Gregory Lee, Ph.D

UBC Centre for Reproductive Health, Vancouver, Canada

^*Corresponding author:Dr. Gregory Lee, 9117 Shaughnessy StreetVancouver, Canada, V6P 6R9, Tel: 778-322-4651;

Abstract

RP215 is a monoclonal antibody generated in mice immunized against OC-3-VGHovarian cancer extract. The cognate cancer-associated antigen, designated as CA215was affinity-isolated from cancer cell extract and subject to tryptic peptide analysis byMALDI-TOF MS method. The unique epitope recognized specifically by RP215 wasshown to locate mainly on the antigen receptors such as immunoglobulin heavy chainsand T cell receptors as well as other glycoproteins expressed by cancer cells, but notthose from normal immune cells. The primary carbohydrate structure associated withRP215-specific epitope was elucidated and suggested to be O-glycans (commonlyknown as Sialyl-T antigen). By extensive IHC studies, RP215 was found to react withmost of epithelial cancers, but rarely with normal ones. By using RP215 as the uniqueprobe for cancerous antigen receptors, extensive in vitro studies are performed withculturing cancer cells. Similar to anti-antigen receptors, RP215 was shown to induceapoptosis and complement-dependent cytotoxicity to almost all cancer cells. Generegulation studies were also performed by using semi-quantitative RT-CCR methodswith selected genes involved in the regulations of immunoglobulins, T cell receptors aswell as toll-like receptors. RP215 and anti-antigen receptors were shown to affectsimilarly the gene expression levels of incubated cancer cells with high mutualcorrelations. Cancerous antigen receptors and RP215 were used separately as theaffinity ligands to identify those human serum proteins or fragments interacting withcancer cells. The results indicate that majority of commonly detected human serumproteins (≥ 85%) are those of known pro-cancer or anti-cancer protein components. Itwas hypothesized from these studies that antigen receptors on cancer cells may serveto interact with those human serum proteins which are essential to growth/proliferation of cancer cells or protection of cancer cells in our normal bodyenvironment. By understanding their functional roles, RP215 may serve as a uniquetool to target cancer cells in immunodiagnostic applications to monitor serum CA215levels in cancer patients or in immunotherapeutic applications for cancer therapy ortreatments.

Key words:RP215 monoclonal antibody, CA215, cancerous antigen receptors, CAT-T celltechnology, cancer immunotherapy and treatment

Introduction

Discovery of antigen receptors in cancer cells

Antigen receptors including immunoglobulins and T cell receptors were known tobe expressed by cancer cells for more than two decades through early molecularbiological studies [1-3]. More comprehensive biological and immunological studieswere reported in early 2000’s [3, 4]. It was observed that immunoglobulins can beexpressed by human cancer cells for essential growth/proliferation. Apoptosis can beinduced through incubations with anti-immunoglobulins or immunoglobulin-relatedSiRNA in vitro and in vivo. However, the functional roles of cancerous immunoglobulinsare still poorly understood when compared with those of our normal immune system[4].

RP215 Monoclonal antibody associated with antigen receptorsexpressed by cancer cells

In 1987, among the monoclonal antibodies generated in mice immunized with OC-3-VGH ovarian cancer cell extract, RP215 was selected and confirmed to react withcarbohydrate-associated epitope localized on immunoglobulin heavy chains expressedby cancer cells [5]. Following MALDI-TOF MS analysis, it was documented that thetryptic peptides of the affinity-isolated cancer-associated antigens, CA215 consistmainly of immune-related glycoproteins (≥ 85%). These includes antigen receptors (≥50%), antigen presenting molecules, cell adhesion molecules and others related toimmunoglobulins superfamily glycoproteins [5, 12]. Judging from this analysis, structure and functional roles of cancerous antigenreceptors in cancer cells should be further explored with extensive molecular andimmunological studies. RP215 may serve as the preferred immune probe for suchinvestigations and similar results compared with those of anti-antigen receptors [5, 6].Understanding of their mechanisms of action as well as gene regulations on cancercells might be essential for future applications in cancer therapy and treatments, whenantigen receptors are selected as targets.

