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Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)–directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Chimeric antigen receptor T (CART) cell immunotherapy has yielded significant clinical success in treating certain hematological malignancies. However, despite high initial response rates, most patients eventually relapse. Resistance to CART cell therapy can stem from tumor cell mutations, T cell defects, and tumor microenvironment (TME) immunosuppression. Tumor cells can downregulate target antigen expression to evade CART cell detection or mutate death receptor pathways to resist CART cell cytotoxicity. Patient T cells can be intrinsically defective, and CART cells often undergo exhaustion. The TME is abundant with immunosuppressive cells and factors which contribute to suboptimal CART cell activity. Collectively, issues originating in tumor cells, T cells, and the TME present significant hurdles to long-term remission after CART cell therapy. Various strategies to combat CART cell resistance have shown promise in preclinical studies and early clinical trials and are crucial to achieving durable responses.

Chimeric antigen receptor T (CART) cells are a promising immunotherapy that has induced dramatic anti-tumor responses in certain B cell malignancies. However, CART cell expansion and trafficking are often insufficient to yield long-term remissions, and serious toxicities can arise after CART cell administration. Visualizing CART cell expansion and trafficking in patients can detect an inadequate CART cell response or serve as an early warning for toxicity development, allowing CART cell treatment to be tailored accordingly to maximize therapeutic benefits. To this end, various imaging platforms are being developed to track CART cells in vivo, including nonspecific strategies to image activated T cells and reporter systems to specifically detect engineered T cells. Many of these platforms are clinically applicable and hold promise to provide valuable information and guide improved CART cell treatment., Graphical abstract, Imaging and tracking adoptively transferred T cells would allow the in vivo characterization of T cell expansion and trafficking to tumor sites. Sakemura and colleagues review the current state-of-the-art modalities for in vivo T cell imaging and their role in the early prediction of outcomes or detection of toxicities.

Introduction: PD-1 inhibits the cytotoxic T-cell functions via the interaction with its ligands (PDL1 and PDL2). Recently, our group showed a selective response (~40%) with PD-1 blocking antibody pembrolizumab in patients with Richter transformation (RT), particularly after prior exposure to ibrutinib (Wei Ding et al, Blood, 2017). Additionally, PD-1 showed an increased expression in tumor B-cells of patients with RT (Rong He et al, AJSP, 2018). Here we investigated the functional implications of the PD-1 signaling axis in lymphoma B-cell pathobiology. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN samples were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). Then, we checked PD-1 expression in 10 lymphoma cell lines and 1 CLL cell line (MEC-1) by both, flow cytometry and Western blot (WB) analysis. Over-expression of PD-1 using pLEX-lentiviral system (Thermo Scientific) was evaluated for in vitro cell cycle regulation and in vivo tumor growth. Overexpression of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Gene expression signatures in CLL and RT patients were also identified by Illumina-based RNA sequencing using Formalin-Fixed Paraffin-embedded (FFPE)-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p < 0.001) [Fig 1A]. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in lymphoma B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable, but at very low-levels in lymphoma lines OCI-LY7 and OCI-LY19 (data not shown). Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold compared to original lines on day 4, respectively, Fig 1BI-II), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% and 24.53% in OCI-LY7 and OCI-LY19 cells (Fig 1B III), respectively. When the luciferase + PD-1 expressing OCI-LY7 cells were injected intravenously (1X106 cells) into NSG mice, increased tumor growth was observed at 22 days of follow up by bioluminescence imaging (p Conclusion: Our data support the notion that PD-1 overexpression in lymphoma cells modifies cell proliferation in vitro and in vivo and regulates the function of phosphatase SHP and p53- pathways. These findings also indicate that aggressive lymphoma or CLL leukemic cells have the ability to hijack the PD-1 pathway and may result in downregulation of the p53 mediated DNA repair. This novel information provide for new strategies to further evaluate the interaction of checkpoint signals with DNA repair pathway, and possibly provide novel targets for Richter's transformation. Disclosures Ding: alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; MEI Pharma: Membership on an entity's Board of Directors or advisory committees. Sakemura:Humanigen: Patents & Royalties. Parikh:Janssen: Honoraria, Research Funding; Merck: Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Liu:Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ansell:Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Bristol Myers Squibb: Research Funding. Kenderian:Sunesis: Research Funding; Tolero: Research Funding; Torque: Consultancy; BMS: Research Funding; Gilead: Research Funding; Kite: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Juno: Research Funding. Kay:Acerta Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.

