CDC: Complement-dependent Cytotoxicity; cIgG: Cancer cells expressed Immunoglobulin G; IgG: Immunoglobulin G; IgM: Immunoglobulin M; MALDI-TOF MS: Matrix Adsorption Laser Desorption Ionization-Time of Flight Mass Spectrometry; CA215:Cancer-associated antigen recognized by RP215 monoclonal antibody; LC-MS/MS: Liquid Chromatography plus analysis by Mass-Spectrometry; DTT: Dithiothreitol; CA215 S+: Human serum protein captured (adsorbed) by CA215-linked immunoaffinity column; cIgG S+:Human serum proteins captured (adsorbed) by anti-human IgG affinity column

Expressions of immunoglobulins in cancer cells were known for decades, even though the mechanisms of action are still not fully understood [1]. This is in contrast to conventional immune system, in which detailed molecular mechanisms of immunoglobulin expressions have been well established [1,2].

Many cancer cells, especially those of the hyperplastic epithelial origins were known to express immunoglobulins of unknown origins [1]. The expressions of immunoglobulins by cancer cells are independent of those by normal B cells [1] and were demonstrated by immunohistochemistry, RT-PCR and IgG-related SiRNA inhibition studies in vitro and in vivo [2,3]. In addition, anti-human IgG were found to induce apoptosis of almost all cancer cells of different tissue origins in vitro [4,5]. Complement-dependent cytotoxicity (CDC) reactions were also demonstrated with anti-human IgG and complement indicating the surface expressions of immunoglobulins [6]. These observations led to a general conclusion that cancer cell-expressed immunoglobulins are essential for the growth/proliferation of cancer cells [7]. Further studies with nude mouse animals also revealed significant reductions in volumes of implanted tumor upon injection of antibodies against human immunoglobulins [8-10]. Additional molecular biological studies also indicated that expressions of cancerous immunoglobulins are quite different for those of immunoglobulins derived from B cells [1]. No class switching and somatic hyper mutation are involved in the V(D)J heavy chain regions of cancer cell-expressed immunoglobulins [11].

To elucidate exact functional roles of cancer cell-expressed immunoglobulins in human environment, interactions between human serum proteins and cancerous immunoglobulins were investigated and analyzed by LCMS/MS and MALDI TOF-MS methods. In 1987, a major breakthrough of this research work came, when a monoclonal antibody designated as RP215 was generated against OC-3-VGH ovarian cancer cell line. It was shown to react specifically with a carbohydrate-associated epitope located mainly on the variable regions of immunoglobulin heavy chains as well as other types of cancerous glycoproteins designated in general as CA215 [8].

Numerous in vitro and in vivo immunological and biochemical studies have been performed during the last decade by using RP215 as the unique probe for studies of cancerous immunoglobulins [8,9]. Based on these studies, it was generally concluded that apoptosis can be induced upon incubation of RP215 or antibodies against antigen receptors (including anti-immunoglobulin and anti-T-cell receptors), which was consistent with the hypothesis that cancerous immunoglobulins are essential for growth/proliferation of cancer cells in vitro, or in vivo [8-10]. Furthermore, in vivo nude mouse experiments with implanted tumors from derived cancer cell line revealed that the volume of the implanted tumor was reduced dose-dependently upon treatments with RP215 or antibodies against immunoglobulins [10,11].

Effects of RP215 and antibodies against antigen receptor on the gene regulations of culture cancer cells were also investigated [12]. Changes of a number of genes including those of toll-like receptors and those required for growth/proliferation of cancer cells were observed. As many as ten to twelve genes were observed to change in response to treatments with RP215 or anti-antigen receptors. High correlations (≥ 90%) in gene regulations of RP215 and other two anti-antigen-receptor ligands were observed and demonstrated. Based on the results of gene regulation studies, it was clearly demonstrated that the blockage of cancer immunoglobulin and/or T-cell receptors by RP215 and other anti-antigen receptors can be well demonstrated by inducing apoptosis to cultured cancer cells in vitro [13]. Therefore, cancerous immunoglobulins are essential for growth/proliferation of cancer cells in vitro and in vivo, and affected by RP215 which reacts mainly with cancerous immunoglobulins on the surface of cancer cells. Therefore, RP215 may have potential therapeutic applications as the anti-cancer drug provided that RP215 is humanized for administration in humans [14]. So far, humanized RP215 has been successfully developed and shown to be bioequivalent to murine RP215 and should be ready for applications in preclinical studies [15].