StructuralElucidations of RP215-SpecificCarbohydrate-Associated Epitope

RP215 was shown to react mainly with protein bands of 55 KDa, when WesternBlot assays were performed either with crude cancer cell extract or with affinity-purified CA215 [5]. Treatments of CA215 or cancer cell extract with NaIO4 resulted in aloss of RP215-immunoactivivity either by Western Blot or other immunoassays,indicating the carbohydrate nature of the RP215-specific epitope. Judging from theanalytical results of MALDI-TOF MS method and Western Blot assay [5, 6], it was clearlysuggested that RP215 reacts mainly with heavy chains of cancerous immunoglobulins.

The exact epitope locations in heavy chains of immunoglobulins was determined byRP215-based sandwich enzyme immunoassays. By using RP215-coated microwells andpurified CA215 as the antigen, RP215-HRP, goat anti-human IgG-Fc, and goat anti-human IgG-Fab were added in dose-dependent CA215 immunoassays. It was clearlydemonstrated that goat antihuman IgG-Fc (1μg/ml) has little effect on CA215 signal,but goat antihuman IgG Fab (1μg/ml) resulted in a total inhibition CA215 binding toRP215 in the immunoassays. Therefore, it was judged that RP215-specific epitope isattached to Fab (variable domains) of heavy chains immunoglobulins, but not in the Fcdomains. Results of such analysis are presented in Figure 1 A.

The carbohydrate-associated epitope recognized by RP215 can either be N-linkedor O-linked glycans. This was determined by using co-incubation of culturing cancercell with tunicamycin which is a known inhibitor of N-glycan [7]. Following 48 hrincubation, with either C33A cervical or OC-3-VGH ovarian cancer cells, it was observedthat CA215 immunoactivity was not affected by tunicamycin(Figure 1B).

Therefore, we assumed that carbohydrate-associated epitope is O-linked glycanattached on the variable regions of immunoglobulin heavy chains.

Through collaborations with Complex Carbohydrate-Research Centre in Athens,GA, the glycosyl linkage analysis was performed with cancerous immunoglobulinswhich were sequentially affinity-purified by affinity columns with RP215 or anti-immunoglobulins as the ligands, respectively. The affinity-purified cancerousimmunoglobulins were subject to glycosyl-linkage analysis. Form the results of suchanalysis, the only O-linked glycan structures associated with RP215- epitope are Core1 structure with 3-linked GalNAcitol and/or 3,6-linked GalNAcitol as presented in Figure 1 C.

The structure of O-linked glycan appears to be Sialyl T antigen which wasknown to be associated with many cancer cells due to aberrant termination oftruncated O-glycans, attached to proteins in cancer cells. For example, O-glycans ofSTn, T, Tn in the mucin 1 (MUC 1) glycoproteins have been reported and used as targetsfor CAR-T cancer therapy [8, 9].

Tissue Specificity of Cancerous AntigenReceptors

From early studies by using RT-PCR for gene expression of cancer cells, it was revealedthat immunoglobulins and T cell receptors can be co-expressed by cancer cells of one singlecell origin [1] as well as over 80-90% of established cancer cell lines [10]. Immunohistochemicaltissue staining studies with RP215 as the immune probe for cancerous antigen receptors alsoindicated widespread expressions of RP215-epitope among various cancer tissues [5, 11].Among these are ovary (n = 87, 64.4%), cervix (n = 51, 84.3%), endometrium (n = 36, 77.8%),stomach (n = 93, 49.5%), colon (n = 87, 43.6%), esophagus (n = 56, 75.7%), lung (n = 58, 31%),and breast (n = 59, 32.2%). In contrast, low percentages of positive tissue staining wasobserved for the cancer of liver (n = 60, 3.5%) and prostate (n = 22, 10%). In the case of T cellreceptors, greater than 80% of the cancer cell lines show positive staining [12]. However, littleor no expressions of co-receptors or co-stimulators such as CD3, CD4, and CD8 were detectedfor almost all cancer cells tested. The results suggest that cancer cells do not have functional Tcells commonly observed in normal immune system [12].

