Immunotherapy and Gastric Cancer
Corresponding author: Dr. Timothy Allen, MD, PhD, Global Allied Pharmaceutical, Center for Excellence in Research and Development, USA, Tel: 321-945-4283; Email: Timothy.Allen@gapsos.com
GC: Gastric Cancer;
US: United States;
H. pylori: Helicobacter pylori;
EBV: Ebstein Bar Virus;
AG: Atrophic Gastritis;
IM: Intestinal Metaplasia;
GEJ: Gastroesophageal Junction;
TCGA: The Cancer Genome Atlas Project;
EBVp: Epstein-Barr Virus positive;
MSI: Microsatellite Instability;
GS: Genomically Stable;
CIN: Chromosomal Instability;
EBVpGC: Epstein-Barr Virus positive Gastric Cancer;
CpG : 5’Cytosine—phosphate—Guanine3′;
DNA: Deoxyribonucleic Acid;
LMP2A: Latent Membrane Protein 2A;
STAT3: Signal Transducer And Activator Of Transcription 3;
DNMT1: DNA Methyltransferase 1;
CDKN2A: Cyclin-dependent Kinase Inhibitor 2A;
MLH1: mutL homolog 1;
PI3K CA: Phosphatidylinositol-4,5-Bisphosphate 3-Kinase
Catalytic Subunit Alpha;
ARID1A: AT-Rich Interactive Domain 1A;
BCOR: BCL6 Corepressor;
JAK: Janus Kinase;
PD-L: programmed cell death ligand;
mTOR : mammalian Target of Rapamycin;
PKB or Akt: Protein Kinase B;
MHC 1: Major Histocompatibility Complex Class I gene;
HER: Human Epidermal Growth Factor Receptor;
EGFR:Epidermal Growth Factor Receptor;
KRAS: Kirsten Rat Sarcoma Viral Oncogene Homolog;
RTK: Receptor Tyrosine Kinases;
CDK6: Cyclin-dependent Kinase 6;
MET: Mesenchymal Epithelial Transition Factor;
FGFR: Fibroblast Growth Factor Receptor;
VEGF: Vascular Endothelial Growth Factor;
RHOA: Ras Homolog Family Member A;
CDH1: Cadherin 1;
CLDN18: Claudin 18;
ARHGAP26: Rho GTPase-activating protein 6;
mRNA: messenger Ribonucleic acid;
mAb: Monoclonal Antibody;
ADCC: Antibody-Dependent Cell-Mediated Cytotoxicity;
FDA: Food and Drug Administration;
MAPK or MEK: Mitogen-Activated Protein Kinases;
TROP2: Tumor-Associated Calcium Signal Transducer 2;
SN-38 : 7-ethyl-10-hydroxycamptothecin;
EGP-1: Epithelial Glycoprotein-1;
HGFR or c-MET: Human Hepatocyte Growth Factor Receptor;
CD: Cluster of Differentiation;
EpCAM: Epithelial Cell Adhesion Molecule;
Fc: Fragment crystallizable;
APC: Antingen Presenting Cells;
DC: Dendritic Cells;
CTL: Cytotoxic T-Lymphocyte;
ADC: Antibody-Drug Conjugate;
GCC: Guanylyl Cyclase C;
MMAE: Monomethylauristatin E;
B7H1: B7 Homolog 1;
LAG-3: Lymphocyte Activation Gene-3;
TIL: Tumor-Infiltrating Lymphocytes;
LAK: Lymphokine-Activated Killer;
NK: Natural killer;
MAGE: Melanoma Associated Antigen;
Erk: Extracellular signal-regulated kinases;
PDGF-R: Platelet Derived Growth Factor Receptor;
RAF: Rapidly Accelerated Fibrosarcoma;
ATP: Adenosine triphosphate;
Ret: Rearranged during transfection;
LCK: Lymphocyte-Specific Protein Tyrosine Kinase;
LYN: Lck/Yes novel;
FLT-3: Fms-Like Tyrosine kinase 3;
G-S:Gap 1/DNA Synthesis;
FKBP-12: Immunophilin FK Binding Protein-12;
CSC: Stemness Cell;
MSC: Mesenchymal Stem Cells;
HSV-TK: Herpes Simplex Virus Thymidine Kinase;
SNAP-25: Synaptosomal-Associated Protein 25;
Gastric cancer (GC) is the fourth prevalent type of cancer (951300 new cases) and the third leading causes of cancer related mortality worldwide (723100 deaths) in 2012 . It is twice more common in men than woman. Developing countries have a higher incidence of GC if compared to developed countries where there is a steady drop in the numbers of new cases. It may be related to different preventive health measures undertaken in the developed countries and a change in life style of their populations . It has also dissimilarities in its geographical distribution whereas it is more widespread in East Asia than North America .
In the United States (US), it is number 15 on the list of most common types of cancers representing only 1,6% . In 2016, it is estimated that there will be 26,370 new cases of stomach cancer (10 /100.000 in men and 5,3/ 100.000 in women) and an estimated 10,730 people (4,5 /100.000 in men and 2,4/ 100.000 in women) will die of this disease . The median age at diagnosis is 69 . In 2013, there were an estimated 79,843 people suffering from GC in the US .
Etiology / Predisposing risk factors:
These are possible etiologies and predisposing risk factors which may contribute to the occurrence of GC:
Helicobacter pylori (H. pylori) infection: H. pylori infection has been linked to peptic ulcer disease and subsequently with GC since 1984. It is a class I carcinogen and it causes chronic inflammation of the gastric mucosa leading to precancerous conditions and cancer of the stomach . It is more prevelant in developing countries and its eradication decreased the incidence of GC in some developed countries .