To maintain growth/proliferation of cancer cells in human circulations, interactions with human serum protein components may play essential roles [4]. Therefore, we believe that cancerous immunoglobulins may be involved in such molecular interactions. Therefore, RP215 which reacts mainly and specifically with cancerous immunoglobulins may be utilized as the unique probe for studies of such interactions. Based on this hypothesis, CA215 and cIgG were initially isolated from culture cancer cell lines in shed medium. CA215 and cIgG can be utilized as affinity ligands to capture human serum proteins in separate affinity columns [6,8]. Through sequential affinity chromatographs, the isolated human serum proteins can then be analyzed by LC-MS/MS methods. From the results of such analysis, the functional roles of cancerous immunoglobulins can be revealed and presented in this review [5].

Affinity-isolation of CA215 and cancerous immunoglobulin as well as human serum protein components

Preliminary studies regarding the affinity isolation of CA215 and cancerous immunoglobulins have been published elsewhere [4,6]. Briefly, CA215 was isolated from shed medium of cultured ovarian OC-3-VGH cancer cells by using RP215-linked affinity column in a typical affinity chromatography . The culture shed medium was first concentrated by adding ammonium sulfate (40% satination) followed by extensive dialysis against PBS, prior to loading on the RP215-linked affinity column. Following extensive wash, the bound CA215 was eluted with 5 mM citrate acid at pH 2.2. After dialysis with PBS, purified CA215 was concentrated and used a ligand to build a CA215-linked immunoaffinity column. Similarly, goat anti-human IgG was utilized as the affinity ligand in an affinity column for purification of cIgG (cancerous immunoglobulins).

To purify human serum protein components which might have affinity or any interactions with CA215 or cIgG were isolated, respectively by CA215- and cIgG linked affinity columns. Following elution, concentration and dialysis in ammonium bicarbonate, purified human protein components which was designated as CA215-S+ and cIgG-S+ were obtained, respectively. They were subjected to analysis by LC-MS/MS methods though a contract service from the University of Victoria Genomic BC Proteomics Centre to be described in the following section.

Analysis of human serum protein components by LC-MS/MS method

Affinity-isolated CA215-S+ or cIgG-S+ (200-500 µg) was rehydrated with a minimum 200 to 500 μl 25 mM ammonium bicarbonate prior to reduction with suitable amount of dithiothreitol (DDT) for 30 min at 37°C in the dark. Alkylation of cysteine sulfhydryl groups was performed with 200 mM DDT and 10 µg trypsin (Promega) was added to each sample for 16 h at 37°C.

A Waters Oasis hydrophilic-lypophilic balanced column (1 ml, 10 mg) was equilibrated with 2 ml acetonitrile and then with 2 ml 0.6% acetic acid. The supernatant was applied to the column by 2 ml 0.6% acetic acid, and peptides eluted with 700 µl 80% acetonitrile plus 0.6% acetic acid. The samples were concentrated via speed vac, acidified and desalted with e19 Stage Tips prior to LC-MS/MS analysis.

The peptide mixtures were separated by online reverse-phase chromatography with a Thermo Scientific Easy-Inc. 1000 system at a flow rate of 300 nl/min. the chromatography system was compared online with an Orbitrap Fusion Tribid mass spectrometer (Thermo Scientific, San Josa, CA). The peptide mixture in each sample was subjected to MS analysis by using standard operation manual. Raw files were created by X Caliber 3.0.63 software from Thermo Scientific and analyzed with Proteome Discoverer 1.4.1.1.4 software from the source.

Human serum proteins recognized by CA215 and cIgG expressed in cancer cells

The expressions of CA215 and/or cancerous immunoglobulins (cIgG) have been well documented through decades of studies [4,6,8-10,12]. The functional roles of cIgG and/or CA215 can be demonstrated by their interactions with major serum protein components. This can be accomplished by the present study with sequential affinity chromatography by using CA215 or cIgG as the respective affinity ligands [4,6].

Molecular identity of CA215 expressed in cancer cells

RP215 was initially shown to react specifically with cancer cell-expressed glycoproteins designated in general as CA215 through the binding of a common but unique carbohydrate-associated epitope. The affinity-isolated CA215 was trypsin-digested and subject to analysis by MALDI-TOF MS method [8].

As shown in Table 1, the molecular identity of affinity-isolated CA215 was listed and categorized according to their basic molecular structure, derived from the detected 124 tryptic peptides. From this table, greater than 40% of detected CA215 were found to be immunoglobulins expressed by cancer cells. In addition, a number of immunoglobulin superfamily proteins (IgSF) were also detected, accounting for as many as 60% of the detected protein species. The remaining 35-40% of the detected CA215 are unrelated to IgSF proteins [16].

