ALBERT

Description: Introduction: There is an unmet need for novel and effective treatments for lymphoma, the fifth most common malignancy in the US. In situ vaccination-an immunotherapeutic maneuver involving local irradiation, intratumoral (i.t.) injections of Flt3L and Toll-like receptor (TLR) agonist-has been shown in recent clinical trials (NCT00185965, NCT00880581, NCT00226993) to induce partial and complete remissions in patients with low-grade lymphoma. The strength of anti-tumor response correlates with the potency of immunogenic dendritic cells (DCs) to efficiently uptake and present tumor antigens to T cells. While the latest clinical trial NCT01976585 employs Poly-ICLC-a synthetic TLR3 agonist-to activate DCs, we hypothesize that "natural" TLR agonists (nTLRa) contained within prophylactic vaccines (e.g. live/attenuated bacteria or viruses) could simultaneously target multiple TLRs and be repurposed as clinical-grade DC activators for the in situ vaccination maneuver. Methods: Twenty-four prophylactic vaccines from our hospital's pharmacy were screened using in vitro assays for TLR ligand activity. DC subsets CD11c+ MHC-II+, CD11c+ CD11b+, and CD11c+ CD103+ were gated for flow-cytometric readouts of: (i.) activation markers (e.g. CD40, CD80, CD86, MHC class I and II), (ii.) ability to co-stimulate T cells in the context of TCR activation, as assessed by T cell activation markers (e.g. CD69, intracellular IFNγ), and (iii.) ability of nTLRa-activated DCs to induce T cell proliferation. TLR knock-out cell lines were used for dissecting the mechanistically-distinct properties of each prophylactic vaccine. Best single and combination nTLRa candidates were evaluated with the in situ vaccination maneuver in an in vivo A20 murine lymphoma model. Results: We identify a combination of vaccines Typhim, BCG-TICE, and MMR that demonstrates the ability to induce high expression of costimulatory molecules on DCs (Figure A) in vitro, as well as possessing the ability to co-stimulate in vitro T cell activation and proliferation. DC costimulation of T cell activation and proliferation is highly dependent on the "live" status of several vaccines, including BCG, MMR, Zostavax, which suggests the involvement of other pattern-recognition receptors in addition to TLRs. Cohorts of mice implanted with A20 lymphoma tumors that received i.t. injections of Typhim, BCG, MMR demonstrated slower A20 growth status post in situ vaccination maneuver, as compared to the control cohort receiving only Flt3L and irradiation (XRT). Conclusion: Prophylactic vaccines contain natural ligands to TLRs and are immediately translatable as sources of clinical-grade stimulators of dendritic cells. Combinations of the best nTLRa candidates display synergistic activation of DCs and demonstrate in vivo anti-A20 murine lymphoma immunity. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Introduction MCL has a poor prognosis. In eligible patients, intensifying frontline, CHOP-like regimens (e.g., cytarabine) as well as high-dose chemotherapy and autologous stem cell transplant (HDT/ASCT) consolidation in first remission have improved progression free survival (PFS) but less so, overall (OS). Preclinical animal models show benefits of adding tumor-specific T-cells to ASCT. CpG (PF-3512676) is a toll-like receptor 9 (TLR9) agonist and an effective vaccine adjuvant that induces costimulatory molecule expression on both antigen-presenting and MCL cells. This phase I/II clinical trial (NCT00490529) adds autologous T-cell transfer, harvested from patients after vaccination with CpG-activated autologous MCL cells - a maneuver termed immunotransplant. This is a planned interim analysis for safety and efficacy triggered by the first 20 patients reaching 1 year post ASCT. Methods Prior to therapy, subjects' tumor cells are collected by biopsy or apheresis and patient-specific vaccine is created by incubating fresh tumor cells with CpG (3 mcg/mL PF-3512676, 72-hr culture), then irradiated and cryopreserved. Patients receive induction chemoimmunotherapy of physician's choice. Patients achieving at least partial response (PR) then receive 3 subcutaneous autologous tumor vaccinations (1 x 108 cells/dose) mixed with CpG (18 mg) every 4-7 days. Primed T-cells (≥ 1 x 1010 CD3 cells) were collected by apheresis 2-4 weeks following vaccine 3 and a rituximab (375 mg/m2) B-cell purge. After standard HDT/ASCT (conditioning = BCNU, cyclophosphamide, etoposide), primed T-cells and a 4th vaccination are given on day+1. A 5th CpG-MCL vaccination followed 3 months post ASCT. The primary endpoint of this study is freedom from minimal residual disease (MRD) at 1 year post ASCT, measured by presence of patient-specific, malignant B-cell VDJ sequence in peripheral blood (ClonoSeq™, analyzed at a sensitivity of ≥ 1 clone/10,000 leukocytes) -an endpoint previously shown to be highly prognostic. Secondary endpoints include PFS, OS, and immune response to vaccine. 