Ebstein Bar Virus (EBV): EBV is typically contracted in childhood and is associated with numerous malignancies. In a meta-analysis involving 15952 GC cases, a low prevelance of EBV was noticed. But, EBV-associated gastric adenocarcinoma was a two-fold higher in men, and it was more likely to arise in the cardia and body .
Lifestyle risk factors: Many daily life habits have been linked to the development of GC such as smoking, drinking alcohol, and consumption of a high meat/low vegetable diet or, elevated salt containing foods. Tobacco use is linked to the development of GC and the cancer risk in past smokers can stay up to 14 years after cessation of smoking . Alcohol consumption alone was not found to be directly correlated with gastric cancer, but probably associated with other confounding factors such as smoking and dietarty habits. Though, it has been observed, the larger the amount you drink over time, the higher the risk of developing GC. Dietary habits such as high in meat, especially processed meat, and low in vegetables and fruits has been associated with increased risk of GC. Consumption of High salt diet or salt preserved foods is linked to GC especially in developing countries where they still depend on salt to long conserve their foods .
Atrophic Gastritis (AG) and Intestinal Metaplasia (IM): AG and IM are both considered as premalignant conditions and they increase the risk of GC . The rate of progression from AG and IM to GC can reach 11% of the cases over a period of 10 years .
Prior Gastric Surgery: Eventhough it is rare, GC have been reported in about 30 cases after bariatric surgery . In the last few decades, there is a growing worldwide obesity epidemic. It is coupled with a surge in the number of bariatric surgeries, which might, similiary, raise the incidence of GC if the link is not properly investigated. It is worth noting that also obesity have been associated with a 55 % increase in cardia GC in non asian populations [3,8-9].
Blood group A: People with blood group A hold a 20% higher incidence rate of having GC, which may indicate some predisposing genetic factors .
Socio-Economic Status: As stated above, GC is less common in developed rich countries than in deprived developing countries. It is possibly related to an elevated consumption of salt preserved foods, unfavorable life style habits/ occupation and lack of proactive preventive measures such as H.Pylori eradication in these parts of the world .
Family history: About 10 % of the cases exhibit a familial, while 1-3% of gastric neoplasms arise from inherited GC predisposition syndromes [11-12], such as hereditary diffuse gastric carcinoma, familial adenomatous polyposis, hereditary nonpolyposis colorectal carcinoma (or Lynch syndrome), juvenile polyposis syndrome, Peutz-Jeghers syndrome, Li-Fraumeni syndrome and gastric hyperplastic polyposis [13-15].
Pathophysiology and molecular basis of Gastric Cancer:
Gasric cancers can be classified according to the site of occurrence in the stomach (gastro esophageal junction, cardia, body, antrum, fundus, pylorus), clinical presentation (early or advanced) and histologic type. There are two methods for the histologic subclassification; the former Lauren’ s criteria (intestinal, diffuse and indeterminate type) and the more recent World Health Organisation 2010 types (tubular, papillary, mucinous, poorly cohesive, and uncommon histologic variants) .
According to Lauren’s classification, that has been widely used in the last decades , GC is divided mainly into 2 types, intestinal and diffuse. Both types have almost similar lifestyle and environmental risks factors but the diffuse type exhibits more genetic grounds. The intestinal type is found more in elderly males, and occurs mainly at the antrum. It initiates from precancerous lesions, such as AG and IM, and is influenced by environmental factors, such as H. pylori infection, obesity, and dietary factors . The histopathologic changes that take place in the gastric mucosa includes AG with the loss of parietal cell mass, IM, and dysplasia, which eventually lead to carcinoma. The metaplasia/dysplasia/carcinoma sequence is more important for the intestinal-type GC . The diffuse-type represents the major histological type in endemic areas, is more frequent in women and younger patients, and is associated with blood group A[10,18].
In developed countries, the incidence of GC originating from the cardia follows that of the esophageal cancer,  suggesting similar behaviors. Gastroesophageal junction (GEJ) separates the lower esophagus from the proximal part of the stomach, typically in the area where the squamous epithelium of esophagus changes into the columnar epithelium of the gastric cardia  Adenocarcinomas of the GEJ represent around 90% of all the GEJ cancers, but still clear evidences for the specific subset of GEJ tumors are lacking.  Other types of GC are squamous cell cancers as well as neuroendocrine tumors that occur in the hormone producing tissues, usually in the digestive system .
The previously mentioned classifications have limited clinical utility. With the recent advances in molecular profiling, a better understanding of the primary gastric carcinoma was made possible. It would build a deeper insight on the optimal classification of this aggressive neoplasm in order to develop a well defined individualized tailored therapies for patients [23,24]. This has been targeted by The Cancer Genome Atlas Project (TCGA) where they developed a newer subtying of gastric cancer divides it into four different genomic tumours: Epstein-Barr virus positive tumours (EBVp), microsatellite instability tumours (MSI), genomically stable tumours (GS) and tumors with chromosomal instability (CIN) [23, 26].
Epstein-Barr virus positive gastric cancer (EBVpGC): EBV is found within malignant epithelial cells in 9% of GCs [23,25]. EBV positivity have been validated as favourable prognostic factors in resected GC  Mostly in men, they present in 81 % of cases in the cardia and fundus. The main molecular feature of EBVpGCs is 5’Cytosine—phosphate—Guanine3′ (CpG) island promoter methylation of GC related genes . EBV The latent membrane protein 2A (LMP2A) expression of EBV may upgrade Deoxyribonucleic acid (DNA) methylation through inducing signal transducer and activator of transcription 3 (STAT3) phosphorylation and subsequent transcription of DNA methyltransferase 1 (DNMT1) . The TGCA reported that DNA hypermethylations is far upregulated in EBVpGCs compared with other groups. Promoter and non-promoter CpG islands are both present in EBV-associated DNA hypermethylations. Cyclin-dependent kinase inhibitor 2A (CDKN2A) promoter hypermethylation was demonstrated in all EBVp- GCs, while mutL homolog 1 (MLH1) hypermethylation was not noted .