Table 1: Molecular analysis of CA215 based on MALDI-TOF MS analysis of tryptic peptides.

Molecular analysis of human serum proteins designated as CA215 S+ and cIgG S+

Following affinity-isolation of CA215 S+ and cIgG S+ from pooled human serum, many distinct human serum proteins or their components were identified by LC-MS/MS methods. The majority of human serum proteins were commonly detected by both CA215 and cIgG –linked affinity columns (80-86%). They were listed in Table 2 for further investigations regarding anti-cancer or pro-cancer nature of these proteins. The remaining proteins which are recognized either by CA215 or cIgG ligands are listed in Table 3 for comparisons.

Table 2: List of human serum proteins or protein fragments which are commonly recognized by CA215 and cIgG.

Table 3: Human serum proteins fragments recognized by either CA215 or cIgG.

Human serum proteins with pro-cancer properties

About 80-86% of the detected human serum protein components were commonly recognized by both CA215 and/or cIgG. Based on the results of previous and ongoing studies by others, part of these proteins were classified to exhibit pro-cancer properties. Further analysis reveals that some are complement-related proteins and serve to prevent complement attack on cancer cells in vitro and in vivo. Among these are C4b-binding protein α chain, complement C3 and complement factor H.

Complement-related components in human serum

1. C4b-binding protein is known to be a soluble complement inhibitor consisting of 7α and one β subunit with a molecular size of 67 kDa [17,18]. By acting as a cofactor, to inhibit complement activation through factor I-mediated degradation of C3b and C4b [19]. This has been demonstrated in SKO-3 and CAOV-3 ovarian adenocarcinoma cell lines, the α-chain of C4b- binding protein is capable of inhibiting the complement classical pathway through the inactivation of C4b. Therefore, it was assumed that interactions of C4b binding protein with C4b-binding proteins in cancer cells would prevent or inhibit complement activation [19]. Therefore, C4b-binding protein α-chain was demonstrated to have pro-cancer function [19].

2. Complement C3 (MWt 187 kDa) and its cleavage C3a and C3b play central roles in the complement cascade and can be engrafted in tumors of mouse animal models to decrease tumor proliferation [17,18]. C3a is the cleavage product of C3 have been found to be associated with activation/proliferation of neoplasma including production of vascular endothelial growth factor (VEGF), extracellular matrix reorganization and disintegration of tumor angiogenesis as well as invasion/migration [20,21]. Complement C3 was also expressed in gastric-cancer related cell lines to promote cancer development and progression [22].

3. Complement factor H is a serum glycoprotein with a molecular weight of 139 kDa, and is key regulator of the alternative pathway in the complement system. Similar to C4b-binding protein, complement factor H acts to prevent complement activation by acting as a cofactor of serine protease factor I to induce cleavage and inactivation of C3b and C4b and to accelerate the degradation of C3 convertase [23]. Complement factor H was found to be expressed in cancer cells of many tissue origins [24-26] rendering them resistant to complement attack and reduce tumor growth. Therefore, it can be hypothesized what complement factor H serves to protect cancer cells from complement activation [24-27].

In summary, the three complement-related proteins or components from human serum were commonly recognized by both CA215 and cIgG expressed by cancer cells. Therefore, certain human serum proteins are important to prevent of complement-inactivation or attack cancer cells in human circulations [27,28].

Serotransferrin and vitronectin

Two human serum components, serotransferrin and vitronectin were also commonly recognized by both CA215 and cIgG [29-36]. Serotransferrin (Serum transferrin) is an iron binding protein of 77 kDa in human serum. It functions to transport iron from degradation to sites of storage and utilization [29-31] and involves in important cellular functions, proliferation in cases of prostate and lung cancer. Therefore, it is believed that blockage of transferrin binding on cancer cell surface will result in tumor growth inhibition and serotransferrin may serve as important growth factor or pro-cancer factor for the proliferation of cancer cells.

Vitronectin is a glycoprotein of 54 kDa produced in liver. Vitronectin is involved in cancer stem cell differentiation of breast and prostate cancer cell. It also interacts with the proteins in the integrin family for the adhesion, differentiation, as well as growth of many cancer cells [35,36].

Therefore, we believe that serotransferrin and vitronectin interacting with cancerous IgG and CA125 are proteins of pro-cancer factors in promoting tumor growth and proliferation [36-38].

