To improve the outcome of multiple myeloma (MM) using a polyvalent tumor vaccine that increase the in vivo effector:target ratio, we have been working on identification and characterization of novel MM antigens. We started by characterizing the 24 immunoreactive clones we isolated from an expression MM cDNA library. Following rapid screening of these clones, it became apparent that they primarily encoded ubiquitously expressed proteins. As a result, the approach was changed to one that relies on bio-informatics from gene databank. Through this approach, we have identified four novel cancer-testis antigens in MM: Sperm protein 17 (Sp17), SPAN-Xb, SEMG1 and SLLP1, all showing a very restricted normal tissue expression and are expressed in the tumor cells from some patients with MM. These antigens could therefore be potentially useful for a polyvalent vaccine. We have proceeded to clone and produce recombinant proteins of Sp17, SPAN-Xb and SEMG1 and used these proteins to generate antigen-specific murine MoAbs. In the present proposal in Specific Aim 1, we will generate SLLP1 recombinant protein and antibodies, use a combination of real time PCR and immunohistochemistry and immunocytochemistry to determine the prevalence and pattern of Sp17, SPAN-Xb, SEMG1 and SLLP1 expression in 200 cases of MM and the frequency of co-expression of these antigens within individual tumor specimens. We will also correlate antigen expression to disease stage. Secondly, we have demonstrated the in vitro immunogenicity to T cells of Sp17, SPAN-Xb and SEMG1 in healthy donors and Sp17 in MM patients. We have also identified the CTL epitope of Sp17 restricted by HLA-A1 and HLA-A2 and SPAN-Xb restricted by HLA-A2.
In Specific Aim 2, we will use the recombinant proteins to determine the in vitro immunogenicity of SPAN-Xb, SEMG1 and SLLP1 by investigating the ability to generate antigen-specific CTLs from healthy donors and patients with MM. We will characterize the nature of the immune responses and identify the CTL epitopes of these antigens that are restricted by HLA-A1 and HLA-A2. Finally, we have demonstrated that Sp17 and SPAN-Xb gene expression is regulated through promoter methylation. Sp17 gene could be upregulated by promoter demethylation and SPAN-Xb promoter activity upregulated by IL-7 and GM-CSF.
In Specific Aim 3, we will determine the ability to upregulate Sp17, SPAN-Xb, SEMG1 and SLLP1 expression in MM cells using pharmacologic agents. Specifically, we will determine the role of promoter methylation in the regulation of antigen expression. We will determine the ability to induce the expression of these antigens using a hypomethylating agent, a histone deacetylase inhibitor and cytokines. We will also determine the relevance of upregulating these antigens for antigen-specific CTL killing. Successful completion of the proposal will provide the basis for the design of potent polyvalent tumor vaccines for MM. ? ?
Showing the most recent 10 out of 29 publications