Furthermore, there is an obvious manifestation of the phosphatidylinositol- 4,5-bisphosphate 3-kinase, catalytic subunit alpha (PI3K CA) mutation EBVpGCs, and nearly 5%–10% of all GCs express a PIK3CA mutation. In addition to PI3K CA mutation, EBVpGCs had frequent AT-rich interactive domain 1A (ARID1A) mutation (55%) and BCL6 corepressor (BCOR) mutation (23%) and rarely had a TP53 mutation. On the other hand, TP53and ARID1A mutations are recognised as markers for high clonality and low clonality subtypes of GC in Chinese patients, respectively. Simultaneously, a novel recurrent amplification locus containing janus kinase 2 (JAK2), CD274, and programmed cell death 1 ligand and 2 (PD-L1/2) was also found in EBVpGCs. These genes encode JAK2 , PD-L1, and PDL2, separately. JAK2 is used by several class I cytokine receptor to activate STAT to regulate gene transcription. PD-L1/2 and their receptors PD-1/2 are involved in immune checkpoints [23, 26].
Therapeutic pathways in EBVpGC:
The potential pathways that can be targeted in this subtype were related to the elevated expression of PD-L1 and PD-L2, JAK2 amplification and PI3K CA mutation by direct inhibition, or alternative mechanism through mammalian Target of Rapamycin (mTOR) and Protein kinase B (PKB) .
MSI gastric cancers
MSI is caused by extenxive replication errors in simple repetitive microsatellite sequences due to the defects in mismatch repair genes, mainly in the major histocompatibility complex class I gene (MHC I). MSI has been identified as an early transformation in GC carcinogenesis . It reprents 21% of GCs. This population was mainly old females with GC . MSI has been correlated with as favourable prognostic factors in resectable GC . Additionally, MSI subtype was characterized
by accumulation of different mutations in PIK3CA, human epidermal growth factor receptor (HER) 3 , HER2, and epidermal growth factor receptor (EGFR), with increase in numbers of tumor specific neoantigens .
Therapeutic pathways in MSI GC:
Possible targetable pathways in this group are immunotherapy which is related to the elevated numbers of tumor specific neoantigens and MHC class I aberrations, drugs directed against HER3, Kirsten rat sarcoma viral oncogene homolog (KRAS), HER2, EGFR and PIK3CA mutation by direct inhibition, or alternative mechanism through mTOR and PKB .
CIN gastric cancers:
CIN is due to the imbalanced division of chromosomes to daughter cells upon mitosis and results in the loss or gain of DNA during cell division . It is more likely to consist of a heterogeneous group of cancers at the clinical, histological and molecular level . GC has been showed to express large number of DNA abnormalities. Copy number gains at 8q, 12q, 13q, 17q, and 20q and copy number losses at 3p, 4q, 5q, 15q, 16q, and 17q are frequently noted in GCs [36-39]. CIN is involved in focal gene amplifications as well as to chromosomal gains and losses. The TCGA project presented the CIN subtype as the largest group (50% of GCs). It has higher occurrence in the GEJ and cardia. Genomic amplifications of genes that encode receptor tyrosine kinases (RTK) were identified in the CIN subtype. A new finding is that elevated phosphorylation of EGFR (pY1068) was observed in the CIN subtype and consistent with amplification of EGFR . In addition, amplifications of cell cycle genes Cyclin E1 (CCNE1), Cyclin D1 (CCND1), and Cyclin-dependent kinase 6 (CDK6) have been noted in CIN tumors. . As a result, CIN subtype was studied abundantly in clinical trials over the past decade 
Therapeutic pathways in CIN GC:
Numerous targetable pathways are available in this subtype. The majority of them are related to RTK gene amplifications, including HER2, EGFR, mesenchymal epithelial transition factor (MET), fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor A (VEGFA)[ 31].
GS gastric cancer
The Authors of TCGA found that GS tumors forms 20% of GCs and were highly associated with diffuse histological variants. It is common at an earlier age at presentation (median 59 years) and a distal localization . Fifteen percent of ras homolog family member A (RHOA) mutations were upregulated in this diffuse GC group. The role of RHOA in cell motility highlights the contribution of RHOA modification to altered cell adhesion in the carcinogenesis of diffuse GCs. In addition, mutations in cadherin 1 (CDH1) have also been detected in diffuse GCs. CDH1 germline mutations underlie hereditary diffuse GCs and are associated with poorly differentiated GCs and poor prognosis.
In the GS subtype, a recurrent interchromosomal translocation between claudin 18 (CLDN18) and Rho GTPase-activating protein 6 (ARHGAP26) was also identified .
Therapeutic pathways in GS GC:
Many therapeutic options can be studies to target different promising pathways such as the RHOA dysregulation, FGFR, VEGFA and PIK3CA mutation by direct inhibition, or alternative mechanism through mTOR and PKB .
The development of gastric cancer is a complex, multistep process, which involves multiple genetic and epigenetic alterations. Angiogenesis is also a plausible target to treat GC that lay outside the TCGA subtypes.
Angiogenesis: Expression of the proangiogenic VEGF was demonstrated to correlate with poor survival in gastric cancer patients. VEGF-A was found to be a significant marker for the presence of tumor cells in the bone marrow, whereas VEGF-D is a useful predictor of the lymphatic spread of tumor cells in gastric cancer patients, indicating that the metastatic spread of gastric cancer could be determined, in part, by the profile of VEGF family members expressed in the primary tumor of gastric cancer patients [40-41].
Immunotherapy in the Treatment of GC:
The treatment protocols to stimulate immunity against gastric cancer are done using: monoclonal antibodies, vaccine based immunotherapy, adoptive cell therapy, Ribonucleic acid (RNA) based therapy, kinase inhibitors, proteasome inhibitors and VEGF inhibitor.