With the development of various strategies of anti-CD19 immunotherapy for treatment of B cell malignancies, it remains to be elucidated whether CD19 targeting with a monoclonal antibody impairs subsequent CD19 targeted chimeric antigen receptor T cell (CART19) therapy. In this study, we evaluated the potential interference between the CD19 targeting monoclonal antibody tafasitamab and CART19 treatment (using a construct similar to the FDA approved therapy, FMC63-41BBζ, tisagenlecleucel) in preclinical lymphoma and leukemia models. For in vitro assays, the CD19 + cell lines JeKo-1 (mantle cell lymphoma), Nalm6 (acute lymphoblastic leukemia), and OCI-Ly7 (diffuse large B cell lymphoma) were used. When CART19 were co-cultured with CD19 + tumor cells in the presence of increasing concentrations of tafasitamab (10-400 µg/mL) or the isotype control, an impairment of CART19 functions at the highest concentration of tafasitamab was observed. However, when tafasitamab was cocultured with tumor cells overnight and removed from the culture by washing, there was no impairment of CART19 anti-tumor activity or effector functions compared to a pre-culture with isotype control. We then aimed to validate these findings in xenograft mouse models by determining whether prior tafasitamab treatment impairs subsequent CART cell function. First, a two-step JeKo-1 xenograft model was created through inoculation of 1x10 6 luciferase + JeKo-1 cells to immunocompromised NOD-SCID-γ -/- (NSG) mice via tail vein injection. One week later, tumor burden was assessed by bioluminescence imaging (BLI) and mice were randomized to receive either 1) PBS (Group 1) or 2) 10 mg/kg of tafasitamab (Group 2) (Fig. 1A). Treatment was performed until the mice reached the endpoint due to the high tumor load. When reaching the endpoint, mice were euthanized and spleen was harvested. Splenic cells were then injected to naïve NSG mice. Two weeks after the inoculation of splenic cells (PBS or tafasitamab treated), mice were imaged with BLI and randomized according to their tumor burden to receive 2x10 6 of 1) untransduced T cells (UTD) or 2) CART19 (i.v.) (Fig. 1B). Mice, treated with CART19 demonstrated potent anti-tumor effects and showed prolonged survival compared to UTD control mice. Interestingly, CART19-treated mice inoculated with Group 2 splenic cells (pretreated with tafasitamab) showed a significantly better tumor control and overall survival compared with mice engrafted Group 1 splenic cells (PBS pretreatment) (Fig. 1C). This finding led us to further investigate the effects of prior tafasitamab therapy on subsequent CART19 treatment. In this context, we studied the sequential treatment with tafasitamab and CART19 in an additional JeKo-1 xenograft model (Fig. 1D). In brief, NSG mice were inoculated with luciferase + JeKo-1 on day -14. On day -8, mice were imaged and randomized to the PBS (Group I) or tafasitamab (10 mg/kg) group. On day -1 tafasitamab treated mice were imaged again, randomized to the tafasitamab discontinuation (Group II) or continuation (Group III). Treatment with PBS was continued in Group I. All groups were administered with 1x10 6 of CART19 on day 0. Cotreatment of JeKo-1 xenografts with tafasitamab and CART19 led to impaired anti-tumor activity presumably as a result of CD19 binding competition between tafasitamab and CART19 (Group III). Mice treated with tafasitamab followed by CART19 had improved tumor control compared to mice treated with PBS followed by CART19 (Fig. 1E-F). Serial peripheral blood analysis demonstrated that mice from Group I showed the highest CART19 expansion on day 10. Strikingly, Group II exhibited weak CART19 expansion at day 10, but strongest expansion on day 23 (Fig. 1G). We therefore hypothesized that prior treatment with tafasitamab results in modulation of CART19 cell activation. To test this hypothesis, spleens of satellite mice were harvested 24 hours after CART19 administration. Flow cytometric analysis revealed that the mice from Group II showed significantly lower CD69 and HLA-DR expression on CART19 cells compared with Group I, and reduced apoptosis as measured by the Annexin assay (Fig. 1H-I). In summary, concomitant treatment of