Frontiers in Anti-Cancer Drug Discovery: Volume 9

Recent Advances and New Insights for the Therapeutic Intervention of Cancer

Nhi Nguyen¹, Stina George Fernandes², Ekta Khattar², Yinghui Li¹, *

¹ School of Biological Sciences (SBS), Nanyang Technological University (NTU), 60 Nanyang Drive 637551, Singapore

² Department of Biological Sciences, Sunandan Divatia School of Science, SVKM's NMIMS (Deemed-to-be University), Vile Parle (West), Mumbai 400056, India

Abstract

Traditionally, anti-cancer treatments mainly focus on chemotherapies, radiation therapy and surgery. However, these treatments are limited in terms of their specificity for targeting only cancer cells. DNA damaging chemotherapy is one of the most common treatment modalities of cancer. Current progress of targeted therapy that relies on DNA damage response (DDR) in cancer offers a vast therapeutic window by specifically targeting DDR functions in patient specific tumours. Recent developments in immunotherapy – therapy that boosts the body’s immune system to fight against cancer cells, have shown promising results. Currently, different approaches of immunotherapies such as cytokine, antibody, oncolytic virus, adoptive cell transfer therapies or cancer vaccination have made significant progress in treating different cancer types. This paper seeks to provide an overview of the recent developments of drugs targeting DDR and various immunotherapeutic approaches, with specific focus on the anti-CD19 CAR-T cell therapy. Finally, the paper will give a perspective on future directions of DDR therapy, CAR-T cell therapy as well as the combination of different cancer therapies for effective cancer treatment regime.

Over the past few years, the decipher of different DNA damage response mechanisms enables the development of novel inhibitors for cancer treatment. Furthermore, recent advances in genomic editing technologies and scientific discoveries reveal important roles of the immune system in the context of cancer development. This also prompts the plethora of drug developments that have vast potential in the treatment of different types of cancer diseases. This paper will give an overview of recent drug developments in chemotherapeutic agents targeting DNA damage response as well as recent immunotherapies that have been successful in the treatment of human cancers. The contents discussed in the paper are summarized in (Fig. 1).

Keywords: Adoptive Cell Transfer Therapies, Antibody Therapy, Anti-CD19 CAR-T Cell Therapy, Combinational Approaches, Cytokine Therapy, DNA Damage Response, DNA Damage Response Inhibitors, Immunotherapy, Oncolytic Virus Approach.

* Correspondence author Yinghui Li: Nanyang Technological University (NTU), School of Biological Sciences (SBS), 60 Nanyang Drive, 637551 Singapore; Tel: 6563162871; Email: liyh@ntu.edu.sg

Fig. (1))

Summary diagram of the chapter.

TARGETING DNA DAMAGE RESPONSE (DDR) IN CANCER

Cells encounter thousands of DNA damaging events every day and various mechanisms have evolved to combat the damage [1]. Induction, detection and resolution of the DNA damage involves various intra and inter-cellular signalling events mediated by enzymes, adaptor proteins as well as other macromolecules. These events are collectively termed as DNA damage response (DDR). The consequences of DDR can be cell cycle arrest, transcription and replication regulation leading to repair or bypass of the DNA damage. Depending on the type of DNA damage, different repair and response mechanisms are activated. The type of DDR signalling also depends on the cell cycle stage during which it is activated. DDR is important for normal human development as it contributes to genome maintenance which is essential for cellular function and health. Loss of efficient DDR may lead to mutations and thus promote cancer initiation and development. While loss of DDR is deleterious to normal cell functioning, it is extremely useful for cancer cells. Cancer cells have to maintain endless replication capacity and overcome high rate of apoptosis to survive, thus they rely heavily on some of the DDR pathways and components making them attractive therapeutic targets [2]. This section discusses the type of DDR and current therapeutic approaches targeting DNA repair proteins and pathways in cancer.