Monoclonal antibody (mAb): This therapy has been shown to be effective in the treatment of gastric cancer. Ther mechanism of action is: blocking growth factor/receptor interactions, down-regulating proteins required for tumor growth, and activating effector mechanisms of the immune system (including complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) .
FDA approved Monoclonal antibodies: The US Food and Drug Administration (FDA)- approved mAb for the treatments of gastric cancer are: trastuzumab and ramucirumab.
Indication and use: Trastuzumab has received FDA approval as first line treatment for HER2-positive metastatic gastric or GEJ adenocarcinoma, which target HER2, in combination with cisplatin and fluoropyrimidine (either fluorouracil or capecitabine). It may also be administered in recurrent cases of gastric cancer overexpressing epidermal growth factor receptor type II in combination with chemotherapy.
Contraindications: It is contraindicated in patients with known history of hypersensitivity reactions to the drug.
Warnings: – This drug is warned in cardiomyopathy, infusion reactions, embryo-fetal toxicity, and pulmonary toxicity.
AE: The most important adverse effects associated with trastuzumab are severe infusion reactions, congestive heart failure, and significant decline in left ventricular cardiac function.
Indication and use: Ramucirumab is a human VEGF receptor 2 antagonist with FDA approval for recurrent or progressive gastric and GEJ cancer after primary treatment with platinium or fluropyrimidine combination chemotherapy, either as a single agent or in combination with paclitaxel.
Warnings: – Increased risk of hemorrhage, including severe and sometimes fatal hemorrhagic events.
AE: The various adverse drug reactions that are reported during the clinical trials are hemorrhage, arterial thromboembolic events, hypertension, infusion-related reactions, gastrointestinal perforation, impaired wound healing, worsening sign and symptoms, as well as liver functions in patients with Child-Pugh B or C cirrhosis and reversible posterior leukoencephalopathy syndrome.
Non FDA approved monoclonal antibodies: The monoclonal antibodies that are under clinical trials have been mentioned in Table-2 below:
Bevacizumab: A recombinant humanized monoclonal antibody directed against the VEGF, a pro-angiogenic cytokine. Bevacizumab binds to VEGF and inhibits VEGF receptor binding, thereby preventing the growth and maintenance of tumor blood vessels.
Cetuximab: A recombinant, chimeric monoclonal antibody directed against the EGFR with antineoplastic activity. Cetuximab binds to the extracellular domain of the EGFR, thereby preventing the activation and subsequent dimerization of the receptor; the decrease in receptor activation and dimerization may result in an inhibition in signal transduction and anti-proliferative effects.
Panitumumab: A human monoclonal antibody produced in transgenic mice that attaches to the transmembrane EGFR. Panitumumab may inhibit autocrine EGFR stimulation of tumor cells that express the EGFR, thereby inhibiting tumor cell proliferation.
Pertuzumab: A humanized recombinant monoclonal antibody directed against the extracellular dimerization domain of the HER2 tyrosine kinase receptor. Binding of the antibody to the dimerization domain of the HER2 tyrosine kinase receptor protein directly inhibits the ability of the HER2 tyrosine kinase receptor protein (the most common pairing partner) to dimerize
with other HER tyrosine kinase receptor proteins; inhibiting receptor protein dimerization prevents the activation of HER signaling pathways, resulting in tumor cell apoptosis.
Trastuzumab emtansine: It is an antibody-drug conjugate consisting of the monoclonal antibodytrastuzumab linked to the cytotoxic agent emtansine.Trastuzumab alone stops growth of cancer cells by binding to the HER2/neu receptor, whereas DM1 enters cells and destroys them by binding to tubulin. Trastuzumab binding to Her2 prevents homodimerization or heterodimerization (Her2/Her3) of the receptor, ultimately inhibiting the activation of Mitogen-activated protein kinases (MAPK) and PI3K/Akt cellular signalling pathways. Because the monoclonal antibody targets HER2, and HER2 is only over-expressed in cancer cells, the conjugate delivers the toxin specifically to tumor cells.
Nimotuzumab: A humanized monoclonal antibody directed against the EGFR with potential antineoplastic activity. Nimotuzumab binds to and inhibits EGFR, resulting in growth inhibition of tumor cells that overexpress EGFR. This agent may act synergistically with radiation therapy.
IMMU-132: An antibody-drug conjugate containing the humanized monoclonal antibody, hRS7, against tumor-associated calcium signal transducer 2 (TROP2) and linked to the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), with potential antineoplastic activity. The antibody moiety of IMMU-132 selectively binds to TROP2. After internalization and proteolytic cleavage, SN-38 selectively stabilizes topoisomerase I-DNA covalent complexes, resulting in DNA breaks that inhibit DNA replication and trigger apoptosis. TROP2, also known as epithelial glycoprotein-1 (EGP-1), is a transmembrane calcium signal transducer that is overexpressed by a variety of human epithelial carcinomas; this antigen is involved in the regulation of cell-cell adhesion and its expression is associated with increased cancer growth, aggressiveness
LY2875358: A humanized IgG4 monoclonal antibody directed against human hepatocyte growth factor receptor (HGFR or c-MET) with potential antineoplastic activity. Anti-c-MET monoclonal antibody LY2875358 binds to c-MET, thereby preventing the binding of HGF to its receptor c-Met and subsequent activation of the HGF/c-Met signaling pathway. This may result in cell death in c-Met-expressing tumor cells. c-Met, a receptor tyrosine kinase overexpressed or mutated in many tumor cell types, plays a key role in cancer cell growth, survival, angiogenesis, invasion, and metastasis.