tafasitamab and CART19 led to impaired anti-tumor activity while, in contrast, sequential treatment of tafasitamab and CART19 did not inhibit CART19 anti-tumor activity but rather promoted anti-tumor effects in xenograft models. Figure 1 Figure 1. Disclosures Sakemura: Humanigen: Patents & Royalties. Augsberger: MorphoSys AG: Current Employment. Schanzer: MorphoSys AG: Current Employment. Patra-Kneuer: MorphoSys AG: Current Employment. Heitmüller: MorphoSys AG: Current Employment. Steidl: MorphoSys AG: Current Employment. Endell: MorphoSys AG: Current Employment. Ding: DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Acerta Pharma: Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Sunesis: Research Funding. Nowakowski: Celgene, NanoString Technologies, MorphoSys: Research Funding; Celgene, MorphoSys, Genentech, Selvita, Debiopharm Group, Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cox: Humanigen: Patents & Royalties. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Owing to the difficulty in isolating T cells from human biopsy samples, the characteristics of T cells that are infiltratinghuman acute graft-versus-host disease (GVHD) tissues remain largely uninvestigated. In the present study, TCR-β deep sequencing of various GVHD tissue samples and concurrent peripheral blood obtained from transplant recipients was performed in combination with functional assays of tissue-infiltrating T cell clones. The T cell repertoire was more skewed in GVHD tissues than in the peripheral blood. The frequent clonotypes differed from tissue to tissue in the same patient, and the frequent clonotypes in the same tissue differed from patient to patient. Two T cell clones were successfully isolated from GVHD skin of a patient. In a cytotoxicity assay, both Tcell clones lysed patient peripheral blood mononuclear cells, but not donor-derived Epstein-Barr virus-transformed lymphoblastoid cells. Their clonotypes were identical to the most and second most frequent T cell clonotypes in the original GVHD skin and accounted for almost all of the skin-infiltrating T cells. These results suggest that human acute GVHD may result from only a few different alloreactive cytotoxic T cell clones, which differ from tissue to tissue and from patient to patient. The characterization of T cells infiltrating human GVHD tissues should be further investigated.

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1(+) CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.

BackgroundIn COVID-19, high levels of granulocyte macrophage-colony stimulating factor (GM-CSF) and inflammatory myeloid cells correlate with disease severity, cytokine storm, and respiratory failure. With this rationale, we used lenzilumab, an anti-human GM-CSF monoclonal antibody, to treat patients with severe and critical COVID-19 pneumonia.MethodsHospitalized patients with COVID-19 pneumonia and risk factors for poor outcomes were treated with lenzilumab 600 mg intravenously for three doses through an emergency single-use IND application. Patient characteristics, clinical and laboratory outcomes, and adverse events were recorded. All patients receiving lenzilumab through May 1, 2020 were included in this report.ResultsTwelve patients were treated with lenzilumab. Clinical improvement was observed in 11 out of 12 (92%), with a median time to discharge of 5 days. There was a significant improvement in oxygenation: The proportion of patients with SpO2/FiO2 < 315 at the end of observation was 8% vs. compared to 67% at baseline (p=0.00015). A significant improvement in mean CRP and IL-6 values on day 3 following lenzilumab administration was also observed (137.3 mg/L vs 51.2 mg/L, p = 0.040; 26.8 pg/mL vs 16.1 pg/mL, p = 0.035; respectively). Cytokine analysis showed a reduction in inflammatory myeloid cells two days after lenzilumab treatment. There were no treatment-emergent adverse events attributable to lenzilumab, and no mortality in this cohort of patients with severe and critical COVID-19 pneumonia.ConclusionsIn high-risk COVID-19 patients with severe and critical pneumonia, GM-CSF neutralization with lenzilumab was safe and associated with improved clinical outcomes, oxygen requirement, and cytokine storm.