DNA Damage Response Pathways

Different types of DNA damage initiate different signalling pathways and repair mechanisms. However, there is no redundancy and different pathways might compensate and cross talk amongst each other to determine the final outcome. The major repair pathways in human cells depending on type of damage are described below.

Base Excision Repair (BER)

DNA bases encounter damage due to various reasons including oxidation, deamination and alkylation. These modifications result in incorrect base pairing and thus lead to mutations in the DNA. Such base lesions which are small and non-helix distorting are repaired by base excision repair pathway. BER is active during all stages of the cell cycle.

The major steps of BER comprise the removal of damaged DNA base by enzymatic action of DNA glycosylase, creating an apyrimidic/apurinic (AP) site. Examples of DNA glycosylases include 3-methyladenine DNA glycosylase (AAG/MAG1), 8-oxoguanine DNA glycosylate (OGG), uracil DNA glycosylase (UDG/UNG) and endonuclease III like DNA glycosylases. AP site is processed by AP endonuclease (APE1) by cleaving the phosphodiester backbone located at 5’ position to the AP site. This generates a 3’ hydroxyl group and a 5’ deoxyribose phosphate moiety (5’dRP). The resulting single-strand break can then be repaired by either short-patch (where a single nucleotide is replaced, predominant mode of repair) or long-patch BER (where 2–10 new nucleotides are synthesized) [3]. The selection also depends on relative concentration of ATP or the nature of 5’dRP intermediates, cell cycle stage, and whether the cell is terminally differentiated or dividing [4].

Short patch BER is catalyzed by polymerase β (pol β) and by pol λ in the absence of pol β. These polymerases insert only one nucleotide and in addition possess a lyase domain that removes the 5’dRP that is generated during AP endonuclease cleavage. DNA ligase III together with its cofactor XRCC1 catalyzes the nick-sealing step and completes the short patch BER. Long patch BER is catalyzed by pol δ and pol ε along with PCNA that acts as a processivity factor. They carry out displacement synthesis in which 5’ end of DNA is displaced to form a flap. This 5’ flap is removed by FEN1 flap endonuclease followed by ligation of the break by DNA ligase I [5].

DNA Mismatch Repair (MMR)

MMR repairs base-base mismatches or insertion/deletion loops that are generated by erroneous replication or recombination. In eukaryotes, the mismatch is recognized by MutS family of proteins. MutSα (Msh2/Msh6) pathway is involved primarily in base substitution and small-loop mismatch repair while MutSβ (Msh2/Msh3) pathway is involved in small-loop repair, in addition to large-loop (around 10 nucleotide loops) repair but does not perform base substitution [6-8]. This is followed by incision through MutLα which is composed of MLH1 and PMS2 proteins [7]. It functions as a DNA endonuclease upon activation by mismatch and other proteins like MutS and PCNA. MutLβ (MLH1 and PMS1) and MutLγ (MLH1 and MLH3) are also known to function in mismatch repair but their roles are not well known. This incision recruits exonuclease which incises the DNA strand 5’ or 3’ depending on the incision. Single strand binding protein RPA (Replication Protein A) coats the region and DNA polymerase III is recruited to fill in the gap which is sealed by DNA ligase. PCNA is also known to play important roles in MMR pathway [9].

Nucleotide Excision Repair (NER)

NER mainly removes DNA damage induced by Ultra Violet light (UV), which results in bulky adducts of DNA typically thymine dimers and 6,4-photoproducts. NER in eukaryotes can be divided into two sub-pathways in the initial damage recognition step: global genomic NER (GG-NER or GGR) and transcription coupled NER (TC-NER or TCR) [10]. Later steps involving dual incision, repair and ligation are common in both sub pathways. In TCR, when RNA polymerase encounters damaged DNA, it stalls and the damaged site recruits XPG and CSB proteins while in GGR, the damaged site is recognized by XPE/DDB2 and XPC/hHR23B. These complexes have distortion recognition properties. Following damage recognition, XPA, RPA, XPG and TFIIH are recruited. TFIIH then unwinds the DNA helix, following which, XPG (uses its endonuclease activity to cut 3’side of lesion) and XPF/ERCC1 (uses its endonuclease activity to cut 5’side of lesion) cut and excise the lesion. Subsequently, DNA polymerase fills the gap and DNA ligase seals the nick to restore normal nucleotide sequence.

