The main objective of this study is to investigate roles of cancerous immunoglobulins in immunology of cancer cells through several biochemical and immunological studies, including affinity-purification of relevant antigen and antibodies, characterization as well as gene regulation analyses. With RP215 as the unique probe, it has become possible to advance our understanding about the potential roles of immunoglobulins expressed by cancer cells [21
Initially, with RP215 as the ligand, it has been possible to isolate CA215 from shed media of cultured cancer cell extract from cell lines derived from many different tissue origins (Figure 1) [22
]. Similarly, cancerous IgG (CIgG) can also be purified with goat anti-human IgG as the affinity ligand and characterized with respect to its affinity to RP215 which recognizes mainly the heavy chains of cancerous IgG (Figure 3) [2
Attempts were made to identify the possible serum antigen (or autoantibodies) which might show specific affinity to cancerous IgG. Purified CA215 or CIgG were used as the affinity ligands to isolate serum antigen or autoantibodies and further characterized with enzyme immunobinding assay [23
]. By using ELISA with purified serum antigen coated on microwells, we have been able to demonstrate specific and significant dose-dependent binding between CA215 or CIgG with purified serum antigen or autoantibodies, when compared to those of the negative control. Judging from the results of enzyme immunobinding assay, it is possible to demonstrate significant binding between CA215 or CIgG and purified serum antigen. The positive binding between CA215 and re-purified serum antigen was also demonstrated following the same binding ELISA as indicated in Table 1. Therefore, the specific binding of re-purified serum antigen to CA215 or CIgG can be documented. However, the re-purified serum antigen was obtained in small quantity with contamination of major serum proteins and difficult to perform molecular characterization at this moment by other means such as Western blot assay and MALDI-TOF MS analysis [1
]. In view of the fact that highly purified serum antigen was found to cross-react with anti-human IgG or IgM, it was therefore suggested that autoantibodies may be present in human circulation against cancerous immunoglobulins and/or CA215 through the expression of RP215-specific immunodominant epitope [15
]. (unpublish observations) Further biochemical and immunological studies of serum antigen or autoantibodies are in progress and would be required to clarify its molecular nature and origins as well as possible biological roles in cancer.
In this study, several functional studies were performed to demonstrate biosimilarity between RP215 and anti-human IgG [3
]. Both were shown to induce apoptosis of cultured cancer cells indicating that cancerous IgG is essential for growth/proliferation of cancer cells [3
]. Both exhibit complement-dependent cytotoxicity reactions to cancer cells indicating the surface nature of cancerous immunoglobulins in cancer cells [3
Previous studies with RP215 and antibodies against antigen receptors strongly suggested that cancerous immunoglobulins play significant roles in innate immunity of cancer cells, as demonstrated in previous studies [9
]. Both receptor ligands, RP215 and anti-human IgG were shown to have strong effects on the regulation of toll-like receptor genes in cancer cells. For example, the up-regulation of TLR-3 gene and down-regulations of TLR-4 and TLR-9 were consistently demonstrated by respective treatments with RP215 and anti-human IgG [9
]. This observation was not totally unexpected, since expressions of cancerous immunoglobulins as well as toll-like receptors were highly regulated by NFκB-1, which is a key transcription factor for both innate and adapted immune system in our human body, including cancer cells [9
]. Based on these studies, it is therefore concluded that expressions of cancerous immunoglobulins may be closely related with the innate immune system of cancer cells by maintaining survival of cancer cells through the functional expression of toll-like receptors.
In addition, cancerous immunoglobulins are expressed with mechanisms which are very different from those of B cells [4
]. For example, the antigen receptors are expressed by B and T lymphocytes in normal immune system [8
], while both can be co-expressed by the same cancer cell clones [32
]. Expressions of immunoglobulins in B cells require a series of activations, differentiation, somatic hypermutation as well as class switching [35
]. Most of these mechanisms in normal B lymphocytes do not exist in cancer cells [38
]. In addition, expressions of cancerous toll-like receptors are strongly influenced or regulated by cancerous immunoglobulins [9
]. Furthermore, cancerous immunoglobulins are known to carry very different O-linked and N-linked glycosylation patterns from those of normal human IgG. [10
]. These observations led us to hypothesize that there are two separate immune systems in our human bodies. One is our normal immune system for the immune surveillance to fight against foreign pathogens or cancer, including the presence of autoantibodies, or specific binding antigen [4
]. Cancer cells, on the other hand, may have their own independent immune system which serves for immune protection of cancer cells. These two systems can operate independently through different mechanisms and objectives within the same body environment. Cancer immunotherapy will work effectively only when the strategy to suppress cancer immune system can be designed.