59, transplant-eligible, MCL patients are targeted for accrual in this 2-stage design. Results In this interim analysis of 24 patients accrued, 20 have surpassed 1 year post ASCT. All patients had Stage IV disease. Median values included (range): follow-up 43.5 months (11.5-60.1), age 60 years (47-70), and MIPI score 5.9 (5.1-7.8). Vaccine was made from biopsy alone (n=12), apheresis alone (n=9), or both (n=1). Frontline therapy included R-CHOP (n=7), R-hyperCVAD (n=14), alternating R-CHOP/R-DHAP (n=2), and R-EPOCH (n=1). 19 patients achieved complete response while another 3 had PR. All responding patients were vaccinated, able to yield sufficient T-cells for adoptive transfer, and proceeded through standard HDT/ASCT. At 1-year post ASCT, freedom from MRD was 90.5% (n=21). 2 patients did not reach 1-year post ASCT. One died from an ASCT complication (metapneumovirus) while the other relapsed 6 months following ASCT (included in MRD analysis). The most common toxicity due to CpG-MCL vaccine was erythematous rash at injection site (90.9%, n=20, each grade 1). No serious adverse events were seen related to vaccines or adoptive T-cell transfer. All patients had successful hematopoietic recovery, but two were delayed and received backup stem cell infusions with eventual recovery. Though median PFS and OS have not been reached, 3-year PFS and OS at interim analysis are 54.5% and 63.6%, respectively (intention to treat). In this data set, higher expression of co-stimulatory molecules (such as CD40, CD80, and CD86) on a subject's MCL in response to CpG was associated with freedom from MRD (p =0.02). Post-ASCT, higher peripheral T-cell granzyme and perforin response to ex vivo re-challenge with autologous MCL cells was also associated with freedom from MRD (p =0.01). Conclusions The addition of CpG-activated, whole MCL vaccination and autologous, adoptive T-cell transfer to standard therapy appears both feasible and safe. At interim analysis, the 1-year freedom from MRD surpasses rates previously reported in other studies and warrants further investigation. To date, 46 patients have either completed and/or are continuing to undergo study treatment and the study remains open to accrual for patients newly diagnosed with MCL. Disclosures Miklos: Pharmacyclics: Research Funding. Rezvani:Pharmacyclics: Research Funding. Faham:Adaptive Biotechnologies: Employment. Levy:Immune Design: Research Funding; Dynavax: Research Funding.

Description: Background Low-grade non-Hodgkin's B-cell lymphomas are generally incurable, with standard therapies inducing only temporary remissions. Preliminary results with anti-PD-1 therapy have yielded low response rates, though tumor-targeted vaccines represent promising, novel treatment strategies. In a pre-clinical mouse model, we attempt to develop and optimize an in situ vaccine combining recruitment of dendritic cells (DC) and low-dose local radiotherapy (XRT) with intratumoral (i.t) administration of a toll-like receptor (TLR) agonist Methods A20 lymphoma-bearing mice were injected i.t. with FMS-like tyrosine kinase-3 ligand (Flt3L) daily for 9 days (30ug/mouse), followed by local XRT (9Gy) and i.t. injections of poly-ICLC (50ug/mouse) for 5 days. Leukocyte accumulation in tumors, lymph nodes, and spleens was analyzed by flow cytometry and animals were monitored for tumor growth and survival. To assess uptake of tumor antigens by DC, mCherry-expressing A20 cells were used. For assessment of systemic anti-tumor response tumors were inoculated on both flanks, but only one site was treated as described before. In some groups, anti-PD-1 blocking antibody was injected systemically during vaccination. Results Injection of Flt3L induced potent accumulation of DC at the tumor site, tumor-draining lymph node (TDLN) and the spleen, with intratumoral injection being superior to systemic injection in increasing intratumoral and TDLN DCs. Interestingly, Flt3L-treatment led to an 8-fold increase in TLR3+ DC in the tumor. Local XRT increased the amount of mCherry+ DC in the tumor, indicating enhanced uptake of dying tumor cells. XRT of A20 cells also induced activation of Flt3L-treated splenic DC in vitro. While combination of FLt3L and local XRT was not able to cure established tumors, the combination of Flt3L and XRT with poly-ICLC induced long-lasting tumor regression in 40% of mice as well as regression of untreated tumors. This was accompanied by induction of