Human serum proteins exhibiting anti-cancer properties and interacting with CA215 and cIgG

Anti-cancer properties of several human serum proteins commonly detected by CA215 and cIgG were noticed and presented in this analysis. Most of these human serum proteins are known to exhibit anti-cancer properties through several previous investigations [39-44]. Among these are anastellin, apolipoprotein A1, fibrinogen β-chain and others such as inter-α-trypsin inhibitor and keratin type 1 cytoskeletal 9 [39-45].

1. Anastellin and Apolipoprotein-A1: Anastellin is known to be a fragment of fibrinogen of the first type III module with a molecular size of 256 kDa [39-42] and shown to react with a variety of proteins, including integrins, proteoglycans, fibronectin and fibrinogen. Upon binding with fibronectin, anastellin can promote changes in organization and assembly of fibrinonectin matrix. Anastellin was also found to inhibit tumor growth by inhibiting angiogenesis [39-42] and by blocking cell cycle growth regulations [39-41].

2. Apolipoprotein A-1 is a major protein component of high-density lipoprotein (HDL) with a molecular size of 31 kDa in human plasma [43]. Apolipoprotein A-1 is known to be cardio protective with anti-inflammatory and anti-oxidant properties. It was also considered as a biomarker of ovarian cancer when combined with CA215 to improve diagnostic sensitivity. Apolipoprotein A-1 was also shown to suppress tumor growth, angiogenesis, metastasis in mouse animal model studies [43,44].

3. Fibrinonectin β chain is one of the peptide chains required for the assembly of fibrinogen involved in the formation of blood clot [45,46]. The first 20 amino acid of N terminus of the fibrinogen beta chain (β43-63) significantly inhibit VEGF-activation of epithelial cells to the extracellular matrix, and tumor vascularization and increase tumor necrosis [45]. Therefore, Fibrinonectin β chain which interacts with cIgG or CA215 is considered to exhibit anti-cancer properties in human body [45,46].

4. Anti-cancer nature of two cIgG-recognized human serum proteins were also identified. They are inter-α-trypsin inhibitor heavy chain 4 (35 kDa) [47] and keratin type 1 cytoskeletal 9 (62 kDa) [20]. The former is a fragment of inter-α-trypsin inhibitor heavy chain which was formed to supress proliferation and migration of tumor cell growth. The latter is a structural protein for intermediate filaments in epidermal cells and formed to suppress tumor growth in drug resistant Human breast cancer tissues.

Human serum proteins recognized separately by CA215 and cIgG

As shown in Table 3, several distinct human serum proteins were recognized separately by CA215 and cIgG ligands. This observation may indicate the differences in the molecular nature between CA215 and cIgG. However, a few of these proteins listed in Table 3 are related to pro-cancer or anti-cancer properties to cancer cells [65-74]. The molecular nature and functions of these captured human serum proteins remain to be explored in the future [49-68].

Human serum proteins recognized by CA215 only

As shown in Table 3, the certain CA215-recognized human serum proteins exhibit pro-cancer or anti-cancer properties. An investigation on identifying predictors of treatment outcome and survival in patients with non-small cell lung cancer treated with docetaxel has shown that α-1-acid glycoprotein appears to be an independent predictor of response and a major objective prognostic factor: the higher the level of α-1-acid glycoprotein, the shorter the patients survive [60]. Among plasma proteomic analysis of patients, apolipoproteins are among the important proteins with anti-cancer in nature. Apolipoprotein D is a promising prognostic and predictive factor in breast cancer and its determination can be done by immunoelectrophoresis in tumor cytosol or immunohistochemistry [62]. Other proteins exhibiting anti-cancer properties are afamin, apolipoproteins and plasminogen. As a vitamin E binding protein, afamin was found to be a potential biomarker of ovarian cancer. Although CA-125 is a widely used biomarker for ovarian cancer, its low sensitivity limits diagnosis for early stage patients. Afamin shows potential complementarity with CA-125 in longitudinal monitoring of patients with ovarian cancer [58]. Mainly associated with high-density lipoproteins, apolipoprotein M is identified as a carrier of the bioactive lipid sphingosine-1-phosphate, through which, it affects a wide range of biological processes, such as lymphocyte trafficking, endothelial permeability in the lung, and even virus suppression and cancer [63]. Judged from effects on a syngeneic tumor model in C57/bl6 mice, histidine-rich glycoprotein is demonstrated as a potent inhibitor of tumor vascularization with anticancer properties. A comparison indicated that after treatment with histidine-rich glycoprotein, the growth of fibrosarcoma, a very aggressive tumor, was reduced by >60% [49]. The nature of plasminogen has been investigated both in vitro and in vivo [67]. The results clearly shown that plasminogen activator inhibitor type 2 acts specifically against prostate cancer cells. The research of its anti-cancer effects on antiproliferation of tumors and prostate cancer lymph node metastasis were carried out. It is proposed that plasminogen could be considered for further development for the therapy of prostate cancer [67].