DNA Double Strand Break Repair

Double strand breaks (DSB) in DNA are extremely harmful if left unrepaired or incorrectly repaired. Normal cells generate DSB through endogenous processes such as V(D)J recombination, DNA replication and exogenous agents like ionizing radiation and chemotherapeutics [11]. Cells can repair DSB through two pathways: the first is homologous recombination (HR) where either a homologous chromosome or sister chromatid is used as a template to repair the DNA break and the second is non-homologous end joining (NHEJ) where DNA breaks are directly joined without or with very little sequence homology.

In HR pathway, following DNA damage, there is an initial resection step by the core complex consisting of Mre11, Nbs1 and Rad50 (MRN) along with nucleases Exo1 and CTIP. This resection creates single stranded DNA which is then bound and stabilized by RPA which activates ATR kinase and also recruits Rad51, BRCA1, BRCA2 and other factors. This complex then searches for the homologous repair template. Once template is found, the DNA polymerases, resolvases and DNA ligase I reseal the repaired break for this high fidelity repair [12].

In mammals, NHEJ is the preferred pathway for DSB repair. NHEJ can be further divided into two biochemically and genetically distinct pathways: classic – NHEJ (C-NHEJ), and alternative NHEJ (A-NHEJ).

The mechanism for C-NHEJ is known in great depth. Seven proteins have been shown to be essential for this pathway namely: Ku70, Ku-86, the DNA dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, X-ray cross complementing 4 (XRCC4), XRCC4-like factor (XLF) and DNA ligase IV [13]. Heterodimer of Ku70 and 86 recognizes and binds to the broken ends of DNA as a ring encircling DNA duplex [14]. This bridge of Ku dimer structurally supports the DNA and also aligns the ends of DNA thus protecting it from degradation. Following this DNA-PKcs is recruited which is then activated when it binds and phosphorylates artemis protein, and the resulting artemis/DNA-PKcs complex is believed to possess nuclease activity that cleaves the 5’ and 3’ DNA overhangs [15]. NHEJ requires two DNA blunt ends in order to perform ligation. Once the blunt ends are placed, the XRCC4/DNA ligase IV ligation complex is recruited to ligate the broken DNA ends. Ligation step is performed by DNA ligase IV, but it requires the binding of XRCC4. XRCC4 protein plays a regulatory role by stabilizing DNA ligase IV, stimulating its ligase activity, and directing the ligase to the site of DNA breaks via its recognition helix and DNA-binding capacity.

A-NHEJ functions when the key proteins in C-NHEJ are less functional or absent. The mechanism and steps of A-NHEJ are not very well known. Major proteins involved are PARP1, DNA Ligase III/XRCC1, DNA Ligase I, MRN, Polynucleotide kinase and FEN1. A-NHEJ pathway utilizes microhomology at the DNA breakpoint regions, consisting of shared sequence (ranging from 5 to 22 bp) between the two DNA ends, hence it is also known as microhomology-mediated end-joining (MMEJ). Recent studies suggest that MMEJ is functional in both normal and cancer cells [16, 17].

DDR Associated Cell Signalling Pathways

DDR simultaneously activates signalling response pathways in the cell. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia Rad3 related (ATR) proteins form the key regulators of DDR and maintain genome homeostasis. Double strand breaks primarily activate ATM kinase while any damage to replicating chromosomes activates ATR kinase although DSB can also activate ATR but that is dependent on ATM and MRN complex [18, 19]. In response to DNA damage, several hundred proteins are phosphorylated majorly by ATM kinase or ATR kinase while DNA-PKcs also regulates a smaller number of target proteins but it is mainly restricted to NHEJ pathway [20-24]. Chk1 and Chk2 represent major downstream kinases of ATR and ATM respectively. Activation of DDR kinase signalling further activates cell cycle checkpoint pathways, mainly cell cycle arrest and DNA repair pathway components.