tumor-reactive, Interferon γ (IFN γ)-producing T cells. Of note, the combination of Flt3L and XRT increased expression of PD-1 and PD-L1 on tumor infiltrating T cells and tumor cells, respectively. Consistently, systemic treatment with a PD-1 blocking antibody significantly enhanced the efficacy of the Flt3L-primed in situ vaccine leading to complete tumor regression at the treated site and a significant survival benefit compared to the in situ vaccine without PD-1 blockade. PD-1 blockade also increased the number of tumor-reactive T cells. Conclusions In situ vaccination combining intratumoral Flt3L injection with local XRT, poly-ICLC and anti-PD-1 induces a potent anti-tumor immune response able to induce long-term regression of established lymphoma tumors. Disclosures Davis: Celldex Therapeutics: Employment. Keler:Celldex Therapeutics: Employment, Equity Ownership. Salazar:Oncovir Inc: Employment. Brody:Gilead: Honoraria, Other: Travel expenses, Speakers Bureau; Acerta Pharma: Research Funding; Immunogen: Equity Ownership; Pharmacyclics: Honoraria, Other: Travel expenses, Speakers Bureau; Novavax: Equity Ownership; Merck: Consultancy, Research Funding; Seattle Genetics: Consultancy; Synergy Pharmaceuticals: Equity Ownership.

Description: Background Autologous transplant has been shown to prolong survival in patients with MCL, a disease which is known to have poor long-term survival. The purpose of this clinical trial is to reduce the relapse rate and prolong survival through the induction of anti-tumor T cells which can effectively survey, expand and eradicate tumor cells when and if they arise. In a murine model, T cells from CpG-tumor vaccinated donors were shown to expand preferentially in recipient mice following a myeloablative regimen and were effective in eradicating tumor in these mice (Goldstein, M. et al., Blood 2011). Consequently, we developed a clinical trial where patients with mantle cell lymphoma (MCL) are given a CpG-based cellular vaccine followed by an autologous stem cell transplant (Immunotransplant). T cells, harvested after vaccination prior to transplant, are reinfused post-transplant. Blood samples are taken prior to vaccine, post the initial vaccination, as well as post immunotransplant to study the CD4 and CD8 anti-tumor T cell responses. Blood samples are also drawn pre and 2 weeks post a vaccine boost given approximately 6 months post-transplant to assess the memory component of the anti-tumor T cells. Methods Peripheral blood lymphocytes (PBL) were cultured for 5 days with and without autologous CpG activated tumor. T cells were re-stimulated over-night with fresh activated tumor then analyzed by flow cytometry for phenotype (CD4+ T cells, CD8+/CD56- T cells and memory marker, CD45RO) as well as functionality (cytokine expression [IFN-g, TNF and IL-2], cytolitic activity [perforin and granzyme B expression] and activation markers, CD137 and ICOS [CD278]). In several cases, tumor-responsive T cells from co-cultures were sequenced to determine their TCRb repertoire. Clonotypes enriched over those found in PBL drawn pre-vaccine were deemed to be antigen-specific. These clones were compared to ones enriched directly in the blood after the original vaccine and later boost. Results To date, 21 MCL patients have been enrolled and have received the immunotransplant as well as final vaccine boost. 15 patients have been analyzed and have shown anti-tumor T cell responses following vaccine and immunotransplant. In most cases, the T cell responses were boosted post immunotransplant suggesting an expansion of anti-tumor T cells as predicted by the murine model. The phenotype of the responding T cells (CD4, CD8, or both CD4 and CD8) varied between patients. (Figure 1.) Also, in 11/15 patients, tumor-responsive T cells were still present at the time of the final vaccine, about 6 months post-transplant. The final vaccine boosted T cell responses, mostly in the CD8 compartment, suggesting an induced memory anti-tumor T cell response, which is durable over time. (Figure 2.) Finally, TCRb clonotypic analyses of the responding T cells demonstrated that these T cell clones were enriched in the blood post vaccine providing further proof that they were vaccine-induced. Conclusion Immunotransplant can induce and expand a population of tumor-responsive memory T cells, both CD4 and CD8, which can be detected as an expansion of specific TCRb clonotypes in whole blood. This response is durable over time and can be boosted suggesting a means by which the immune system can be enticed to maintain anti-tumor surveillance and eradicate tumor cells in the future. Disclosures: Faham: Sequenta, Inc.: Employment, Patents & Royalties.

Description: Background A clinical trial is ongoing at Stanford for MCL patients in first remission that interdigitates an autologous CpG-stimulated tumor cell vaccine with autologous peripheral blood stem cell transplant (PSCT) (NCT00490529). In this trial, blood samples collected before and after vaccination and serially post-transplant are assayed for minimal residual disease (MRD) and for T cell repertoire using the LymphoSIGHT™ sequencing method (Faham et al., Blood 2012). We identified a set of T cell clones that appear to be responding to the vaccine, and therefore we investigated whether the number of these clonotypes was correlated with MRD status. Methods Using universal primer sets, we amplified rearranged IgH variable (V), diversity, and joining (J) gene segments from genomic DNA. Amplified products were sequenced to obtain 〉1 million reads. The B cell tumor-specific sequence was identified for each patient based on its high frequency in the original tumor biopsy. The presence of the tumor cells was then monitored in serial blood samples with a sensitivity of 1 cell per million leukocytes. The same blood samples were used for amplification, sequencing and analysis of the entire TCRβ repertoire. To facilitate identification of tumor vaccine-induced TCRβ clonotypes, we sequenced the TCRβ repertoire immediately before and after the administration of both the priming vaccination and a booster vaccination. We developed a metric called the vaccine response score (V score). This metric is calculated for each clonotype and reflects the increase in frequency after the initial vaccination AND after the boost. The formula for calculating V score is: V = F1 x F2 x square root [1/ (|F1 – F2| + 1)], where F1 and F2 represent the fold-change of the priming and boost vaccinations, respectively. Clonotypes with a V score 〉10 were deemed to be vaccination-induced by virtue of these frequency changes. Results In a series of 12 vaccinated patients, the number of clonotypes with V score ≥ 10 ranged between 0 and 262, with a median of 57. We utilized an antigen-specific analysis to validate that clones with high V scores (≥ 10) were in fact tumor-specific. For this analysis, we incubated peripheral blood mononuclear cells (PBMCs) with the tumor and then sequenced the TCRβ repertoire from cells obtained after culture. Clones that were enriched after culture compared to pre-stimulation PBMCs were deemed to be antigen-specific. These clones that are antigen-specific are highly likely to have a high V score compared to a random frequency-matched set of clones (P two tailed = 1.8 x 10-10), providing further evidence that clones with a high V score are tumor-specific. We then analyzed the relationship between V score and clinical outcome. Patients could be stratified into two groups with “high” (〉 25) or “low” (

Description: Abstract LBA-2 Background: CD4+CD25+FOXP3+ regulatory T-cells (Tregs) protect cancers from the immune system. They express important functional molecules such as CTLA-4 and Ox40 on their surface. Anti-CTLA4 monoclonal antibody (mAb) is the first drug to improve survival of refractory metastatic melanoma. Its recent approval by the FDA/EMEA inaugurates a paradigm shift in cancer therapy where the immune system is targeted rather than the tumor itself. Scientific question: Does local immunomodulation of tumor specific Tregs trigger a systemic anti-tumor immune response and cure disseminated lymphoma? Results: The Tregs that infiltrate the tumor site preferentially express CTLA4 and OX40 compared to their counterparts in the blood and other lymphoid organs, both in mice and in humans. Upon injection of low doses of anti-CTLA4 and anti-OX40 together with CpG, a TLR-9 agonist, directly into the tumor, tumor-specific Tregs are depleted from the tumor-infiltrate. This immunomodulatory combination therapy triggers an anti-tumor immune response able to cure mice with established disseminated disease. The triple combination is uniquely required as neither CpG alone nor mAbs without CpG are effective. The local Treg immunomodulation with anti-CTLA4+anti-OX40+CpG in a single sub-cutaneous tumor eradicates disease in mice with established CNS lymphoma, even with leptomeningeal and spinal cord metastases, whereas chemotherapies or mAb therapy directed against the tumor cells have little effect in the CNS site. Moreover, these cured mice have a long term intra-cranial anti-tumor immunity since they are protected against late intra-cranial re-challenge. Significance: Immunomodulatory antibodies are currently under clinical development for cancer treatment, and their major side effect is the triggering of auto-immune diseases. We show here that injecting very little doses of these antibodies in combination with CpG at one tumor site is sufficient to trigger a systemic anti-tumor response able to eradicate distant sites, including in the CNS which is usually considered a sanctuary site for conventional systemic therapies. Impact: We recently have published positive results of intra-tumoral CpG in patients with follicular Lymphoma (Brody,Levy, et al. JCO, 2010). Anti-CTLA4 has just been FDA approved in patients with metastatic melanoma, and anti-Ox40 antibodies are currently being tested in phase I/II clinical trials. Therefore, the combination described here can be tested in patients with injectable sites of lymphoma, even if they have CNS disease. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND: Lymphomas are the 5th most common cancer in the U.S. and most are incurable with standard therapy. Previously, we completed three trials of ‘in situ vaccination’ - combining low-dose radiotherapy (XRT) with intratumoral administration of TLR9 agonist (CpG). We demonstrated induction of anti-tumor CD8 T cell responses and clinical remissions of patients’ non-irradiated sites of disease, lasting up to 4+ years. One limitation may have been the paucity of intratumoral dendritic cells (DC). DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells. METHODS: Flt3L– the predominant DC differentiation factor– induces tumor leukocyte infiltration and regression of lymphoma tumors pre-clinically and a new formulation of this cytokine -CDX-301- was shown to mobilize BDCA-1 and BDCA-3 DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DC (the CpG-responsive DC subset). We initiated a phase I/II study of a new iteration of the in situ vaccine, adding Flt3L-priming and replacing the prior TLR9 agonist with the TLR3 agonist poly-ICLC (Fig 1A). The vaccine consists of: -intratumoral Flt3L administration to increase DC within the tumor -low-dose XRT to induce immunogenic tumor cell death and release tumor-associated antigens, and -intratumoral poly-ICLC administration to activate tumor antigen-loaded DC. RESULTS: Six patients have been enrolled, two patients have completed therapy. Treated patients had 2-200-fold increases in BDCA1 and BDCA3 intratumoral DC after Flt3L administration and marked DC activation after XRT and poly-ICLC. Both treated patients have had partial remissions of untreated sites per Cheson criteria, persisting or improving for 〉6 months after vaccination. These include regressions of bulky lymph nodes (Fig 1B), as well as peripheral blood (Fig1C) and bone marrow disease. A patient with significant peripheral blood tumor burden experienced 〉10-fold decrease in malignant B cells with concurrent increase in non-tumor B cells, suggesting a degree of cell specificity in the tumor-killing mechanism. Adverse effects have been mild. CONCLUSIONS: Preliminary results suggest that the Flt3L-primed in situ vaccine is feasible, safe and immunologically and clinically effective, warranting further study. Figure 1 Figure 1. Disclosures Off Label Use: CDX-301 - the purpose of which is to mobilize dendritic cells to an injected tumor site.. Crowley:Celldex Therapeutics Inc: Employment. Keler:Celldex Therapeutics, Inc.: Employment. Davis:Celldex Therapeutics Inc: Employment.

Description: Abstract 144 Introduction: Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma. Although newer treatment regimens including intensive chemo-immunotherapy followed by stem cell transplant have improved survival to a median approaching 6 years, MCL is still incurable in many cases. The use of flow cytometry to investigate stimulation induced signaling at the single cell level represents an opportunity to obtain each patient's signaling profile as well as to discover tumor cell heterogeneity and identify signaling events potentially responsible for poor outcomes. We recently used this method to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a lymphoma negative prognostic (LNP) cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome (Irish et al., PNAS 2010). In the present study we used the same approach to identify signaling responses with large variation within MCL patient samples, and tested whether these signaling events might predict patients' clinical outcomes. By understanding the biology based on lymphoma signaling profile, we might identify prognostic significant factors such as the LNP subset observed in FL. Method: Single cell flow cytometry measurements of signaling were acquired for samples of MCL (n=25). Of these, 21 MCL specimens were obtained prior to any treatment. Median age was 61 years, and follow up time ranged from ½ until 15 years. Treatment regimens varied. Samples from diffuse large B cell lymphoma (DLBCL, n=12), FL (n=14), chronic lymphocytic leukemia (CLL, n=14) and tonsils from healthy donors (n=4) were also included for comparison. Phosphorylation of 14 signaling proteins was measured under 12 different stimulation conditions in every cell within the patient specimens, including lymphoma B cells and in tumor-infiltrating T cells. Stimulation conditions included BCR crosslinking, CD40 ligand, CpG oligodeoxynucleotides (CpG ODN) and several cytokines (IL-4, IL-10, IL-21, IFN-γ). Result: The magnitude of most cytokine induced signaling responses in lymphoma cells from MCL patients was higher than in lymphoma cells from FL. This included IL-10 induced phosphorylation of STAT3 (p

Description: Key Points B-cell–specific overexpression of SOX11 promotes oncogenic proliferation of B1a B cells and drives an MCL-like phenotype. SOX11 overexpression is associated with increased signaling through the BCR pathway that can be reversed by pharmacological BTK inhibition.

Description: Abstract 3068 Mantle cell lymphoma (MCL) has a poor long-term prognosis. Though autologous transplant prolongs survival, novel and mechanistically distinct therapies are needed to target residual, myeloablation-resistant tumor cells that result in relapse. Trials of CpG-based vaccines for low-grade lymphoma have shown induction of anti-tumor T cells and clinical responses [Brody J. et al, J Clin Oncol. 2010 Oct 1;28(28) :4324–32]. In a pre-clinical model, we developed the immunotransplant maneuver combining: 1) CpG-based vaccination, 2) harvest of vaccine-primed T cells, 3) myeloablation with stem cell rescue, and 4) T cell re-infusion. Immunotransplant amplifies the proportion of anti-tumor T cells by an order of magnitude and cures even bulky, systemic lymphoma burden [Brody J. et al, Blood. 2009 Jan 1;113(1) :85–94]. METHODS: We initiated a phase I/II study of immunotransplant for newly diagnosed MCL patients to test the hypothesis that immunotransplant will amplify anti-tumor T cells as in the pre-clinical model. Anti-tumor T cells are assessed by co-culturing autologous tumor with peripheral blood T cells and measuring their production of: IFNg, TNF, IL2, CD137, perforin and granzyme by multiplex surface and intracellular flow cytometry. A secondary endpoint is measurement of molecular residual disease (MRD) using both standard allele-specific oligonucleotide (ASO) qPCR as well as high-throughput sequencing (HTS) of the entire IgH repertoire. The study is powered to detect a 50% improvement in sustained molecular remission rate compared to recent trials of standard transplant [Pott C. et al, Blood 2010 Apr 22;115(16) :3215–23, Geisler C. et al, Blood 2008 Oct 1;112(7) :2687–93]. Using the same HTS technology, we have also initiated studies of the entire TCR β repertoire as an alternate approach of tracking the amplification of vaccine-induced T-cells. RESULTS: Accrual has been rapid with 25 patients enrolled in 22 months and 13 patients completing the complete protocol so far. Flow-cytometric immune response testing has demonstrated that immunotransplant amplifies the proportion of tumor-reactive T cells in 83% of patients thus far. Notably, we have observed some patients with primarily CD8 T cell responses, some with CD4 T cell responses, and some with a combination of the two. In some cases, tumor-reactive T cells have been tested for reactivity to autologous, non-malignant B cells and have demonstrated a significant proportion that are tumor-specific. TCR β repertoire sequencing has also demonstrated instances of significant clonal amplification after immunotransplantation, some exceeding three orders of magnitude. In extreme cases, these have yielded dominant clones comprising as much as 50% of a patient's entire peripheral blood T cell repertoire post-transplant. HTS of the IgH repertoire has been an effective measurement of MRD bypassing the assay individualization of ASO qPCR and has been shown to be more sensitive than conventional flow cytometry. CONCLUSIONS: Pre-clinically, amplification of anti-tumor T cells correlates with cure of even myeloablation-resistant disease. The reiteration of anti-tumor T cell amplification in our preliminary patient data raises the possibility that immunotransplant may improve clinical outcomes. Ongoing MRD testing should suggest whether certain patterns of T cell response –measured functionally per flow cytometry or clonally per HTS- correlate with clinical benefit and whether the cohort has a better-than-expected molecular remission rate. Disclosures: Moorhead: Sequenta, Inc.: Employment. Zheng:Sequenta, Inc.: Employment. Klinger:Sequenta, Inc.: Employment. Faham:Sequenta Inc: Employment, Equity Ownership. Advani:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding.