Ten Year Survival in Patients with Myeloma Is Characterized by the Persistence of Immunological Control Which Is Detected by Immunological Biomarkers

Abstract 1818 There is clinical evidence for the presence of a limited degree of host-tumor control in patients with multiple myeloma (MM) but the exact mechanisms involved are not known. Patients who survive for more than ten years are likely to have the most active immunological host-tumour control and are an ideal cohort to study. We now add to our preliminary observations using a range of immunological biomarkers in the 29 of these patients who attend our clinic. 51% of MM patients (n=264) had expanded CD3+CD8+ TCRVβ+ CD57+T cell clones detected by TCRVβ analysis (Beckman, BetaMark). Clonality was confirmed by IgH CDR3 sequencing. These clones accounted for 14.3% (median) of the CD3 cells (range 4–49%). CFSE tracking demonstrated the anergic nature of these clonal T cells (median 6% proliferation) compared with other CD8 cells (70% proliferation) while Geneset analysis of mRNA microarrays (Affymetrix U133) identified that anergy was caused by upregulated RAS, CSK, TOB and suppressed ERK pathways. Unlike recent reports for T-LGL, microarray analysis suggested that there was no evidence of STAT3 upregulation in the MM T cell clones. Functional studies suggested that these non-proliferating clones have split anergy as interferon-γ production was normal. In contrast, all ten year survivors had expanded T cell clones and serial studies demonstrated a >8 year persistence of the same clone in 8 out of 12 patients studied. More importantly, unlike the other MM patients, the T cell clones in 19 out of 21 of the ten year survivors studied were not anergic. The Treg/Th17 ratio in the ten year survivors was significantly lower than other MM patients (median 1.9 vs 12.0; p<0.0002) and even lower than age matched normals (median 5.6; p<0.006). This difference in the ten year survivors was due to an increased absolute Th17 cell (p<0.005) number and a reduced absolute Tregnumber (p<0.05). MM patients had a reduced number of absolute 6-Sulfo-LacNAc (SLAN)-DCs, a major source of IL-12, compared to both age matched controls and long term survivors (p < 0.01). Allo SLAN-DCs stimulated a higher proliferative response by MM T cells than could be achieved with mononuclear preparations. In conclusion, MM patients have expanded clones of cytotoxic T cells that exhibit split anergy and these cells are potential candidates for restoring immunological control of MM and other cancers. The normalised Treg/Th17 ratio suggests that the induced tolerance associated with increased Treg cell numbers is also absent in these patients. Our observations that the ten year MM survivors do not have anergy in their clonal T cells and have less Treg suppression offers a unique cohort for future studies. Disclosures: No relevant conflicts of interest to declare.

First Generation Chimeric Antigen Receptor Display Functional Defects In Key Signal Pathways Upon Antigen Stimulation

Abstract 2088 Introduction: A promising strategy for tumor therapy is the adoptive transfer of tumor specific T cells which are endowed with chimeric antigen receptors (CAR). First generation CARs are constructed by single chain antibodies and as signal domain the ζ chain of the CD3 complex. However, clinical trials are disappointing as adoptive transferred T-cells showed only modest persistence in patients resulting in limited clinical activity. We there for hypothesized that CAR expressing T-cells in comparison to unmodified T-cells display signaling defects when stimulated via their CARs. Methods: Cytomegalovirus(CMV)pp65 MHC I restricted CD8+ T-cells were generated, isolated by tetramer selection and modified with first generation CAR targeting CD19 and purified based on their receptor expression to more than > 95% purity. T-cell receptor (TCR) and CAR expression were quantified by Quantibright beads. Effector function of both T-cell populations were analyzed for specific lysis, cytokine production (IFN-g, TNF-a) and proliferation (CSFE) upon target cell stimulation. Phosphorylation of Erk, Jnk, p38 and PLC-γ was measured and analyzed with CBA Flexsets from BD. All statistical analyses have been performed using the statistical software package R. Signal peak intensities have been compared using the nonparametric wilcoxon rank sum test. Results: CMV-specific MHC-I restricted TCR as well as the CARs are expressed at same density levels and T-cells show equally lysis of targets either in the time of lysis onset as well the maximal lysis. In contrast, cytokine production (IFN, TNF-a) as well as antigen driven proliferation was reduced in CAR expressing T-cells when compared to CMV-specific CD8+ T-cells upon target exposure. PLC-γ was phosphorylated within minutes after target contact by CMV-specific CD8+ T-cells whereas CAR transduced CMV-specific CD8+ T-cells showed no significant phosphorylation of PLC-γ to target cell exposure. T-cell activated via CAR's demonstrated a statistically significant reduction of maximal phosphorylation in comparison to CMV-specific T-cells for ERK, for JNK and for p38. To exclude that CAR modification of CMV-specific CD8+ T-cells may impair signaling, CAR-CMV-specific CD8+ T-cells were exposed to CMVpp65 expressing targets. Killing, cytokine production and signal intensity were restored in comparison to parental CMV-specific CD8+ T-cells. Conclusion: CAR expressing T-cells show functionally signs of split anergy by efficient target elimination but fails to produce significant levels of cytokines and do not proliferate in response to target stimulation. Split anergy is not due to reduced expression of the CAR's but due to a complete lack of phosphorylation of PLC-γ as well as reduced phosphorylation of MAP-kinases ERK, p38 and JNK. These results potentially explain why primary CAR expressing T-cells fail to show significant clinical efficacy. Analysis of adequate phosphorylation, as proposed here, may be a powerful tool to identify the most promising second generation CARs for clinical studies. Disclosures: No relevant conflicts of interest to declare.