DDR as an Anticancer Drug Target

DDR in cancer cells is different as compared to that in normal cells and that provides a rationale for drug targeting. The major differences include: loss of one or more DDR pathways resulting in dependency of cancer on remaining pathways, elevated levels of replication stress and also higher levels of endogenous DNA damage.

DDR Inhibitors in Clinical Development

A DDR deficiency in cancer cells that results in dependency on a particular DDR pathway or target presents an opportunity to use inhibitors of the pathway or target so as to block repair in such cancer cells. This concept is known as synthetic lethality. In the context of cancer DDR therapeutics, synthetic lethality exploits the possibility that, in cancers, one loss-of-function event is genetic and distinct to the tumor whereas the second loss-of-function event can be triggered pharmacologically using a DDR inhibitor. An example of such synthetic lethality is observed in the case of PARP inhibition. PARP enzyme activity is important for chromosome relaxation following which PARP dissociates from the DNA for SSB repair to proceed. PARP inhibitors are designed with a NAD+ mimetic core to compete with NAD+ such that they stall PARP1 onto SSBs and these PARP1-DNA complexes block replication. Such trapped PARP1-DNA complexes will further result in toxic DSBs by virtue of stalled and collapsed replication forks. In normal cells, HR would be activated to repair these DBS, however HR deficiency in cancer cells would direct the cells to initiate less efficient NHEJ, resulting in increased genomic instability and thus cell death.

This has been investigated in the cancers with defective forms of the tumor suppressor genes BRCA1 and BRCA2, whose normal function involves repair of DSBs by HR. It has been demonstrated that PARP inhibitor activity in BRCA homozygous mutant (BRCA-/-) is almost 1000-fold as compared to BRCA heterozygous (BRCA-/+) and WT BRCA (+/+) because PARP inhibition in BRCA-deficient tumor cells will exhibit greater effects than in normal cells which are WT or heterozygous for BRCA1 or BRCA2 [25]. In cases of germline BRCA mutations, the tumors will lack both copies of functional BRCA and normal cells will harbour at least one functional copy. In non-BRCA, HR deficient cancers, numerous approaches like DNA sequence analysis of HR genes, analysis of DNA rearrangements or mutational patterns due to loss of HR pathway, an immunohistochemistry approach, etc., are being exploited for selection of a single non-BRCA, HR-associated deficiency.

Further examples of inhibitors targeting HR by exploiting synthetic lethality phenotype include Rad52 inhibitors in BRCA1/2 cancers, ATR inhibition combined with PARP inhibition and CDK inhibition with PARP inhibition [26-28].

The first PARP inhibitor to be clinically tested was olaparib (AZD2281). Currently, there are several PARP inhibitors in clinical trials (Table 1).

One important challenge is the identification of agents that are truly synthetically lethal, because if the agent only functions to increase sensitivity of the lesion to another agent, monotherapy may not be an option.

Table 1 DDR Inhibitors in Clinical Development [328].

Replication stress represents one of the several hallmarks of cancer wherein dissociation of DNA polymerase from the replisome complex creates stretches of single-stranded DNA at the replication fork and this results in recruitment of replication protein A (RPA) which triggers DDR, and predominantly activates ATR kinase pathway [29]. Insufficient concentration of nucleotides or other replication factors can also result in replication stress [30]. For example, G1/S checkpoint defect in cancer cells due to pRB deficiencies coupled with CDKN2A deletion or Cyclin D1/Cyclin E amplification can cause premature entry into S-phase and then stalling of DNA replication due to insufficient replication resources [31]. Cyclin E and MYC amplification result in rapid replication origin firing causing collision of replication and transcription machinery [32, 33]. MYC overexpression also generates high levels of ROS that causes oxidative damage to DNA [34]. Since cancers are associated with deprivation of cell-cycle checkpoints, increased expression of oncogenes, elevated levels of intrinsic ROS, etc., cancer cells will experience greater replication stress as compared to normal cells upon chemotherapy. This elevated replication stress coupled with the loss of DDR mechanisms can help design cancer-specific and DDR target-specific strategies for a number of proteins functioning in replication stress.

