regime in near future. The issue of development of resistance toward these mABs can be managed by using bi-specific antibodies which can target more than one cancer antigen or signaling pathway at the same time. Along with targeting cancer antigen, same bsABs can also be used for cytotoxic drug delivery or apoptosis activation and multiple such combinations can be made. However, more research is required to address of the risk factors which lead to development of severe infections and accurate screening to prevent HBV reactivation. Immune check point blocking is one of the most efficient techniques in terms of its potency and ability to deal with a range of cancers. However, this therapy faces a serious drawback of life threatening side effects which, at present, is hindering its development as a major immunotherapeutic. Combinatorial therapies can culminate the advantages of two therapies and can generate a heightened response against cancer. However, recent trials which involved combination of ipilumumab with therapeutic vaccine or radiotherapy, didn’t generate promising results. Further, the combination of two immune check point blockers might have given positive results due to the expected synergy but the issue of increased toxicity has to be answered. Hence these combinatorial therapies call for extensive research to develop a combinatorial therapy which can cater to the needs, is cost effective and least toxic. An important aspect with immunotherapies is choosing the appropriate therapy for a particular type of cancer. Biomarkers can provide insights about genetic makeup and immune system interactions of a particular type of cancer in individual patient. They are also used to monitor the response of a patient INTERNATIONAL REVIEWS OF IMMUNOLOGY toward a particular treatment regime. Serum proteins, tumor micro-environment factors, circulating tumor cells, immune cells are some of the important biomarkers among many others [149]. A recent clinical trial involving patients suffering from gastrointestinal stromal tumor showed that circulating tumor DNA (ctDNA) indicated disease activity in these patients presenting its potential as prospective biomarker for future [150]. Similarly, a study predicted plasmatic biomarkers in patients suffering from NSCLC who were being treated with nivolumab [151]. These biomarkers are a rapidly developing focus in the field of immunotherapy which holds the key to more efficient and safe treatments. A special mention is required here for those chemotherapeutics which boost the natural immunity against cancer forming the very basis of immunotherapy. Myeloid Derived Suppressor Cells (MDSCs) can mount up in tumors during its growth and can help in cancer immune tolerance by inhibiting function of CD8þ T cells. Experiments in mice and cell lines showed that Gemcitabine and 5-Flurouracil are selectively cytotoxic to MDSCs allowing effector T cells to function more efficiently [152]. Regulatory T cells (Tregs), suppress high avidity tumor-specific T cells. Cyclophosphamide can be used to supress Tregs for effective anti-tumor immune response [153]. Hence, depletion of the immunosuppressive activities of Tregs and MDSCs should lead to an effective induction of anti-tumor T cell response [115]. The future prospects of immunotherapy seem very bright. Given their specificity and efficiency, these can be widely used in future to deal with a range of cancers. However, along with the concerns like cost and toxicity, other parameters like heterogeneity in tumors as well as in cancer patients have to be taken in consideration. As cancer is unstable genetically, next generation sequencing and other bioinformatics tools are of utmost importance in the identification of neoantigens with high immunogenic potential as well as for the development of safer and efficient therapies in future. Similarly, oncolytic viruses hold a great potential in differential killing of cancer cells. Combinatorial treatments have shown a high level of synergy, especially by using two immune check point inhibitors. If future research can take care of the added toxicities in these combinatorial therapies, a major outbreak in the cancer treatment can be reached. 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