Functional Assessment of Cancerous AntigenReceptors ThroughStudies of Induced Apoptosis and Gene Regulations

Induced apoptosis and complement-dependent cell lysis byRP215 and anti-antigen receptors

Antigen receptors are known to be expressed on cancer cells and apoptosis can beinduced upon 24 and 48 hr incubation of culturing OC3-VGH ovarian cancer cells and manyothers with RP215 or with anti-antigen receptors such as goat anti-human IgG and anti-T cellreceptors [6, 12]. In the case of RP215 at 1 µg/ml or 10 µg/ml, as much as 30% to 50% of cancercells underwent apoptosis following 24 to 48 hr incubation, when TUNEL assays wereemployed [6]. When goat anti-human IgG (1 and 10 µg/mL) was used instead as the ligand,apoptosis of incubated cancer cells was induced to a similar extent as that of RP215. In contrast,the negative control with normal mouse or rabbit IgG of the same concentrations did not resultin significant induced apoptosis to cancer cells. Purified anti-T cell receptors β (rabbit IgGfractions) were under the same assay conditions, apoptosis of incubated cancer cells was alsoinduced at concentration of 10 µg/mL. The apoptosis was similarly induced when several othercancer cell lines, namely, DU145 (prostate), A549 (lung), C33A (cervix) and MDA-MB 435(breast) were used instead for studies under similar conditions of TUNEL assays [6, 12]. RP215,antihuman IgG or anti-T cell receptors gave similar results of induced apoptosis, when differentcell lines were employed for similar studies. In conclusion, RP215 and the two anti-antigenreceptors were confirmed to react with the antigen receptors on cancer cells, and similardegrees of induced apoptosis can be observed to cancer cells. Complement-dependentcytotoxic reactions (CDC) could result in cell lysis in the presence of complement following 2hr co-incubation with all different types of cancer cells [6, 12]. In contrast, normal mouse orrabbit IgG of the same concentrations resulted in little or no complement-dependent cell lysis.

Effects of RP215 or anti-antigen receptors on gene expressions of culturing cancer cells

By semi-quantitative RT-PCR, the levels of gene expression change significantly upon thetreatments of RP215, or anti-antigen receptors to cancer cells in culture for 24 hours or 48hours [13]. More than ten different genes were selected for gene regulation analysis. They aregenerally involved in gene expressions of immunoglobulins and T cell receptors in normalimmune systems as well as toll-like receptors in innate immunity. The selected genes are listedas follows: NFĸB-1, IgG, TCR, P21, ribosomal P0, ribosomal P1, cyclin D1, c-fos and EGFR [14, 15]as well as toll-like receptors (TLR-2, -3, -4, -6, -7, and -9) [16,17]. Comparative results of therelative gene regulations changes of cancer cells are given in Table 1.

Generally speaking, upontreatments of cancer cells with RP215 or anti-antigen receptors, up-regulations (20 to 25%) ofgenes expressed by NFĸB-1, IgG, TCR, P21, and ribosomal P1 were observed. On the other hand,down regulation of Cyclin D1, c-fos, EGFR, and ribosomal P0 were also consistently observed(10-30%). Gene regulation patterns did not change significantly when C33A cervical cancer cellline was employed instead for similar gene regulation studies [13].