Catumaxomab: A trifunctional bispecific monoclonal antibody with potential antineoplastic activity. Catumaxomab has two antigen-recognition sites: one for human Cluster of differentiation 3 (CD), a T cell surface antigen; and one for human epithelial cell adhesion molecule (EpCAM), a cell surface antigen expressed by a variety of epithelial tumor cells. In addition, the modified fragment crystallizable (Fc) portion of this antibody binds Fc receptors on antingen presenting cells (APC) such as macrophages and dendritic cells (DC). Catumaxomab brings T cells, EpCAM-expressing epithelial tumor cells and APCs together into tricellular complexes, which may result in a potent cytotoxic T-lymphocyte (CTL) response against EpCAM-expressing epithelial tumor cells. Fc-mediated binding of APCs in the tricellular complex potentiates EpCAM antigen presentation to T cells and the activation of anti-tumor cytotoxic T cell functions.
MLN0264: An antibody-drug conjugate (ADC) containing a monoclonal antibody directed against guanylyl cyclase C (GCC) conjugated to monomethylauristatin E (MMAE), an auristatin derivative and a potent microtubule inhibitor, with potential antineoplastic activity. The monoclonal antibody moiety of MLN0264 selectively binds to GCC, a transmembrane receptor normally found on the intestinal cells and dopamine neurons in the brain, but is also overexpressed on the surface of gastrointestinal cancers. Upon internalization and proteolytic cleavage, MMAE binds to tubulin and inhibits its polymerization, resulting in G2/M phase arrest and tumor cell apoptosis in GCC-expressing tumor cells.
AMG 102 (Rilotumumab): A fully human Immunoglobulin(Ig) G2 monoclonal antibody directed against the HGF with potential antineoplastic activity. Rilotumumab binds to and neutralizes HGF, preventing the binding of HGF to its receptor c-Met and so c-Met activation; inhibition of c-Met-mediated signal transduction may result in the induction of apoptosis in cells expressing c-Met. c-Met (or HGFR), a receptor tyrosine kinase overexpressed or mutated in a variety of epithelial cancer cell types, plays a key role in cancer cell growth, survival, angiogenesis, invasion, and metastasis.
BMS-986148: An antibody-drug conjugate composed of a monoclonal antibody directed against the cell surface glycoprotein mesothelin and conjugated to an as of yet undisclosed cytotoxic drug, with potential antineoplastic activity. The monoclonal antibody moiety of anti-mesothelin ADC BMS-986148 targets and binds to the tumor-associated antigen mesothelin. Upon internalization, the cytotoxic agent kills or prevents cellular proliferation of mesothelin-expressing tumor cells through an as of yet undescribed mechanism of action. Mesothelin is overexpressed by all mesotheliomas and a variety of other cancers, while it is minimally expressed in normal tissue.
Non-FDA approved checkpoint inhibitors: The checkpoint inhibitors that are under clinical trials are given in the Table-3 below:
Pembrolizumab: A humanized monoclonal IgG4 antibody directed against human cell surface receptor PD-1 with potential immunopotentiating activity.
Table 1 . Non FDA approved monoclonal antibodies [45-57]
Nivolumab(ONO-4538): A fully human monoclonal antibody directed against the negative immunoregulatory human cell surface receptor PD-1 with immunopotentiation activity. Nivolumab binds to and blocks the activation of PD-1, an Ig superfamily transmembrane protein, by its ligands PD-L1 and PD-L2, resulting in the activation of T-cells and cell-mediated immune responses against tumor cells or pathogens. Activated PD-1 negatively regulates T-cell activation and effector function through the suppression of P13k/Akt pathway activation.
MEDI4736: A monoclonal antibody directed against B7H1 (B7 homolog 1; PDL-1) with potential immunostimulating activity. Upon intravenous administration, MEDI4736 binds to the cell surface antigen B7H1, thereby blocking B7H1 signaling. This may activate the immune system to exert a CTL response against B7H1-expressing tumor cells. B7H1, a member of the B7 protein superfamily and a negative regulator of cytokine synthesis, is overexpressed on certain tumor cell types.
PDR001: PDR001 is an anti-PD1 monoclonal antibody that inhibits the PD-1 immune checkpoint, by blocking the interaction between PD-1 and its ligands, PD-L1 and PD-L2, which activates an antitumor immune response by activating effector T-cells and inhibiting regulatory T-cells.
LAG525 +/- PDR001: A humanized monoclonal antibody directed against the inhibitory receptor lymphocyte activation gene-3 (LAG-3), with potential immune checkpoint inhibitory and antineoplastic activities. Upon administration, the anti-LAG-3 monoclonal antibody LAG525 binds to LAG-3 expressed on tumor-infiltrating lymphocytes (TILs) and blocks its binding with MHC class II molecules expressed on tumor cells. This activates antigen-specific T-lymphocytes and enhances cytotoxic T-cell-mediated tumor cell lysis, which leads to a reduction in tumor growth. LAG-3, a member of the Ig superfamily and expressed on various immune cells, negatively regulates cellular proliferation and activation of T-cells. Its expression on TILs is associated with tumor-mediated immune suppression. It is being tested as a single agent and in combination with PDR001 to adult patients with solid tumors.
Adoptive cell therapy:
The segregation of antigen-specific cells, their ex-vivo expansion and consequent activation, with subsequent autologous administration is considered the cornerstone of adoptive cell transfer therapy and antitumor immune responses. The molecular identification of tumor antigens and the ability to display the persistence and transport of transferring cells has provided the ability to apply the mechanisms of tumor immunotherapy. The efficacy associated with the cell-transfer therapies for the treatment of patients with GCs is well appreciated .
Adoptive cell treatment focuses on passive transfer of tumor specific T cells into a tumor-bearing host, which focuses on deterring the tumor.