RPA recruitment to the ssDNA at the stalled replication fork shields the ssDNA from cleavage and engages ATR and SMARCAL1 (helicase). By activating Chk1 and ribonucleotide reductase M2 (RRM2), ATR inhibits new replication firing and accumulation of ssDNA respectively. Accumulation of ssDNA and subsequent consumption of RPA needs to be prevented because exhaustion of all RPA will result in the remainder ssDNA being converted to lethal DSBs. Apart from the ATR-CHK1 pathway, WEE1-CDK pathway also needs investigation for designing replication stress-driven therapies in cancer [35]. WEE1 and CDKs are involved in replication origin firing and inhibition of WEE1 prevents inhibition of CDK1 and CDK2 via phosphorylation by WEE1, thereby allowing increased replication origin firing, reduction in nucleotide levels and an increase in Mus81-Eme1 endonuclease-mediated DSBs [36]. As CDK1 functions as a G2/M checkpoint protein, its upregulation will force early entry of cells into mitosis eventually leading to cell death in the absence of completely replicated DNA.

Studies reveal that Chk1 inhibition causes much higher levels of γH2AX induction as compared to ATR inhibition, suggesting that Chk1 inhibition kills cells at much lower levels of replication stress whereas ATR inhibition selectively destroys cells that exhibit stress levels beyond a certain threshold [37]. It has been demonstrated that ATR-Chk1 may not always follow a linear pathway with respect to replication origin firing, which can be prevented even in the presence of ATR inhibitor wherein alternative pathway may be utilized involving DNA-PK and ATM [31]. Table 1 enlists several targets and compounds with which clinical trials are being conducted.

Combining DDR Inhibitors with DNA Damaging Radiotherapy and Chemotherapy

Over the course of many years, ionizing radiation and systemic chemotherapy have been used for cancer treatment apart from surgery. 1 Gy of ionizing radiation can generate about 1000 SSBs and 35 DSBs due to the oxygen free radicals produced, radiation contributes to around 40% of cures [38, 39]. However, one major disadvantage is the collateral damage to normal healthy cells. One approach to overcome this problem is to identify and utilize a tumor’s intrinsic sensitivity to radiation and generate a patient-specific treatment plan. In this way, more sensitive tumors can be given lower doses of radiation whereas resistant tumors can be approached more aggressively.

Another approach is combining DDR inhibitor treatment with radiotherapy to sensitize the tumor and to increase therapeutic index of the treatment. A preclinical trial evaluated radiosensitisation effects of PARP inhibitor olaparib in BRCA2-deficient and BRCA2-complemented isogenic cell lines and several human head and neck squamous cell carcinoma cell lines [40]. This study demonstrated that radiation followed by 7-hour exposure to olaparib, at concentrations much lower than those required to induce cell death unassisted, were adequate to radiosensitise the cells. Also, olaparib could radiosensitise the BRCA2-deficient cells at lower doses than in BRCA2-complemented samples. However, it still needs to be ascertained if this kind of combination therapy clinically helps to enhance therapeutic index. One major challenge faced by research of this nature is the requirement of a syngenic or orthotopic immune-competent host model in which antitumour activity and normal tissue toxicity in response to radiation can be studied simultaneously. Another challenge is the duration of the clinical study as dose escalation studies (to gauge chronic radiation-induced toxicities) require months of follow up in the cohort drug design. Also localized disease control is not a true measure of overall survival of a patient.