Effects on gene regulations of toll-like receptors expressed by cancer cells

RP215 and anti-antigen receptors were also demonstrated to affect the gene regulationpatterns of those involved in innate immunity such as toll-like receptors. Gene regulationchanges were studied with same cancer cell lines and compared [13]. Similar changes in generegulation levels were observed for either of these ligands. Generally speaking, upontreatments with RP215 or anti-antigen receptors, significant changes in gene expressions wereobserved to all of the toll-like receptors (10% to 90%). The up-regulation of TLR-3 gene was found to increase by 226%, 143%, and 219%, respectively for RP215, anti-IgG and T-cellreceptors [18]. On the other hand, down-regulations of TLR-4 and TLR-9 genes wereconsistently observed [19]. Results of such a semi-quantitative RT-PCR analysis of generegulation changes are semi-qualitatively presented in Table 1 [13].

Similar gene regulation profiles of cancer cells by RP215 and cancerous antigen receptors

Based on results of semi-quantitative RT-PCR analysis, changes in levels of geneexpressions by treated cancer cell lines revealed a high degree of similarity among the threeantigen receptor ligands. For example, in response to RP215 treatment of cancer cells, changesin gene expression levels are well correlated with those of anti-human IgG (R2 = 0.9135) or withthose of anti-T cell receptors (R2 = 0.9071). They are demonstrated in Figure 2 [13].

Theseobservations strongly support our initial assumptions that RP215 and anti-antigen receptorsmay affect the treated cancer cells through more or less the same molecular mechanisms of

Action [13]. Therefore, cancerous antigen receptors can be targeted by RP215 with similareffectiveness and specificity to cancer cells [13].

Toll-like receptors (TLRs) are known to be a family of receptors in innate immunityinvolved in pathogen-associated molecular patterns for host-defense against infections [12,16]. They also play important roles in carcinogenesis and proliferation of cancer cells which areconsistently regulated by cancerous antigen receptors as demonstrated in this study. Since toll-like receptors are important components for innate immunity, expressions of cancerousimmunoglobulins and/or T cell receptors may in part explain their essential requirements forgrowth and survival of cancer cells [18, 19]. The involvements of both adaptive and innateimmunity in gene regulations of cancer cells are drawing more attentions lately and may havefar reaching implications in cancer immunology; not only for basic understanding of cancer,but also for their potential therapeutic applications as well.

Dual and Distinct functional Roles of AntigenReceptors as Expressed by Cancer CellsInteractions between Cancer Cells and Human Serum Proteins

Under normal human body environments, cancer cells acquire the ability to survive andproliferate and at the same time they are capable of eliminating hostile protein elements inhuman circulations. Based on this hypothesis, immunology of cancer cells may be differentfrom that of the conventional one [20, 21]. In conventional immune system, the coordinationsof separate B cells and T cells are required to generate antibodies and cellular immunity toeliminate or neutralize hostile foreign pathogens in adaptive immunity [20, 21]. However, thismay not be the case in cancer cells. Both immunoglobulins and T cell receptors are co-expressed in one single cancer cell to perform somewhat different immunological/biologicalobjectives. We believe that surface bound cancerous antigen receptors are also required toeliminate or neutralize any hostile human serum components or proteins for their survival. Atthe same time, they are capable of interacting with useful protein components for growth andproliferation of cancer cells. Based on these assumptions, simple experiments were designedto identify those human serum components (anti-cancer or pro-cancer proteins) to meet theexpected purposes. Results of such experiments might help us resolve the specific functionalroles of these antigen receptors in cancer cells.

CA215 was initially affinity-purified from the shed medium of culturing OC-3-VGH ovariancancer cells with RP215 as the affinity ligand. Parallelly, cancerous immunoglobulins were alsoaffinity-isolated from the shed culture medium with anti-human IgG as the ligand. Followinginitial purifications, CA215 and cancerous immunoglobulins were used as separate affinityligands in affinity chromatography. These two affinity columns were then used to capturehuman serum proteins or components which may interact with or can be adsorbed by eitheraffinity ligands from pooled human serum samples. The affinity-purified human serum proteinsfrom either affinity column were then subject to separate analysis by LC-MS/MS methodsfollowing MS analysis of tryptic digests of the affinity-purified human serum proteins orcomponents. Many distinctive serum proteins or their components were unanimouslyidentified by LC-MS/MS methods for further elucidation of mechanisms of action [20, 21].