Table 2. N on-FDA approved Checkpoint inhibitors [58-62]
These treatments are tailored for every patient. Lymphokine-activated killer (LAK) cells in patients with advanced cancer were produced by culturing peripheral lymphocytes in high concentrations of Interleukin -2 (IL-2); that resulted in the generation of cytotoxic cells, which could directly lyse tumor cells [63,64]. Adoptive cell treatment with autologous TILs works with lymphocytes. Adoptive cell treatment with TILs, separated from resected tumors, when administered in combination with IL-2 patients has shown a 50% longer survival in gastric cancer patients. TILs have been isolated from various gastrointestinal tumors, like colon adenocarcinoma and may be deemed as effective in patients with GCs [65,66].
Non FDA approved adoptive cell therapy: The adoptive T-cells that are under clinical trials has been shown in Table-4 below:
Naturakl Killer (NK) Cell: NK cells are a type of cytotoxic lymphocyte critical to the innate immune system. The role NK cells play is analogous to that of cytotoxic T-cells in the vertebrate adoptive immune response. NK cells provide rapid responses to viral-infected cells and respond to tumor formation, acting at around 3 days after infection.
Allogeneic Lymphocytes: A population of lymphocytes therapeutically administered to a recipient individual who is genetically distinct from a donor of the same species.
Table 3. Non FDA approved adoptive T-cell therapy [67,68]
Vaccine Based immunotherapy:
Generally, vaccines activate and expand tumor-specific T-cells as effector T-cells. They may act by increasing already existing immunity, by inducing new immunity, or by antitumor response. The tumor-specific T-cell can be brought upon by peptides stemmed from tumor-related antigens at the T cell sites. Effective vaccination requires these peptides to be presented by a professional antigen-presenting cell, such as a DC. Immature dendritic cells with a high phagocytic capacity, which are gathered in peripheral tissues, or more localized to tumor growing sites take up antigens, which are digested into smaller oligopeptides. These antigens are bound to a significant MHC class I/II elements for presentation to CD8+ and to CD4+ helper cytotoxic T Cells .
Tumor antigen-pulsed DC-based antibodies have been confirmed to activate both CD8+ and CD4+ T-cell reactions in patients
with advanced malignancies. The clinical trials utilizing DC-based immunizations in patients with advanced malignancies have encouraged into positive immunologic endpoints. Both protein and peptide targets have been used to stimulate a specific immune response in gastric cancer. The experiments based on peptides derived from the tumor associated antigen HER2/neu-derived peptide and MAGE (melanoma associated antigen), which are restricted to MHC class I have been shown to induce cytotoxic T cells against tumors. Gastric cancers typically overexpress HER2/neu and vaccination using DCs pulsed with HER2/neu peptide, which results in decreased tumor .
Non FDA approved vaccines: The vaccines that are under clinical trials have been given in Table 4 below:
Autologous gp96 vaccination: An autologous cancer vaccine derived from tumor-specific gp96 heat shock proteins. Heat shock protein chaperone peptides through the endoplasmic reticulum, are key regulators of dendritic cell maturation, migration and antigen processing, and are involved in T-cell activation.
OTSGC-A24: OTSGC-A24 is a protein-based compound, comprising of five unique vaccines. Active vaccination with tumor specific antigens and VEGFR1 Human Leucocyte Antigen-A24 epitopes can improve survival of patients with advanced Gastric Cancer.
S-1 plus DC-CIK: It uses Cytokine-induced Killer (CIK) cells, which are induced and transfected by DC. DC presents the tumor antigen to the CIK cells and enhance its ability to recognize and kill the cancer cells.
Table 4. Non FDA approved vaccines [71-74]
RNA-based vaccine therapy:
Incubation of dendritic cells with mRNA has the capability to represent the encoded antigen. Thus, mRNA based gene transfer vaccine offers an attractive possibility for immunotherapy in GC . The initiation of immune responses with naked, but stabilized mRNA has been seen to be proficient in mouse models and clinical trials have been encouraging in GC .The RNA based vaccines doesn’t have any side effects, such as the growth of autoimmune disease or anti-DNA antibodies .
Non FDA approved kinase inhibitors: The kinase inhibitors that are under clinical trials have been given in Table-6 below:
Gefitinib: An anilinoquinazoline with antineoplastic activity. Gefitinib inhibits the catalytic activity of numerous tyrosine kinases, including the EGFR, which may result in the inhibition of tyrosine kinase-dependent tumor growth.
Erlotinib: A quinazoline derivative with antineoplastic properties. Competing with adenosine triphosphate, erlotinib reversibly binds to the intracellular catalytic domain of the EGFR tyrosine kinase, thereby reversibly inhibiting EGFR phosphorylation and blocking the signal transduction events and tumorigenic effects associated with EGFR activation.
Lapatinib: A synthetic, orally-active quinazoline with potential antineoplastic activity. Lapatinib reversibly blocks phosphorylation of the EGFR, HER2, and the extracellular signal-regulated kinases (Erk) -1 and-2 and AKT kinases; it also inhibits cyclin D protein levels in human tumor cell lines and xenograft.
Pazopanib: It selectively inhibits VEGFR1, -2 and -3, c-kit and platelet derived growth factor receptor (PDGF-R), which may
result in the inhibition of angiogenesis in tumors, in which these receptors are upregulated.
Dovitinib: It strongly binds to FGFR3 and inhibits its phosphorylation, which may result in the inhibition of tumor cell proliferation and the induction of tumor cell death.
Sorafenib: It blocks the enzyme RAF kinase, a critical component of the RAF/MEK/ERK signaling pathway that controls cell division and proliferation; in addition, sorafenib inhibits the VEGFR-2/PDGFR-beta signaling cascade, thereby blocking tumor angiogenesis.
