Contributions
Abstract: PB2128
Type: Publication Only
Background
Our group has previously described that high frequency of serum monoclonal immunoglobulins (M-Igs) of G and A class (M-IgG, M-IgA) from patients with Multiple Myeloma (MM) exhibit polyreactivity, which is a common feature with Natural Antibodies (NAbs) (Abs present in the serum of healthy subjects, able to recognize self- and non self- antigens). This and other findings support the view that in MM, certain clones producing M-Igs originate from NAb-producing clones occurring under normal conditions. Antibodies able to penetrate living cells (CPAbs) have been detected and well characterized in patients with systemic lupus erythematosus and in mouse models; recognize DNA and accumulate in the nucleus. Their penetration ability seems to be directly related to their polyreactivity. Our laboratory has recently described the existence of Cell-Penetrating Antibodies (CPAbs) in healthy conditions, derived either from BALB/c mice (monoclonal CPAbs), or from intravenous immunoglobulin (IVIg) (polyclonal human IgG). The development of human monoclonal IgG-CPAbs is of major importance in drug delivery and in cancer therapy.
Aims
The aim of the present study was to investigate the cell-penetrating ability of M-IgG exhibiting NAb–like activity, as well as their intracellular biological functions in neoplastic cells.
Methods
Seventy-one sera of patients with IgG-MM (IgG M-peak >5 g/l: 42 IgGκ/29 IgGλ) were studied by in-house ELISAs against a panel of self and non-self antigens (actin, tubulin, myosin, carbonic anhydrase, thyroglobulin, DNA and the hapten trinitrophenyl (TNP). We selected and purified five polyreactive IgG and one non-polyreactive IgG (negative control) by protein-G affinity chromatography. We then established the optimum conditions in terms of concentration, temperature and time-course in order to examine their ability to: 1) penetrate FcγR+ (Raji & MDA-MB-231) and FcγR- (NIH-3T3 & HeLa) cells by immunofluorescence, 2) induce apoptosis on the aforementioned cells by flow cytometry (FACS), and 3) hydrolyze plasmic and genomic DNA.
Results
The five purified M-IgGs tested (3 IgGκ and 2 IgGλ) were able to penetrate all cell types, either FcγR+ or FcγR-, at 37oC in a dose- and time-dependent mode of entry, while three of them were able to also penetrate cells at 4oC (energy-independent mode of entry); optimum conditions of cell-penetration: 300 μg/ml, 2h-incubation. All M-IgGs: 1) were polyreactive, exhibiting a unique polyreactivity profile with the antigens of the panel used, 2) accumulated in the cytoplasm, 3) induced apoptosis especially in MDA-MB-231 cells, and 4) were able to hydrolyze plasmid DNA isoforms, while one of them also hydrolyzed genomic DNA.
Conclusion
We demonstrate herein that the sera of MM-patients with M-IgG represent an excellent source of human monoclonal IgGs exhibiting cell-penetrating ability and intracellular functionality. These human monoclonals can be exploited as a potential therapeutic tool in several disorders including malignant diseases, using them either per se, or as carriers for intracellular drug delivery, or even both.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Immunoglobulin, Monoclonal gammopathy, Myeloma
Abstract: PB2128
Type: Publication Only
Background
Our group has previously described that high frequency of serum monoclonal immunoglobulins (M-Igs) of G and A class (M-IgG, M-IgA) from patients with Multiple Myeloma (MM) exhibit polyreactivity, which is a common feature with Natural Antibodies (NAbs) (Abs present in the serum of healthy subjects, able to recognize self- and non self- antigens). This and other findings support the view that in MM, certain clones producing M-Igs originate from NAb-producing clones occurring under normal conditions. Antibodies able to penetrate living cells (CPAbs) have been detected and well characterized in patients with systemic lupus erythematosus and in mouse models; recognize DNA and accumulate in the nucleus. Their penetration ability seems to be directly related to their polyreactivity. Our laboratory has recently described the existence of Cell-Penetrating Antibodies (CPAbs) in healthy conditions, derived either from BALB/c mice (monoclonal CPAbs), or from intravenous immunoglobulin (IVIg) (polyclonal human IgG). The development of human monoclonal IgG-CPAbs is of major importance in drug delivery and in cancer therapy.
Aims
The aim of the present study was to investigate the cell-penetrating ability of M-IgG exhibiting NAb–like activity, as well as their intracellular biological functions in neoplastic cells.
Methods
Seventy-one sera of patients with IgG-MM (IgG M-peak >5 g/l: 42 IgGκ/29 IgGλ) were studied by in-house ELISAs against a panel of self and non-self antigens (actin, tubulin, myosin, carbonic anhydrase, thyroglobulin, DNA and the hapten trinitrophenyl (TNP). We selected and purified five polyreactive IgG and one non-polyreactive IgG (negative control) by protein-G affinity chromatography. We then established the optimum conditions in terms of concentration, temperature and time-course in order to examine their ability to: 1) penetrate FcγR+ (Raji & MDA-MB-231) and FcγR- (NIH-3T3 & HeLa) cells by immunofluorescence, 2) induce apoptosis on the aforementioned cells by flow cytometry (FACS), and 3) hydrolyze plasmic and genomic DNA.
Results
The five purified M-IgGs tested (3 IgGκ and 2 IgGλ) were able to penetrate all cell types, either FcγR+ or FcγR-, at 37oC in a dose- and time-dependent mode of entry, while three of them were able to also penetrate cells at 4oC (energy-independent mode of entry); optimum conditions of cell-penetration: 300 μg/ml, 2h-incubation. All M-IgGs: 1) were polyreactive, exhibiting a unique polyreactivity profile with the antigens of the panel used, 2) accumulated in the cytoplasm, 3) induced apoptosis especially in MDA-MB-231 cells, and 4) were able to hydrolyze plasmid DNA isoforms, while one of them also hydrolyzed genomic DNA.
Conclusion
We demonstrate herein that the sera of MM-patients with M-IgG represent an excellent source of human monoclonal IgGs exhibiting cell-penetrating ability and intracellular functionality. These human monoclonals can be exploited as a potential therapeutic tool in several disorders including malignant diseases, using them either per se, or as carriers for intracellular drug delivery, or even both.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Immunoglobulin, Monoclonal gammopathy, Myeloma