Pro-cancer and anti-cancer nature of identified human serumproteins

Out of hundreds of human serum proteins which were isolated and identified by LC-MS/MS, about 85% of these serum proteins were commonly detected from those isolated by either affinity columns [20, 21]. They were selectively listed in Table 2 [20]and classified in general, according to their pro-cancer or anti-cancer nature which has been known through previous studies by other investigators.

For example, among the selected human serumproteins of pro-cancer nature are C46-binding protein [26], complement C3 [22], complementfactor H [23, 24], serotransferrin [25, 26], and vitronectin [27]. C46-binding can prevent or protect cancer cells from complement attacks [22]. Complement C3 is capable of promotingcancer cell development and progression through various tumorigenic effects [22].

Complement factor H can protect cancer cells from complement-mediated lysis [23, 24].Serotransferrin has been shown to be a growth factor for cancer cell proliferation, whereasvitronectin was known to be inducer of cancer cell differentiation, spreading, migration, andgrowth [25-27]. Among the anti-cancer human serum protein (components), anastellin wasshown to inhibit tumor growth and metastasis in vivo [28]. Inter-α-trypsin inhibitor heavy chain4 can lead to down regulation of tumor initiation and progression in multiple solid tumors [29].

Apolipoprotein A was known to suppress tumor growth and metastasis [30, 31]. Fibrinogen βchain has been shown to inhibit tumor vascularization in mouse models [32] whereas keratintype 1 cytoskeleton 9 is known to reduce drug resistance in breast cancer [33]. In summary,from these studies, it can be established that quite a few human serum proteins werecommonly detected or identified with known pro-cancer or anti-cancer properties. Theinteractions between human serum proteins (components) and CA215 or cancerousimmunoglobulin ligands could simply be antigen-antibody binding reactions or may bereceptor-ligand mediated. The mechanisms of action of these interactions should beindividually investigated in the future [20].

Based on the results of these studies, the dual functional roles of cancerous antigenreceptors or its associated CA215 can be clearly demonstrated [20, 21]. The hypothesis of dualfunctional roles of cancer antigen receptors may help to explain why cancer cells under ournormal human environment can survive/proliferate and eliminate/neutralize hostile humanserum components. Therefore, we believe that there are essential requirements forexpressions of cancerous antigen receptors to fulfill specific functional roles for cancer cells.

Potential Clinical Applications of RP215Monoclonal Antibody

The universal appearance of antigen receptors on cancer cell surface has been welldocumented in this review and constituted to be one of important components in cancer cells.The functional roles of antigen receptors have been extensively studied by using RP215 as aunique immune probe for anti-antigen receptors [20, 21]. Therefore, cancerous antigenreceptors are unique targets for cancer diagnostic and therapeutic applications, when RP215was used as the unique probe.

RP215 is specific to carbohydrate-associated epitope of CA215, consisting mainly ofcancerous antigen receptors [5]. RP215-based sandwich enzyme immunoassay wasestablished for quantitation CA215 in serum specimens of cancer patients [34]. CA215 antigenstandards were prepared from partially purified shed medium of culturing OC-3-VGH ovariancancer cells. As cancer-associated antigen, CA215 was calibrated and determined in arbitraryunits per mL (AU/mL). Typical RP215-based sandwich enzyme immunoassay is a two-hoursingle step procedure at 37°C with a sensitivity of 0.02 AU/mL [35, 36].

Serum levels of CA215 among patients with ovarian and cervical cancers were evaluatedseparately by the established enzyme immunoassays [34, 35]. In the case of ovarian carcinoma,positive rates of CA215 were determined to be 58% (n = 24), 86% (n = 7), and 70% (n = 40) fordiseases of Stages I, II, and III, respectively [35]. By comparisons, the positive rates of CA215for those patients with cervical carcinoma were determined to be 66% (n = 35), 94% (n = 18),and 71% (n = 7), for disease of Stage I, II, and III, respectively. In the case of benign tumors,such as endometriosis, and uterine myoma, much lower positive rates of 10% (n = 10) and 26%(n = 23) were obtained, respectively [35]. Statistical analysis revealed that stage dependenceof serum CA215 levels was demonstrated for either cancer, especially at Stage I vs. Stage II orIII (P < 0.001). However, serum CA215 levels between Stage II and Stage III are not as clearlydefined. Results of these clinical evaluations also revealed that serum CA215 levels remainedat relatively high levels during the initial preoperative stages or within one week after surgicaloperations or after chemo- or radiation therapy [35]. In contrast, serum CA215 levelsdecreased significantly when determined seven days after surgical operations. The results mayindicate that serum CA215 levels reflect the tumor burden of individual cancer patients.

RP215 was shown to react with carbohydrate-associated epitope on cancerous antigen receptors expressed by most of epithelial cancers [5, 6], RP215-based immunoassay should enable us to detect or determine CA215 levels from many cancers of diversified tissue origins [36]. In a large-scale clinical evaluation, serum levels of CA215 were determined from about 500 cancer patients which had been confirmed with either one of twelve cancers [36]. The same clinical specimens were also used to determine marker levels of other known cancer biomarkers including alpha-fetoprotein (AFP), carcino-embryonic antigen (CEA), cancer antigens such as CA125, CA15-3, and CA19-9, β2-microglobulins and cytoskeleton 19 fragments (cyfra-21) [36]. To given specimens, the positive rates of CA215 were determined and compared with those of other biomarkers for different types of cancer including: lung, colon, liver, ovary, kidney, espophagus, pancreas, breast, and stomach [36]. The results of comparative studies with positive rates of individual biomarkers and those combined with CA215 are summarized and presented in Table 3.

Based on such a comparative study, the positive rates of serum CA215 levels are also listed according to different types of cancer: lung (52%), colon (44%), ovary (59%), breast (38%), pancreas (51%) esophagus (61%), stomach (60%), kidney (38%), and lymphoma (83%). Positive rates of serum CA215 levels are also compared with other known cancer biomarkers, each of which is more tissue-specific than CA215 [35, 36]. Among these markers, CA125 is commonly used for detection and monitoring of ovarian cancer [37]. CA15-3 is suitable for cancer of breast [37], whereas CA199 is generally used for monitoring of pancreas, stomach and liver cancer [38]. CEA and β2-microblubulins are cancer biomarkers of broad tissue origins [38]. Cyfra-21-1 is commonly used for monitoring of lung cancer [40].

In Table 3, positive rates of CA215 and other biomarkers, singly and combined are summarized with six types of human cancer (lung, liver, ovary, esophagus, breast, and stomach). It was generally observed that the combined use of CA215 and either one of these cancer biomarkers resulted in higher detection rates to a given cancer [36]. For example, in the case of ovarian cancer, positive detection rates of individual biomarkers are 59% each (n = 68 and 66, respectively). However, the positive rates of two biomarkers combined could increase to 82%. In the case of lung cancer, CA215 and Cyfra 21-1 assays gave positive rates of 52% (n = 112) and 50% (n = 52), respectively. When both biomarkers were combined for determinations, the combined positive rate could reach 77% (Table 3).

The clinical utility of CA215 and other known biomarkers was demonstrated from theimmunoassay results of all of these available biomarkers [36]. Combinations of CA215 withother cancer biomarkers are certainly beneficial in increasing positive detection rates duringroutine monitoring of a given cancer patient. Therefore, potential clinical utility of CA215 as apan cancer biomarker can be established for routine cancer monitoring [35, 36].