Apatinib: An orally bioavailable, small-molecule receptor tyrosine kinase inhibitor with potential antiangiogenic and antineoplastic activities. Apatinib selectively binds to and inhibits VEGF 2, which may inhibit VEGF-stimulated endothelial cell migration and proliferation and decrease tumor microvessel density. In addition, this agent mildly inhibits c-Kit and c-Src tyrosine kinases.
Poziotinib: An orally bioavailable, quinazoline-based, mall-molecular and irreversible pan-epidermal growth factor receptor (EGFR or HER) inhibitor with potential antineoplastic activity. Poziotinib inhibits EGFR (HER1), HER2, HER4 and EGFR mutants, thereby inhibiting the proliferation of tumor cells that overexpress these receptors. EGFRs, cell surface receptor tyrosine kinases, are often upregulated in a variety of cancer cell types and play key roles in cellular proliferation and survival.
Table 5. Non FDA approved kinase inhibitors [78-91]
KX2-391: An orally bioavailable small molecule Src kinase inhibitor with potential antineoplastic activity. Unlike other Src kinase inhibitors, which bind to the Adenosine triphosphate (ATP) -binding site, Src kinase inhibitor KX2-391 specifically binds to the peptide substrate binding site of Src kinase; inhibition of kinase activity may result in the inhibition of primary tumor growth and the suppression of metastasis. Src tyrosine kinases are upregulated in many tumor cells and play important roles in tumor cell proliferation and metastasis.
PF00299804: An orally bioavailable, highly selective, second- generation small-molecule inhibitor of the pan-EGFR family of tyrosine kinases with potential antineoplastic activity. Dacomitinib specifically and irreversibly binds to and inhibits human EGFR subtypes, resulting in the inhibition of proliferation and induction of apoptosis in EGFR-expressing tumor cells. EGFRs play major roles in tumor cell proliferation and tumor vascularization, and are often overexpressed or mutated in various tumor cell types.
Regorafenib: An orally bioavailable small molecule with potential antiangiogenic and antineoplastic activities. Regorafenib binds to and inhibits VEGFRs 2 and 3, and RET, Kit, PDGFR and Raf kinases, which may result in the inhibition of tumor angiogenesis and tumor cell proliferation. VEGFRs are receptor tyrosine kinases that play important roles in tumor angiogenesis; the receptor tyrosine kinases RET, Kit, and PDGFR, and the serine/threonine-specific Raf kinase are involved in tumor cell signaling.
Nintedanib: An orally bioavailable, indolinone-derived, RTK inhibitor with potential antiangiogenic and antineoplastic activities. Multitargeted tyrosine kinase inhibitor BIBF 1120 selectively binds to and inhibits VEGFR, FGFR and PDGFR tyrosine kinases, which may result in the induction of endothelial cell apoptosis; a reduction in tumor vasculature; and the inhibition of tumor cell proliferation and migration. In addition, this agent also inhibits members of the Src family of tyrosine kinases, including Src, Lck, Lyn, and FLT-3 (fms-like tyrosine kinase 3). VEGFR, FGFR and PDGFR RTKs play key roles in tumor angiogenesis.
Dacomitinib: It is a selective pan-EGFR inhibitor. It is an irreversible inhibitor of EGFR, HER2 and HER4 tyrosine kinases. Dacomitinib targets multiple receptors of the HER pathway, whereas currently marketed EGFR inhibitors.
Neratinib: it is an irreversible tyrosine kinase inhibitor of the HER-2 receptor with potential antineoplastic activity. Neratinib binds to the HER2 receptor irreversibly, thereby reducing autophosphorylation in cells, apparently by targeting a cysteine residue in the ATP-binding pocket of the receptor. Treatment of cells with this agent results in inhibition of downstream signal transduction events and cell cycle regulatory pathways; arrest at the G1-S (Gap 1/DNA synthesis)-phase transition of the cell division cycle; and ultimately decreased cellular proliferation. Neratinib also inhibits the EGFR kinase and the proliferation of EGFR-dependent cells.
BKM120 (Buparlisib): An orally bioavailable specific oral inhibitor of the pan-class I PI3K family of lipid kinases with potential antineoplastic activity. Buparlisib specifically inhibits class I PIK3 in the PI3K/AKT kinase signaling pathway in an ATP-competitive manner, thereby inhibiting the production of the secondary messenger phosphatidylinositol-3,4,5-trisphosphate and activation of the PI3K signaling pathway. This may result in inhibition of tumor cell growth and survival in susceptible tumor cell populations. Activation of the PI3K signaling pathway is frequently associated with tumorigenesis. Dysregulated PI3K signaling may contribute to tumor resistance to a variety of antineoplastic agents.:
Non FDA approved proteasome inhibitors: The preoteasome inhibitors that are under clinical trials have been given in Table 6 below:
Bortezomib: A dipeptide boronic acid analogue with antineoplastic activity. Bortezomib reversibly inhibits the 26S proteasome, a large protease complex that degrades ubiquinated proteins. By blocking the targeted proteolysis normally performed by the proteasome, bortezomib disrupts various cell signaling pathways, leading to cell cycle arrest, apoptosis, and inhibition of angiogenesis.
Carfilzomib: it is a selective irriversable proteasome inhibitor. it has an antiproliferative and proapoptotic activities delaying tumor growth.
Table 6. Non FDA approved proteasome inhibitors [92-93] Growth Factor Receptor inhibitors:
Non FDA approved Growth Factor Receptor inhibitors: The growth factor receptor inhibitors that are under clinical trials have been given in Table-8 below:
Ziv-aflibercept: A protein comprised of segments of the extracellular domains of human VEGFR 1 and 2 fused to the constant region Fc of human IgG1 with potential antiangiogenic activity. Afilbercept, functioning as a soluble decoy receptor, binds to pro-angiogenic VEGFs, thereby preventing VEGFs from binding to their cell receptors. Disruption of the binding of VEGFs to their cell receptors may result in the inhibition of tumor angiogenesis, metastasis, and ultimately tumor regression.