Combining DDR inhibitors with DNA-damaging chemotherapy also bears its own challenges as they involve systemic delivery of the drug and many of the drugs show overlapping toxicities with DDR inhibitors. Frequently used DNA-damaging chemotherapies are:

Platinum salts for induction of covalent crosslinks of DNA bases e.g. carboplatin, cisplatin, oxaliplatin, etc.

Alkylating agents for modification of DNA bases e.g. temozolomide

Topoisomerase inhibitors e.g. Topoisomerase 1-camptothecin, topotecan and irinotecan and Topoisomerase 2- etoposide, doxorubicin.

Topoisomerases function to relax the DNA supercoil by nicking the DNA, rotating the strands and then relegating them. Topoisomerase inhibitors produce non-productive Topoisomerase-DNA cleavage complexes (Topcc) post DNA cleavage but before relegation. Topoisomerase 2 nicks both strands of DNA and its inhibitors produce DSBs while Topoisomerase 1 nicks one strand and its inhibitors produce SSBs which get converted to DSBs due to stalled replication forks. Topoisomerase inhibitors differ from PARP inhibitors as their target enzymes normally have error-free activity and are not part of any DDR machinery and also that their therapeutic index is so much smaller than PARP inhibitors.

The choice of chemotherapy to be used in conjunction with a DDR agent depends on the type of DNA damage. For instance, Top1 inhibitors can be used in combination with ATM, ATR or PARP inhibitors as all three operate in DDR resulting from Top1 inhibition [41]. Also, a combination of PARP inhibitor and Temozolomide can be used as PARP is required for repair of TMZ-generated SSBs [42]. Identifying an effective combination of drug dosage and schedule is still a major challenge in multiple studies that are being conducted. However, generating such data holds clinical significance as it has been shown that combination therapy is able to re-sensitize chemo-resistant tumors.

One approach to alleviate the toxicity of chemotherapy plus DDR agent in healthy cells is to allow a gap of two to three days between chemotherapy and treatment with DDR agent. This gapped schedule approach exhibited differential effects of platinum-induced DNA damage in tumor against bone marrow. In the bone marrow, resolution of DNA damage took place within 48 hours following therapy whereas in tumor cells, gamma-H2AX foci could be detected even after 72 hours. An alternative strategy is to use targeted delivery agents like nanoparticles which is currently being explored.

Future Prospects of DDR Targeting Agents in Cancer Treatment

Synthetic Lethality

Initially, combination therapy of DDR inhibitors began with chemotherapy, with MGMT inhibitor O⁶-benzylguanin being used in conjunction with alkylating agent BCNU [43]. However, this combination was poorly tolerated. A major development came in 2005, when PARP inhibitor olaparib treatment in BRCA-deficient cancers helped to signify the concept of synthetic lethality. In the last decade, DDR inhibitors targeting essential replication proteins, inhibitors that enhance differential S-phase replication stress between cancers and normal cells, G2/M checkpoint inhibitors, inhibitors that exploit genetic deficiencies in cancers, etc., have been developed. Thus, identifying novel genetic combinations with synthetic lethality holds therapeutic potential.

DDR with Epigenetic Compounds

Epigenetic modulators are also being tested with DDR inhibitors. For example, histone lysine methyltransferase (HKMT) inhibitors have been shown to cooperate with PARP inhibitor activity, thereby preventing retention of BRCA1/BARD1 complex at DSBs and inhibiting cancer cell growth [44].