Immunotherapeutic applications of RP215 monoclonal antibody development of RP215-based anti-cancer drugs

Functional roles of cancerous antigen receptors have been described in this review. Anti-cancer properties of RP215 with carbohydrate-associated epitope of O-linked ST glycan hasbeen elucidated through biological studies in vitro and models of nude mouse experiments[5,11]. Similar to anti-antigen receptors, RP215 was shown to induce apoptosis andcomplement-dependent cytotoxic reactions to culturing cancer cells [13]. Therefore, genes ofmurine RP215 were humanized to avoid complications arising from the immunospecificityduring human applications [41]. Generally speaking, the genes expressing the Fc domains of murine RP215 (mRP215) are replaced with human IgG, Fc. Modifications of FR domains in theFab regions of murine RP215 should be made for compatibility of humanized forms, whereasthose of CDR domains should be minimized to maintain binding specificity. Genes ofhumanized RP215 (hRP215) were reconstructed and expressed in CHO cells of high productionyield. MRP215 and hRP215 were demonstrated to be bioequivalent or biosimilar in many invitro biofunctional assays [41]. Basically, hRP215 can be produced in large quantity for infusionto cancer patients and to achieve anti-cancer efficacy, similar to those available and utilized inmany clinical applications.

RP215 Applications in CAR-T Cell Constructs for CancerTreatments

The methods described [41] for application of hRP215 as anti-cancer drugs would requireinfusion of gram or subgram quantities of antibodies as antibody-based anti-cancer drugs.Alternatively, the technology of CAR-T (chimeric antigen receptor – transfected T cells) are alsobeing developed for therapeutic treatments of different human cancer [42, 43]. Generallyspeaking, genes of hRP215 in the Fab domains are engineered as ScFv and packaged intolentiviral vector for final formulations as chimeric antigen receptor (CAR). Patient’s T cells arethen isolated and transfected with hRP215-ScFv-linked CAR incorporated in lentiviral vector.Following in vitro expansion of CAR-T cells, the hRP215-CAR-transfected T cells are theninfused to the autologous cancer patient for the induction of target-specific cytotoxic killingsof cancer cells in vivo and to achieve expected therapeutic objectives [44]. HRP215 (ScFv)-linked CAR-T cells have been successfully constructed. The efficacy of cytotoxic killing of cancercells was demonstrated through a model study in vitro with C33A cervical cancer cell line.

Typical results of this cytotoxic cell killing assay with concurrent cytokine releases are presented in Figure 3 (A, B, C, and D), respectively.

Briefly, C33A cervical cancer cells (Target, T) were cultured for 8 hours with three differentconcentrations of hRP215-linked CAR-T cells (Effector, E). Under these experimental conditions,dose-dependent lysis of cancer cells was observed as shown in Figure 3A. The validation ofhRP215-ScFv-CAR-T cell therapy was further documented by cytokine release assays of IL-2, IL-7, and INF α, respectively (Figure 3B, C, and D).

By using the same technology, CD19-CAR-T cells have been successfully utilized in thetherapeutic treatments of liquid tumor, such as malignancy of blood cells [45]. High degrees ofremission rates (50-90%) were observed for different types of lymphomas. However, it remainsto be a major challenge during treatments of solid tumors by using this technology. The cancer treatments of solid tumors which consist of the majority of human cancer have only limited success. This is mainly due to the intrinsic problems associated with the immunotherapy ofsolid tumors [43-46]. With the introduction of hRP215 (ScFv) CAR-T cells, additional progressmay be made in the future, when the clinical studies are permitted through FDA’s formal INDapproval.