AZD 4547: An orally bioavailable inhibitor of the FGFR with potential antineoplastic activity. FGFR inhibitor AZD4547 binds to and inhibits FGFR, which may result in the inhibition of FGFR-related signal transduction pathways, and, so, the inhibition of tumor cell proliferation and tumor cell death. FGFR, up-regulated in many tumor cell types, is a receptor tyrosine kinase essential for tumor cell proliferation, differentiation and survival.
Table 7. Non FDA approved Growth Factor Receptor inhibitors [94,95]
Non FDA approved mTOR inhibitors: The mTOR inhibitors that are under clinical trials have been given in Table-9 below:
Everolimus (RAD001): A derivative of the natural macrocyclic lactone sirolimus with immunosuppressant and anti-angiogenic properties. In cells, everolimus binds to the immunophilin FK Binding Protein-12 (FKBP-12) to generate an immunosuppressive complex that binds to and inhibits the activation of the mTOR, a key regulatory kinase. Inhibition of mTOR activation results in the inhibition of T-lymphocyte activation and proliferation associated with antigen and cytokine (IL-2, IL-4, and IL-15) stimulation and the inhibition of antibody production.
Table 8. Non FDA approved mTOR inhibitor 
Cancer cell stemness inhibitor:
Non FDA approved cancer cell stemness inhibitor: The cancer cell stemness inhibitors that are under clinical trials have been given in Table-10 below:
BBI608: An orally available cancer cell stemness inhibitor with potential antineoplastic activity. Even though the exact target has yet to be fully elucidated, BBI608 appears to target and inhibit multiple pathways involved in cancer cell stemness. This may ultimately inhibit cancer stemness cell (CSC) growth, as well as heterogeneous cancer cell growth. CSCs, self-replicating cells that are able to differentiate into heterogeneous cancer cells, appear to be responsible for the malignant growth recurrence and resistance to conventional chemotherapies.
Table 9. Non FDA approved cancer stemness inhibitor 
Mesenchymal stem cell based therapy:
Non FDA approved mesenchymal stem cell based therapy: The mesenchymal stem cell based therapy that is under clinical trials has been given in Table-11 below:
MSC apceth 101: Human autologous mesenchymal stem cells (MSCs) harvested from the bone marrow of a patient and genetically modified with a self-inactivating retroviral vector expressing the suicide gene herpes simplex virus thymidine kinase (HSV-TK), that can be used to activate synthetic acyclic guanosine analogues, when co-administered. Upon intravenous administration of autologous mesenchymal stem cells apceth_101, the cells are actively recruited to the tumor stroma, differentiate into more mature mesenchymal cells, and become part of the tumor microenvironment. When a synthetic acyclic guanosine analogue, such as ganciclovir, is co-administered, the HSV-TK within the HSV-TK-transduced MSCs will monophosphorylate this prodrug. Subsequently the monophosphate form is further converted to the diphosphate form and then to its active triphosphate form by cellular kinases. The active form of ganciclovir kills the HSV-TK-transduced MSCs and leads to a bystander effect, which eliminates neighboring cancer cells. Therefore, synthetic acyclic guanosine analogues are activated only at the tumor site, which increases their local efficacy and reduces systemic toxicity
Table 10. Non FDA approved mesenchymal stem cell based therapy 
Non FDA approved neurotoxins: The neurotoxin that is under clinical trials has been given in Table-11 below:
Botulinum Toxin: An injectable formulation of a neurotoxin derived through the fermentation of the Hall strain of Clostridium botulinum type A with neuromuscular transmission inhibitory and analgesic activities. Upon injection into the affected muscle, the heavy chain portion of on a botulinum toxinA binds to the cell membrane of the motor nerve and is internalized via endocytosis. Upon entry, the light chain portion of the toxin is activated and cleaves the protein Synaptosomal-associated protein 25 (SNAP-25), thereby preventing the fusion of acetylcholine (ACh)-containing synaptic vesicles with the cell membrane and, so, the release of ACh into the neuromuscular junction; subsequent binding of ACh to motor end-plate nicotinic acid receptors and ACh-mediated muscle contraction are thus blocked. In addition to ACh, onabotulinumtoxinA may inhibit the release of neuropeptides, such as substance P and glutamate, which may contribute to its analgesic activity.
Table 11. Non FDA approved neurotoxin
Non FDA approved:
Aldesleukin: A recombinant analog of the endogenous cytokine IL-2 with immunoregulatory and antineoplastic activities. Aldesleukin may stimulate lymphocytes to kill tumor cells. Treating lymphocytes with aldesleukin in the laboratory may help the lymphocytes kill more tumor cells when they are put back in the body after chemotherapy.
Table 12. Non FDA approved neurotoxin 
The exact mechanisms underlying gastric cancer are not yet completely understood and there is a vital need for novel treatment options, such as immunotherapy. The new molecular classification by TCGA has helped to define each subtype of GC as a different disease entity with potential specific targeted pathways. Some therapeutic drugs have been proven to make a difference in the Event free survival and overall survival in patients with GC; such as trastuzumab and ramucirumab. Many others immunotherapeutic agents are being tested, using multiple targeted pathways, with a large hope for breakthroughs in the management of the cancer of the stomach. Vaccine based immunotherapy with DCs pulsed with HER2/neu-peptides represent a potential candidate for the novel treatment of patients with gastric cancer. The efficacy associated with the cell-transfer therapies and mRNA based gene transfer vaccine for the treatment of patients with gastric cancers is also well appreciated. We are still in n early phase to perceive a curative therapy for Gastric cancer, but with the current advances in molecular biology and immunotherapeutic agents, we are definitely on the right path.
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