Background Dasatinib has shown promising anti-leukemic activity against chronic myeloid leukemia (CML). However, patients receiving dasatinib frequently require dose reductions and treatment interruptions (treatment alteration). Methods We prospectively analyzed the frequency and significance of treatment alteration during dasatinib therapy in patients with CML. In all patients, trough plasma concentrations of dasatinib (Cmin) at steady state were assessed on day 28 of therapy. Results 28% of patients had their doses reduced at a median of 42 days, and 25% of patients had temporarily interrupted at a median of 54 days after treatment initiation. The overall dasatinib treatment alteration-free rate at 1 year was 66%. Age was significantly correlated with Cmin on day 28 (p = 0.014), and the correlation remained significant after adjusting dasatinib dose (g), body weight (kg) (Cmin/D/W) (p = 0.026). In the univariate analysis, deep molecular response, advanced PS, higher Cmin/D/W were associated with a significantly higher risk of treatment alteration (HR 4.19, 95% CI: 1.06–16.60, p = 0.041; HR 5.26, 95% CI: 1.33–20.80, p = 0.018; and HR 10.15, 95% CI: 2.55–40.48, p = 0.001, respectively). In the multivariate analysis, advanced PS and higher Cmin/D/W were correlated with the incidence of treatment alteration (HR 4.78, 95% CI: 1.01–22.70, p = 0.049; HR 6.17, 95% CI: 1.17–32.50, respectively). Conclusion Current data demonstrate that patients treated with dasatinib who displayed a high Cmin/D/W value and/or advanced PS were at a high risk for altered treatment.

Recent successes and FDA approvals of CD19 redirected chimeric antigen receptor T cell (CART19) therapy have been achieved in pivotal clinical trials in patients with B cell malignancies. However, durable response rates in non-Hodgkin B cell lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) are low. Tumor-associated macrophages have emerged as key mediators of tumor-induced CART cell immunosuppression. Axl receptor tyrosine kinase (Axl-RTK) is overexpressed in various human cancers including B-NHL or CLL and correlates with poor overall survival. Thus, several Axl-RTK inhibitors are currently being investigated in clinical trials. We have previously reported that the Axl-RTK inhibitor TP-0903 modulates CART19 function by Th1 polarization. Moreover, we observed a significant reduction in myeloid-derived cytokines in preclinical models. This led us to hypothesize that Axl-RTK inhibition also modulates monocytes which in turn affects CART function. To test our hypothesis, we interrogated the interactions between Axl-RTK inhibition, monocytes, and CART. First, we isolated CD14+ monocytes from peripheral mononuclear cells (PBMCs) from healthy donors by magnetic negative selection. Flow cytometric analysis revealed a robust Axl expression on monocytes (Fig.1A). Monocytes were maintained in ImmunoCult Macrophage Medium (STEMCELL Technologies) and differentiated with lipopolysaccharide and interferon-γ for M1-type and interleukin (IL)-4 for M2-type macrophages. After confirming the macrophage phenotypes, cells were assessed for Axl expression. Flow cytometric analysis demonstrated that M2-type macrophages expressed higher Axl compared to M1 macrophages (Fig.1B). Then, we treated PBMCs from healthy donors with TP-0903 for 24 hours to determine the specific cell types affected by Axl-RTK inhibition. Flow cytometric