Conclusions

In this review, we highlight the initial discovery of a gene expressions of antigen receptorsincluding immunoglobulins and T cell receptors in cancer cells of non-hematological origins [1-4]. In contrast to conventional immunology, both immunoglobulins and T cell receptors are co-expressed on the same cancer cells and do not play the identical functional roles in the cancerimmunology as those of the former. These cancerous antigen receptors were demonstrated tobe essential for growth/proliferation and survival of cancer cells through interaction studieswith human serum proteins [20, 30, 46]. The discovery of RP215 which reacts withcarbohydrate-associated epitope (-ST antigen) located in cancerous antigen receptors mayrepresent a major breakthrough for further investigations regarding their respective functionalroles. Extensive biological and immunological studies were performed to explore functionalroles of cancerous antigen receptors. RP215 has become a unique probe to target cancerousantigen receptors, not only for functional studies, but also for potential immunodiagnostic andimmunotherapeutic applications in cancer monitoring and therapy [46]. Therefore, humanizedRP215 can be an ideal ligand to suppress vital functions of cancerous antigen receptors, eitherused as antibody-based anti-cancer drugs or cancer therapy with hRP215 (ScFv)-CAR-T cellsconstructs in targeting sold tumors in the future [46].

Abbreviations

A549: lung cancer cell line from ATCC; AFP: alpha fetoprotein; C33A: cervical cancer cell line from;ATCC: CA125: cancer marker 125; CA15-3: cancer marker 15-3; CA19-9: cancer marker 19-9; CA215:tumor-associated antigen 215 recognized by RP215; CAR: chimeric antigen receptor; CAR-T:chimeric antigen receptor transfected T cell; CD3: core receptor of T-Cell; CD4: core receptor of T-Cell; CD8: core receptor of T-Cell; CDC: complement-dependent cytotoxicity reactions; CEA:carcinoembrionic antigen; c-foss: onco gene; CHO: Chines hamster ovary; cIgG: IgG expressed bycancer cells; cyclin D1: A 36-kD protein which regulates cyclin-dependent protein kinase activity;cyfra-21-1:lung cancer marker; Du145: prostate cancer cell line; E/T: effector / target ratio; EGF:epitermal growth factor; EGFR: epidermal growth factor receptor; EIA: enzyme immunoassay;ELISA: enzyme-linked immunosorbent assay; Fab: fragment of antigen-binding domains; Fc: Fc

fragment, constant domain of immunoglobulins; GalNAcitol: N-acetyl galactose-linked glycan;hRP215: humanized RP215; IFNγ : Interferon γ; IgSF: immunoglobulin superfamily proteins; IHC:immunohistochemical staining; IL2: Interleukin 2; IL7: Interleukin 7; IND: initial new drugapplications; LCMS/MS: liquid chromatography tandem mass spectrometry; LDH: lactatedehydrogenase; MALDI-TOF MS: matrix-assisted laser desorption ionization time-of-flight massspectrometry; MB 231: breast cancer cell line from ATCC; mRP215: murine RP215; MUC 1: mucin1 proteins; NFĸB-1: Nuclear Factor Kappa-B, Subunit 1; OC-3-VGH: ovarian cancer cell line of serusorigins from Dept CBS/GYN, VGH, Taipei Taiwan; Po: ribosomal protein; P1: ribosomal protein; P21:cell cycle regulator; PCR: polymerase chain reaction; RT-PCR: reverse transcriptase polymerasechain reactions; RP215: a monoclonal antibody generated against ovarian cancer cell extract; ScFv:Single-chain variable fragment of IgG; SiRNA: small RNA of interference; TCR: T cell receptors; TLR:toll like receptors; TUNEL: terminal deoxynucleotidyltransferasedUTP nick end labeling.

Acknowledgement

The review of this research project was supported in parts by NRC-IRAP (#821-901) of Canada.Helpful comments and suggestions by Dr. Joseph Han is acknowledge.

Conflict of Interest

The author is co-founder of Vancouver Biotech Ltd. In Vancouver. No conflict of interest regarding the content is involved.

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Received: August 25, 2018;
Accepted: September 07, 2018;
Published: September 12, 2018.

To cite this article : Gregory Lee. Functional Roles of Antigen Receptors Expressed by Cancer Cells and Clinical Applications. British Journal of Cancer Research. 2018: 1:4.

© Lee G. 2018.