ALBERT

Description: Abstract 2979 Allogeneic stem cell transplantation (allo-SCT), alone or followed by donor lymphocyte infusion (DLI), is a potentially curative treatment for various hematological malignancies. In an HLA-matched transplantation setting, the therapeutic graft-versus-tumor (GvT) effect is mediated by donor T-cells directed at minor histocompatibility antigens (mHags), which are HLA-bound polymorphic peptides. Since mHags are also involved in the induction of graft-versus-host disease (GvHD), the immunotherapeutic potential of a mHag depends on its hematopoietic tissue restriction. The identification of more relevant mHags, which are presented by frequent HLA molecules and display an equally balanced population frequency, is imperative to enable broad implementation of mHag-based immunotherapy. We now report the discovery of a novel mHag that fulfills all these criteria and thus has evident clinical and immunotherapeutic relevance. The mHag was identified by analysis of a cloned CD8+ cytotoxic T lymphocyte (CTL), isolated from a multiple myeloma (MM) patient at the peak of his clinical response. This response started just after a second DLI following an HLA matched sibling transplantation and resulted in a complete remission that at present persists for over 8 years, without other treatment. The CTL 503A1 was specifically selected for analysis since it displayed strong cytotoxic activity toward a mHag which was presented by the frequent HLA-molecule HLA-A2*0101. Phenotype analyses revealed a phenotype frequency of around 40% in the Caucasoid population, providing a possibility to apply this mHag in immunotherapy in nearly 12% of allo-SCT patients. Using genome wide zygosity-genotype correlation analysis, followed by fine epitope mapping with synthetic peptides, we identified the antigen recognized by this clone as a nonameric peptide encoded by the SNP rs2166807 on the gene C12orf35. This new mHag was designated as UTA2-1. Quantitative PCR experiments confirmed the hematopoietic tissue restriction of gene C12orf35, with high expression on malignant B and T cells. In a detailed analysis exploring the clinical relevancy of UTA2-1, CTL 503A1 lysed not only benign hematopoietic cells but also primary and immortalized multiple myeloma cells without affecting fibroblasts, keratinocytes or stromal cells derived from the original patient. Tetramer analyses executed on original recipient peripheral blood samples taken after allo-SCT and the DLIs, demonstrated significant expansion of UTA2-1 specific T-cells, coinciding with strong clinical responses after allo-SCT and the second DLI. In summary, UTA2-1 has an ideally balanced population frequency, HLA-A2 restriction, hematopoietic-specific tissue distribution, optimal expression on malignant cells and the clinical capacity to evoke effective T-cell responses during an anti-myeloma effect. With these properties, UTA2-1 is the most clinically relevant mHag identified so far, next to the well-studied mHag HA-1, and has potential of expanding mHag-based adoptive immunotherapy for a great number of patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1333 Poster Board I-355 The curative Graft versus Tumor (GvT) effect of allogeneic stem cell transplantation and donor lymphocyte infusions is mainly mediated by donor derived T cells recognizing minor Histocompatibility antigens (mHag) presented by malignant cells. Traditionally, CD8+ cytotoxic T cells (CTLs) are considered as “the” effector cells of these anti-tumor responses, whereas a sole helper role is attributed to CD4+ T cells. CD4+ T cells often possess killer capacities in vitro, raising the possibility that they may also mediate anti-tumor effects without the need for CD8+ T cells. Hence, we here explored the feasibility of adoptive immunotherapy with sole CD4+ CTLs by testing the therapeutic capacity of a mHag-specific, CD4+ CTL (3AB11) in a human GvT model. Rag2−/−γc−/− mice were inoculated with BLI detectable, mHag+ Multiple myeloma cells. Treatment of established tumors with 3AB11 but not with control CD4+ T cells by triple consecutive injections of 30-40×106 cells/day rapidly reduced the medullary growing tumors, illustrating for the first time the feasibility to establish a significant GvT effect by targeting a sole mHag recognized by CD4+ CTLs. The therapy was less effective at a higher tumor load and unsuccessful by a single injection of 20×106 cells, underscoring the critical importance of T cell dose-to-tumor load ratio to establish an efficient anti-tumor effect. In further exploration, tumors were also significantly reduced by treatment with “dual antigen-specific” T cells, which were generated by transduction of the T cell receptor (TCR) of clone 3AB11 into recall antigen (Tetanus Toxoid; TT)-specific T cells. Finally, the in vivo persisting dual-specific T cells could be boosted by administration of TT loaded mHag negative B cells, demonstrating for the first time the feasibility and potential advantages of immunotherapy with TCR-transduced “dual antigen-specific” CD4+ T cells. We conclude that potent GvT effects may be achieved in clinical trials by targeting a sole mHag antigen with original or TCR-redirected CD4+ CTLs. Disclosures No relevant conflicts of interest to declare.

Description: Minor histocompatibility antigens (mHags) are highly immunogenic, HLA-bound polymorphic peptides playing prominent roles in the development of the graft vs host disease (GvHD) as well as the therapeutic graft vs tumor (GvT) effect of allogeneic stem cell transplantation (allo-SCT). To date, a small subset of mHags has been identified as genuinely hematopoietic system-specific. This small number of conceptually GvT-associated mHags are currently being tested in various clinical trials for the feasibility and efficacy of inducing an exclusive GvT effect without GvHD in patients with relapsing haematological malignancies after allo-SCT. Nonetheless, a broader and timely application of mHags as therapeutic tools in allo-SCT, as well as the development of appropriate GvHD prevention strategies still requires more insights that can be acquired through the identification of a much larger set of mHags presented by various HLA molecules in different ethnic populations. Recently, the introduction of genome-wide association (GWAS)-based strategies, despite a number of drawbacks, significantly accelerated the identification of mHags. To develop a strategy that can overcome the drawbacks of these earlier developed strategies, we now integrated our previously developed GWAS methodology [1,2] with the comprehensive genomic databases from the 1000 Genomes Project [3]. We show that the data set of the 1000 Genomes Project is suitable to identify all theoretical possible mHags, hereby enabling us to delineate important yet unknown characteristics of mHags. Furthermore, we demonstrate the actual power of this novel approach by the rapid and unambiguous identification of the HLA-DP4 restricted mHag UTDP4-1, which -despite extensive efforts in the past 15 years- could not be identified using any of the previously developed biochemical, molecular biological or genetic strategies. The 1000 Genomes Project-based identification of mHags thus represents a very rapid, convenient and robust method for the identification of new mHags, that will not only improve the understanding of the yet unknown facets of mHags but may also significantly enlarge the arsenal of targets for immunotherapy in haematological cancers. Spaapen et al., J.Exp.Med. 2008 Nov 24;205(12):2863-72. Oostvogels et al., Leukemia 2013 Mar;27(3):642-9. Abecasis et al., Nature 2012 Nov 1;491(7422):56-65. Disclosures Lokhorst: Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Mutis:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding.