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Introduction: Novel therapies have increased the complete response (CR) rates in multiple myeloma (MM) with corresponding improvement in progression free survival (PFS). However, relapse occurs in a significant number of patients. Emerging evidence strongly correlates minimal residual disease (MRD) negativity with improved clinical outcome. Nonetheless, definitive clinical guidance on the management of MRD-positive MM is currently lacking. Here we present the results of a phase II trial evaluating an allogeneic GM-CSF vaccine in combination with lenalidomide (Len) in patients with MM in near CR (NCT01349569). Methods: To be enrolled, patients required a sustained near CR (defined as no measurable M-spike and positive immunofixation, IFE) for at least four months while on a Len-containing regimen. The vaccine consisted of two allogeneic, commercially available MM cell lines (U266 and H929) coupled to a GM-CSF producing leukemia cell line (K562, GVAX®). Patients received four vaccinations over 6 months in combination with Len maintenance for at least 1 year, although in most patients Len was continued until disease progression. The primary endpoints of this trial were eradication of detectable disease and conversion to CR. The secondary endpoints were safety, time to response and immune monitoring of vaccine- and MM-specific T cell responses. Bone marrow (BM) and peripheral blood (PB) samples were collected at pre-established timepoints and cryopreserved for subsequent analyses. The ImmunoSEQ® assay (AdaptiveBiotechnologies, Seattle, WA) was used to sequence T cell receptors (TCR) and the resulting data was used to analyze TCR repertoire metrics. The ImmunoSEQ® assay was also used to sequence the IGH and IGK/L B-cell receptors for MRD quatification. Deep phenotyping of T cells and immune monitoring were performed by flow cytometry. Results: Fifteen MM patients were enrolled. The primary enpoint of the trial was met as 53% (n = 8/15) of patients converted to a true CR (negative IFE) at a median time of 11.6 months (p = 0.011 when compared to the 25% threshold for futility). With a median follow-up of 5.13 years from enrollment, the median PFS could not be estimated as only 6 patients (40%) experienced disease relapse. At the time of the analysis, the median OS was 7.8 years from enrollment (95% CI: 4.2-7.8 years, n = 6/15, 40%). MRD testing was performed on 7 patients. The disease burden threshold of 105/106 cells was arbitrarily used to evaluate the clinical significance of high-level and low-level MRD positivity. Interestingly, all 3 patients with high-level MRD experienced relapse within 1 year of vaccination (median = 4.8 months, range: 2.8 - 9.5 months), while median PFS for low-level MRD patients was significantly prolonged (median = 84.15 months, range: 51.9 - 97.3 months, p = 0.01). Consistently, high-level MRD was associated with increased likelihood of clinical relapse (hazard ratio, HR = 25.79, 95% CI: 2.17 - 306.4). Immune phenotyping of BM CD8+ T cells identified a CD27- DNAM1-/low subpopulation whose abundance was associated with prolonged MM remission. This subset included senescent, effector and exhausted CD8+ T cells and was nearly absent in patients with high-level MRD. Analysis of T-cell repertoires identified vaccine-specific T-cell clonotypes that expanded in both BM and PB of all patients and persisted for up to seven years. Accordingly, polyfunctional T cell responses against vaccine-specific and MM-related antigens were increased and persisted 7 years after enrollment. Despite the correlation between MRD negativity and improved clinical outcome, 47% of patients subsequently developed a detectable M-spike that did not meet criteria for disease relapse nor required any change in treatment. We identified a bone marrow-resident T cell population likely responsible for the establishment of an immune equilibrium mediating long-term disease control. Conclusions: Collectively, these data demonstrate that an allogeneic MM vaccine in combination with Len effectively stimulates antitumor immunity and supports long-term remissions. Although MRD negativity is a critical factor in maintaining remission, the establishment of a MM-MGUS immune equilibrium is likely crucial in the setting of MRD+ MM to prevent disease progression. To our knowledge, this is the first study attempting to treat MRD+ MM in an effort to further improve disease response as well as prevent relapse. Disclosures Sanders: Adaptive Biotechnologies: Current equity holder in private company. Ali:Celgene: Membership on an entity's Board of Directors or advisory committees. Noonan:Aduro: Patents & Royalties. Borrello:WindMIL Therapeutics: Other: Founder , Research Funding; Aduro: Patents & Royalties; Celgene: Research Funding.

Introduction: Novel therapies have significantly increased the complete response (CR) rates in multiple myeloma (MM) with corresponding improvement in progression free survival (PFS). However, the emergence of treatment resistant sub-clones leads to disease relapse in a significant number of patients. Despite continuous improvements, cancer vaccination strategies have not been effective in preventing disease progression. Possible reasons include suboptimal timing of administration, antigen selection, disease burden at the time of vaccination and lack of the appropriate adjuvant. Here, we present the results of a phase I/II trial evaluating an allogeneic MM cellular vaccine administered in the context of a minimal disease burden in combination with lenalidomide (Len) to prevent MM progression. Methods: The vaccine consists of two MM cell lines (U266 and H929) coupled to a GM-CSF producing leukemia cell line (K562, GVAX® platform). After irradiation, the vaccine was cryopreserved until further use. Patients achieving a near CR (nCR), defined as absent M-spike and positive urine/serum immunofixation, were observed for at least 4 months to monitor response stability before enrollment. Vaccination was administered at 1, 2, 3 and 6 months together with the PrevnarÒvaccine in patients continuing on Len maintenance. Bone marrow (BM) and peripheral blood (PB) samples were utilized for immune monitoring, T-cell receptor (TCR) sequencing, and B-cell receptor (BCR) sequencing for minimal residual disease (MRD) determination using the immunoSEQ®Assay (Adaptive Biotechnologies, Seattle, WA)at enrollment, 3 months, 1 year and long-term follow-up timepoints. Results: Of the 30 patients that were initially observed, 15 patients maintained a stable nCR for at least 4 months and were vaccinated at the established timepoints. Patients who either progressed or improved their clinical response transitioning to a CR were not considered eligible. TCR immunoSEQ analysis revealed both the appearance and expansion of new T-cell clones without overall changes in repertoire clonality, suggesting active response to vaccination in both BM and PB compartments. Persistence of expanded clones was detected by immunoSEQ up to 1 year after the first vaccine dose in all patients. Phenotypic analyses and antigen-stimulation assays on BM T cells demonstrated maintenance of MM-specific T-cell reactivity at all examined timepoints. Polyfunctional MM-specific T-cell responses were detected in follow-up samples up to 8 years after the first vaccine dose and surprisingly showed persistence of MM-specific T-cell immunity when compared to the 1-year time point. Although negative MRD status and depth of response directly correlated with disease remission, some patients developed a low, but persistent MRD and M-spike ( Conclusions: These data demonstrate that an allogeneic MM vaccine effectively stimulates antitumor immunity in the setting of low disease burden with Len. Moreover, polyfunctional antitumor immune responses were persistent up to 8 years post vaccination. It is likely that the diversity of antigens in the vaccine supported polyclonal MM-specific immune responses, thus establishing long-term MM control. Although depth of response to treatment is a critical factor in maintaining MM remission, the long-term persistence of functional MM-specific T-cell clones highlights the importance of an active immune response to prevent MM relapse. Overall, these findings suggest that allogeneic MM vaccination could be an integrative therapy to support persistent antitumor immunity and increase PFS, especially in patients who responded to treatment without achieving an MRD-negative status. Disclosures Huff: Member of Safety Monitoring Board for Johnson and Johnson: Membership on an entity's Board of Directors or advisory committees; Karyopharm, Sanofi, MiDiagnostics: Consultancy. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. Gittelman:Adaptive Biotechnologies: Employment, Other: Financial Interest. Johnson:Adaptive Biotechnologies: Employment, Other: Financial Interest. Ali:Celgene: Research Funding; Poseida: Research Funding. Noonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX. Borrello:BMS: Consultancy; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau.