IMMUNOTHERAPY

The essential role of the immune system as the natural defence against cancers has been studied for years. It has also been observed in clinical researches and epidemiological studies that immunosuppressed patients have higher risks of developing different types of cancers. Amongst them, the incidence of Kaposi’s sarcoma, a type of cancer developed from the opportunistic infection of human herpes virus type 8 (HHV-8) in lymph node and skin, has been shown to be 400 to 500 higher in HIV-positive or organ transplant patients compared to the control population [45]. The termination of immunosuppressive treatments reduces the risk for Kaposi’s sarcoma, suggesting a possible association between immunosuppression and tumour development in certain individuals [45, 46]. Furthermore, studies among patients showing spontaneous tumour regression after episodes of bacterial infections present convincing evidence that the immune system plays a significant role in combating cancer development [47-49]. Among them, the injection of attenuated Baccillus Calmette-Guerin bacterium for the treatment of superficial bladder cancer has been in practice since 1976 [49]. This suggests that the enhancement of one’s immune system has the potential to counter tumorous growth. These observations build the basis for continuous efforts in harnessing the immune system for effective cancer treatments.

In recent years, immunotherapy has shown promises as a powerful and specific treatment for cancers. Immunotherapy is considered as a biological therapy, which makes use of substances produced by living organisms to counter cancerous growth. There are several advantages of immunotherapies in cancer treatments. Unlike chemotherapy and radiotherapy, immunotherapy is more specific and does not target normal rapidly dividing cells such as hair follicles, bone marrow or intestinal epithelial cells [50-52]. Due to the specificity of immunotherapy, damages to adjacent normal tissue can be reduced as compared to the standard chemotherapy and radiation therapy. Furthermore, immunotherapy can be modified to target non-dividing cancerous cells such as rare cancer stem cells, which are associated with cancer recurrence. Location-wise, modified immune cells can target areas that are unreachable by surgery. Given the heterogeneous nature of tumour, there are variations in responses to general treatments among different patients having the same type of tumour. Therefore, the flexibility of immunotherapy allows greater specific and effective targets of cancers. Personalized treatments of immunotherapy designed to target neoantigens unique to a certain population have the potential to reduce the problem of heterogeneity among patients [53]. Considering these advantages, immunotherapy presents a paradigm shift in cancer treatment routines in addition to the traditional methods. The regular use of immunotherapy in combination with standard radiation therapy, chemotherapies and surgery is foreseeable in standard cancer treatment of the future.

Approaches to immunotherapy are categorized into five broad branches: adoptive cell transfer approach, vaccination approach, antibody therapy, cytokine approach and oncolytic virus approach [54, 55]. In the past few years, there have been numerous clinical trials and FDA-approved drugs in response to positive findings highlighting the effectiveness of immunotherapy in the treatments of different cancer types. This review aims to: (i) give a brief overview of these immunotherapeutic approaches by discussing their mechanisms of action, their advantages and disadvantages as well as highlighting currently approved drugs in each area (Table 2), (ii) discuss in detail the newly approved anti - CD19 chimeric antigen receptor (CAR) T cells therapy and its vast potential.

Table 2 Current FDA-approved immunotherapeutic drugs.

Common Immunotherapeutic Approaches

Cytokine Therapy

Cytokines are signalling molecules that cells use to communicate with one another. They play a major role in the development of both innate and adaptive immune systems. For most immune cells, cytokines are essential in their proliferation, differentiation and prolonged survival necessary for effective functioning of the immune system. Harnessing the role of cytokines in immunotherapy enhances or inhibits certain immunologic responses in leukocytes. Various clinical trials with interferon (IFN)-α, interleukin – 2 (IL-2), IL-12, IL-15, IL-21, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factors (TNFs) suggest their roles in effective treatment of cancer [56]. Among them, IFN-α and IL-2 cytokines have been approved by the FDA for the treatment of different cancer types [56].

IFN-α was the first cytokine therapy to be approved by the FDA for the treatment of hairy cell leukemia (HCL) in 1986 [57]. Subsequently, IFN-α was also permitted as a treatment for several cancer types such as Kaposi’s sarcoma, metastatic melanomas [58] and chronic myelogenous leukemia for instance [59], [60-63]. IFN-α belongs to type I IFN - cytokines that are produced upon viral invasion by various immune cells [64, 65]. Through tyrosine kinase (Tyk) and Jak/Stat signalling, IFN-α activates antigen specific B and T cells, which aids in the maturation of dendritic cells (DCs) as well as promote cancer cell apoptosis [56]. Among HCL patients treated with IFN-α, 77% showed significant improvements in red blood cell count and granulocyte level [66]. After the success of IFN-α, IL-2 was also approved by the FDA in 1992 to treat melanoma and renal cell cancer [67, 68]. IL-2 is a globular glycoprotein cytokine that acts through Janus kinase signal transducers and activators of transcription (JAK/STAT) signalling to promote natural killer (NK) cells, antigen-specific CD8+ and CD4+ T cell expansion [69-72]. IL-2 also mediates antibodies secretion from IgM-stimulated B cells. High doses of IL-2 therapy against metastatic renal cancer used over different durations have shown moderate responses of 14% with 5% of the patients having complete remissions (CR) [73-75]. In addition, low dose of IL-2 treatment has been shown to increase the amount of CD56+ CD3- NK population from 450% to 900% [76].

Even though some positive results have been observed in many clinical trials, the use of cytokine therapy as monotherapy is limited in its potential in cancer treatment. In terms of effectiveness, IL-2 and IFN-α administrations have induced many side effects in patients [77]. This is because cytokines act within a short distance, which results in high concentrations of cytokines being administered peritoneally or subcutaneously to achieve desired effects [78-83]. This large dosage of cytokines causes flu-like symptoms like nausea, fever or neuropsychological effects like mania or depression. In fact, FDA immediately terminated all IL-12 trials after a phase II clinical trials of IL-12 therapy caused severe side effects to 15 over 17 patients, resulting in the death of two patients [84]. Flu-like symptoms caused by severe toxicities of IFN-α are also well documented [85, 86]. Furthermore, some cytokines used in immunotherapy have been implicated in immunosuppression. In fact, IL-2 plays a role in the maintenance of peripheral regulatory T cells (Tregs) – T cell subpopulation that mediate immunosuppression and immune escape of cancerous cells [87]. This compromises on the effectiveness of IL-2 as a cytokine therapy.

Antibody Immunotherapy

Monoclonal Antibody Immunotherapy

Monoclonal antibody (mAb) therapy has been used a long time ago as a useful tool to target specific cancer-related antigens. These cancer-specific antigens can be expressed on cancer cells or as active members in cancer proliferation pathways, blood vessel formation or immune checkpoint mechanism [88, 89]. Furthermore, conjugation of radioactive substances or other anticancer drugs like cytokines and toxins with mAbs has been shown to enhance the effectiveness of tumour targeting in mAb therapy [90]. The most commonly used mAb is IgG mAb, which has two regions: a variable region and a constant fragment. The variable region is antigen-specific while the constant fragment helps bring together antigen-specific cells and leukocytes for elimination. Furthermore, the constant fragment of mAb facilitates the immune role of mAb against antigen-specific cells like antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or antibody-dependent phagocytosis (ADCP) [91, 92]. Hence, mAb functions as a useful tool in cancer immunotherapy by facilitating the killing of cancer cells using ADCC, CDC and ADCP, inhibiting specific ligand-receptor interaction on cancer cells or regulating checkpoint molecules on immune cells [91, 92].

MAbs Targeting Cell Proliferation Pathways

Spontaneous mutations of genes involved in proliferation pathways lead to uncontrolled divisions of cancer cells. Among them, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor HER2, members of ErbB family of receptor cytokine kinases [93, 94] are cell surface receptors that have been widely implicated in the autonomic growth of various cancer types [95, 96]. They have extracellular ligand binding domains, through which specific ligands bind and activate growth-related pathways [97]. Therefore, several mAbs have been approved by the FDA to specifically target these EGFR and HER2 extracellular domains involved in the downstream proliferation pathways.

EGFR-targeting mAbs

EGFR is a transmembrane tyrosine kinase receptor protein, which is the most extensively researched member of the ErbB family [94, 98]. Overexpression of EGFR