Human serum proteins recognized by cIgG only

On the right column of Table 3, gelsolin exhibits pro-cancer properties; but Inter-alpha-trypsin inhibitor heavy chain H2 and complements exhibits anti-cancer properties. Tumor gelsolin is associated with protein overexpression of breast cancer patients. Although gelsolin alone is not a prognostic factor, gelsolin coexpression may be an important additional prognostic factor in some breast cancer patients [64]. As a central part of the innate immune system, complements such as complement 3, 5, and 9 provides a highly effective means for destruction of invading microorganisms and elimination of dead and apoptotic cells [61]. While the complements are activated, they can suppress the growth of cancer cells. However, cancer cells seem to be able to establish a convenient balance between complement activation and inhibition. Some attempts have been already made to modulate antibody-mediated complement activation to increase the complement-fixing capacity of the therapeutic antibodies . With the introduction of monoclonal antibodies in cancer immunotherapy, complement has come into play a great potential as anticancer proteins [61]. The study on inter-alpha-trypsin inhibitor heavy chains molecules has been shown to play a particularly important role in inflammation and carcinogenesis [55]. Inter-alpha-trypsin inhibitor heavy chain genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. The results indicate that inter-alpha-trypsin inhibitor heavy chains may represent a family of putative tumor suppressor or anti-cancer proteins [55].

Potential application of RP215 in cancer immunotherapy

Expressions of surface immunoglobulins in cancer cells are almost universal with few exceptions and can be targeted for cancer immunotherapy [65,70]. Since the discovery in 1987, RP215 has been used as a unique probe for studies of immunoglobulins expressed on the surface of cancer cells [10]. Biological and immunological studies in vitro during the last decade indicated that RP215 can serve as the substitute for anti-immunoglobulins to target this surface bound molecules to induce apoptosis and complement-dependent cytotoxicity reactions to cancer cells [71]. In vitro nude mouse experiments also suggested that volumes of implanted tumors where reduced significantly in model studies [12,71-73]. Therefore, RP215 has been humanized to make it suitable for human applications in immunotherapy of almost all human cancer following appropriate preclinical and clinical studies [74] in the near future.

In this study, quite a few human serum proteins were shown to interact with both cIgG and CA215 and exhibit either pro-cancer or anti-cancer properties. As many as 80-86% of the affinity isolated human serum proteins were commonly captured by both CA215 and cIgG affinity ligands and identified by LC-MS/MS methods. This is not totally unexpected since the majority of identified CA215 glycoproteins are found to be cancerous immunoglobulins (≥ 40-60%) (Table 1).

From this study, the functional roles of cIgG were clearly demonstrated. CA215 and cIgG were found to recognize a number of human serum proteins which exhibit either pro-cancer or anti-cancer properties in nature (Table 2). Therefore, the surface bound cIgG in cancer cells are capable of interacting with human serum protein components, which can be isolated by affinity chromatography. On the other hand, serum proteins with pro-cancer properties were captured by cIgG to promote the growth/proliferation of cancer cells in circulation. At the same time, serum proteins with anti-cancer properties can also be neutralized by cIgG or CA215 on the surface of cancer cells to minimize damages to cancer cells. In view of the fact that CA215 consist mainly of heavy chains of cIgG, both exhibit similar functional roles in cancer cells. In previous studies [9], both were found to induce apoptosis and complement-dependent cytotoxity to cancer cells upon binding with antibodies against cIgG or RP215 which reacts specifically with CA215 or cIgG [4,6].

The exact mode of interactions between CA215 or cIgG and human serum proteins are currently unknown. It could be simply antigen-antibody bindings. It may also be receptor-ligand-mediated interactions. This remains to be further investigated with each specific case.

Therefore, in this study, we have shown that cIgG, cancer-cell-expressed immunoglobulins and CA215 were able to recognize human serum proteins, some of which were shown to exhibit pro-cancer or anti-cancer properties. Therefore, dual functional roles of cIgG in cancer cells can be clearly demonstrated through their mutual interactions with human serum proteins. These observations may help to explain why cancer cells are able to survive and proliferate under our natural human environment with the widespread expressions of surface-bound cIgG.