Major histocompatibility complex (MHC) class I presentation of exogenous antigens is the mechanism enabling professional antigen-presenting cells (APCs) to induce CD8+ T-cell responses against viruses and tumors that do not have access to the classical MHC class I pathway. We have characterized the uptake, processing, and MHC class I cross-presentation by human dendritic cells (DCs) of cell-associated antigens derived from physiologically relevant sources, namely, vaccinia virus-infected apoptotic and necrotic cells. We show that cross-presentation is a rapid process, detectable within 2 to 4 hours after uptake of dead cells, and that proteolysis by cathepsin D in an acidic endosomal compartment is essential for cross-presentation. The presentation is abolished when the phagocytic or macropinocytic functions of the cells are inhibited and is dependent on transporter associated with antigen processing, sensitive to brefeldin A, and requires functional proteasomes. Altogether, these data suggest that antigens derived from apoptotic and necrotic cells require access to the cytosol to intersect with the conventional MHC class I pathway for presentation of cytosolic proteins. (Blood. 2003;102:4448-4455)

BACKGROUND. The detection of minimal residual disease (MRD) at the level of 0.01%/10-4 or above is a strong independent predictor of reduced progression-free (PFS) and overall survival (OS) in patients with CLL treated with chemoimmunotherapy. Although newer agents such as B-cell receptor pathway inhibitors can result in prolonged survival without achieving complete response, there remains a important role for MRD analysis in assessing therapeutic strategies aimed at disease eradication and cure. This is particularly important in front-line trials for fit patients which now require at least five years of follow-up if PFS is used as an endpoint. The feasibility of using MRD as a surrogate or intermediate endpoint for accelerated approval of new treatments is under review by regulatory agencies but further prospective validation is required. At the same time technology is rapidly evolving and high-throughput sequencing (HTS) technologies now detect MRD at the 0.0001%/10-6 level. It is therefore important to determine the most effective approaches for quantifying MRD that are compatible with previous studies but sufficiently sensitive for current treatments. AIMS. This collaborative project had two objectives. First, to identify the simplest and most flexible flow cytometry panel capable of detecting MRD at the 0.01%/10-4 or lower, that is compatible with published data and independent of instrument/reagent manufacturer. Second, to compare the flow cytometry approach with HTS analysis using the ClonoSEQ assay (Adaptive Biotechnologies, Seattle, WA). METHODS AND RESULTS. A core panel of antibodies for MRD detection was identified by testing an 8-marker combination in 52 samples (27 post-treatment and 25 dilution study) and re-analysing data with serial exclusion of single markers to determine redundancy. A 1-tube core panel of CD19, CD20, CD5, CD43, CD79b, and CD81 was identified and validated against the previously published 2-tube 6-marker and 4-tube 4-marker ERIC panels in 76 samples (19 post-treatment and 57 dilution study). The results showed good concordance (for log-transformed data above the LoQ, linearity=0.977, Pearson correlation co-efficient=0.983, average difference=0.026 log, 95% limit of agreement 0.20log) and a limit of detection of 0.001%/10-5 for the 1-tube core panel. Inter-operator variation was similar to CML MRD monitoring with both experienced operators, or inexperienced cytometrists after ~1 hour of specific education, achieving a 95% limit of agreement less than 0.3log in samples with MRD levels ranging from 0.0001 – 100%. The flow cytometry approach was compared with the ClonoSEQ HTS assay in 109 samples (21 dilution study and 88 post-treatment samples, complete data currently available on 13/88). The assay was applicable to the vast majority CLL patients, often with two clonal markers. There was 94% concordance at the 0.01% (10-4) threshold (15 samples with ≥0.01% CLL by both methods, 14 samples with CONCLUSIONS. The 1-tube 6-marker flow cytometry core panel is compatible with published studies, manufacturer-independent and flexible, providing directly quantitative results to 0.001%/10-5 without requiring a pre-treatment sample. HTS requires further work to standardise the quantitative analysis and prospective validation but the ClonoSEQ assay is applicable to >95% of CLL patients, does not require viable cells and is extremely sensitive, detecting residual disease in a significant proportion of cases with Disclosures Rawstron: Roche: Honoraria; Biogen Idec: Consultancy; Gilead: Consultancy, Honoraria; Abbvie: Honoraria; BD Biosciences: Intrasure reagent Patents & Royalties; Celgene: Honoraria; GSK: Honoraria. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Sanders:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Hallek:Celgene: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; GSK: Honoraria; Gilead: Honoraria. Hillmen:Roche: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

Abstract 4193 T cell repertoire diversity is generated by recombination of variable (V), diversity (D) and joining (J) segments in the T cell receptor (TCR) locus. Further variability and antigen recognition capacity is introduced by nucleotide insertion (NI) in the recombined sequences resulting in a complex repertoire, the organization of which is poorly understood. We postulate that TCR b D, J and V gene segment usage in an individual would result in a TCR repertoire with a fractal, self-similar frequency distribution of T cell clones with respect to gene segment usage. To determine this, the TCR repertoire of donors and recipients of HLA matched-related and unrelated allogeneic stem cell transplantation (SCT) was evaluated by high-throughput (HT) sequencing of the CDR3 region of TCR b. Ten SCT donor-recipient pairs were selected for HT-TCR b sequencing. cDNA was isolated from T cells obtained from the donors at baseline and recipients at day 100, 1 year post SCT or at the time of graft-versus-host disease (GVHD) diagnosis. HT-TCR β sequencing was performed and analyzed using the ImmunoSEQ analyzer tool (Adaptive Biotechnologies, Seattle, WA). TCR b clone frequencies were used to determine the TCR self-similarity score (SSS = log clone frequency × log scale), evaluating clonal frequency at each gene segment-scale (J, VJ and VJ+NI). This revealed a TCR SSS with a relatively narrow distribution of 1.64 ± 0.1 (mean ± SD) for J, 1.69 ± 0.2 for VJ, and 1.41 ± 0.01 for VJ+NI, which was consistent between all normal stem cell donors analyzed. Relative proportional distribution (RPD) graphs were then generated to allow for the depiction of the TCR b D, J, V distribution for simultaneous comparison at an individual level. Representative data in Figure 1 shows the relatedness across donors at the TCR b J segment level. Relative clonal frequency was determined for each unique TCR clone at the specified segment level and plotted according to frequency-rank. Slope of the resulting linear regression lines from log-log plots of rank-frequency was used to determine self-similarity and fractal dimension. These plots were comparable among donors with resulting slopes of 1.6 ± 0.01 and 1.8 ± 0.1, respectively for TCR J and VJ containing clones (Figure 2). The plots also revealed a hierarchy of T cell clones, with few dominant clones occupying the high ranks and a multitude of clones in the later ranks. For donor-recipient comparisons, we examined the dominant ranking clones which would be most likely to be involved in GVHD and response to infection. The ordered TCR clonal frequency distribution seen in donors was perturbed in recipients following SCT, with recipients demonstrating a lower level of complexity in their TCR repertoire, and a large shift in the frequency distribution of the dominant T cell clones compared to the donor. When dominant clones were compared between donors and recipients, recipients shared only a small proportion of TCR clonotypes with their donors despite full donor T cell chimerism. This difference did not change over time, suggesting an alternate T cell clonal hierarchy and repertoire develops in transplant recipients when compared with their donors. Using simple mathematical analysis, we demonstrate that the TCR b repertoire has a fractal, self-similar pattern with a hierarchy of dominant and minor clones. We note that the complexity of the TCR repertoire is diminished and TCR b hierarchy is altered following SCT. Restoration of this order may serve as a marker for post-SCT immune reconstitution. Further, by demonstrating shifts in TCR clonal dominance, fractal analysis comparing donor and recipient T cell repertoire may allow for more accurate monitoring of immunotherapy of malignancies in general, beyond allogeneic SCT. Figure 1. Graphical representation of self-similarity in TCR b J gene segment usage across segments and individuals. Relative-proportional-distribution (RPD) graph depicting TCR b J segment usage in ten hematopoietic stem cell donors. Each ring in the graph represents J segment usage frequency in a single donor, showing similarity in J segment frequency across ten different donors. Figure 1. Graphical representation of self-similarity in TCR b J gene segment usage across segments and individuals. Relative-proportional-distribution (RPD) graph depicting TCR b J segment usage in ten hematopoietic stem cell donors. Each ring in the graph represents J segment usage frequency in a single donor, showing similarity in J segment frequency across ten different donors. Figure 2. Log-Log plot depicting linear distribution of frequency ranked TCR b DJ (top panels) and VDJ (bottom panels) clones in two stem cell donors. Figure 2. Log-Log plot depicting linear distribution of frequency ranked TCR b DJ (top panels) and VDJ (bottom panels) clones in two stem cell donors. Disclosures: No relevant conflicts of interest to declare.