analysis revealed that monocytes were significantly inhibited by TP-0903 compared to B cells or T cells (Fig.1C). Having demonstrated that TP-0903 strongly suppresses monocytes and that M2-type macrophages express Axl on their surface, we then performed a cytotoxicity assay against CD19+ mantle cell lymphoma cell line, JeKo-1, to study the impact of Axl-RTK inhibition on monocyte-induced CART suppression. We used CART19 generated in our laboratory (FMC63-41BBζ). CART19, JeKo-1, and M2-type macrophages were co-cultured at 1:2:1 ratio and incubated for 3 days. The data revealed that M2-type macrophages strongly suppress CART19 killing (Fig.1D) which could be overcome with TP-0903 (Fig.1E). Next we studied monocyte-induced CART19 suppression with antigen-specific proliferation. CART19, JeKo-1, and monocytes were co-cultured at 1:5:1 ratio and incubated for 5 days. Flow cytometric analysis revealed that in the presence of monocytes, antigen-specific proliferation of CART19 was significantly suppressed, which was reversed in the presence of TP-0903 (Fig.1F). To assess how Axl inhibition modulates myeloid cell-derived cytokines/chemokines, we performed Multiplex (Millipore-Sigma) with the supernatants obtained from these co-culture assays. The presence of monocytes resulted in a significant increase in IL-17a, IL-6, IL-1 receptor agonist, IL-1β, and soluble CD40 ligand. However, Axl-RTK inhibition resulted in selective reduction of those cytokines/chemokines (Fig.1G) while preserving other effector cytokines. Finally, we tested whether Axl-RTK inhibition with TP-0903 could overcome the negative effects of monocytes in vivo. Here, immunocompromised NSG mice were injected with 1x106 luciferase+ JeKo-1 cells intravenously. Ten days later, mice were injected with freshly isolated monocytes derived from a healthy donor. Two weeks after JeKo-1 inoculation, tumor burden was assessed with bioluminescence imaging. Mice were then randomized according to the tumor burden to receive 0.5x106 CART19 as a monotherapy or in combination with 20 mg/kg/day of oral TP-0903. The combination therapy resulted in superior anti-tumor effects and overall survival compared to CART19 monotherapy (Fig.1H-I). In summary, Axl-RTK inhibition with TP-0903 demonstrates a direct suppression of inhibitory monocytes, which in turn enhances CART cell function. Our findings support the strategy of further refining and testing the novel and potent combination of Axl-RTK inhibition and CART cell therapy for the treatment of B cell malignancies. Figure 1 Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Mouritsen:Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Foulks:Tolero Pharmaceuticals, Inc.: Current Employment. Warner:Tolero Pharmaceuticals, Inc.: Current Employment. Ding:Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees. Parikh:GlaxoSmithKline: Honoraria; Verastem Oncology: Honoraria; Genentech: Honoraria; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding. Kay:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Abbvie: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees. Kenderian:Mettaforge: Patents & Royalties; Lentigen: Research Funding; MorphoSys: Research Funding; Sunesis: Research Funding; Tolero: Research Funding; BMS: Research Funding; Juno: Research Funding; Gilead: Research Funding; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents & Royalties, Research Funding.

Although CD19-directed chimeric antigen receptor T cell (CART19) therapy is highly effective and was FDA approved for certain B-cell malignancies, most patients relapse after CART infusion within the first 1-2 years due to inadequate CART expansion in vivo. Vesicular stomatitis virus (VSV) has the ability to infect and lyse cancer cells. Clinical trials of VSV oncolytic therapy indicate that VSV efficiently infects cancer cells as well as innate immune cells. Therefore, we hypothesized that in patients who achieve suboptimal response to CART19, VSV engineered to express CD19 will augment anti-tumor activity through 1) direct lysis of cancer cells and 2) infecting cancer cells and innate immune cells with CD19 to further stimulate CART19. To test our hypothesis, human CD19 or GFP (control) was engineered between the glycoprotein and large-protein (Fig.1A) in a modified VSV backbone. A matrix inactivating mutation (M51R) rendered it incapable of suppressing anti-viral reactions of infected targets, potentially promoting its immunogenicity. First, we tested the anti-tumor activity of VSV-CD19 and VSV-GFP against the luciferase (luc)+CD19+ acute lymphoblastic leukemia cell line NALM6 and the luc+CD19- acute myeloid leukemia cell line MOLM13. VSV-CD19 and VSV-GFP successfully lysed NALM6 (Fig.1B) or MOLM13, both in vitro and in vivo (data not shown). Next, we investigated the efficiency of VSV-CD19 in infecting tumor and immune cells. 24 hours after exposure to VSV-CD19 or VSV-GFP, we analyzed the surface expression of CD19 on MOLM13 and revealed efficient CD19 delivery (Fig.1C). Next, we assessed VSV infection of peripheral blood mononuclear cells (PBMCs) from healthy donors (HDs). Freshly isolated HD PBMCs were infected with VSV-CD19 for 6 hours and subsequently assessed for CD19 expression. Consistent with findings from clinical trials, VSV-CD19 selectively infected and induced CD19 expression on monocytes while other cells were not affected (Fig.1D). To exclude potential toxicities against CART19, we co-cultured CART19 with VSV-CD19 or VSV-GFP using second-generation 4-1BB costimulated CART19. Both VSV-CD19 and VSV-GFP did not infect CART19 as evident by preservation of CART19 viability and lack of CD19 or GFP expression (Fig.1E). Having demonstrated that VSV-CD19 specifically delivered CD19 to monocytes, we next tested whether the infected monocytes stimulated CART19. VSV-CD19 infected monocytes induced potent antigen-specific proliferation of CART19 (Fig.1F) and resulted in enhanced anti-tumor activity against luc+NALM6 in vitro (Fig.1G). Next, we aimed to confirm these findings in vivo. We generated luc+CART19 to track CART19 expansion in vivo. Freshly isolated HD monocytes were infected with VSV-CD19 ex vivo. After 4 hours, VSV-CD19 was washed away and immunocompromised NSG mice were intravenously injected with VSV-CD19 infectedmonocytes. After 24 hours, 3.5x106 of luc+untransduced T cells (UTD) or luc+CART19 were injected intravenously. The T cell expansion was assessed by bioluminescence imaging (BLI). VSV-CD19 infected monocytes specifically stimulated and expanded CART19 (Fig.1H). Finally, we tested whether VSV-CD19 can stimulate and rescue suboptimal anti-tumor effects of CART19 in vivo using a NALM6 relapsed model. Here, 1x106 luc+NALM6 were injected intravenously into NSG mice on day -6. At day -1, mice were imaged and randomized according to tumor burden to receive 1x106 UTD or CART19 on day 0. Subsequently, at day 4, mice were re-imaged and randomized. At day 5, HD monocytes were injected intravenously. Three hours after administering monocytes, mice received 1x107 VSV-CD19 or VSV-GFP (Fig.1I). BLI revealed that CART19 plusVSV-CD19 showed better tumor control than CART19 monotherapy or CART19 plus VSV-GFP (Fig.1J-K). Furthermore, CART19 plus VSV-CD19 exhibited long-term survival (Fig.1L). In summary, VSV-CD19 not only demonstrated direct anti-tumor effects but also specifically delivered CD19 to monocytes and tumor cells, thereby re-stimulating and enhancing the anti-tumor activity of CART19. This work provides a rationale to study VSV-CD19 in patients who demonstrate only suboptimal response to CART19. This approach could also be applied to augment CART therapy in other tumors. Figure 1 Disclosures Sakemura: Humanigen: Patents & Royalties. Eckert:Genentech: Current Employment. Cox:Humanigen: Patents & Royalties. Parikh:Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; Verastem Oncology: Honoraria; MorphoSys: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Merck: Research Funding; Janssen: Honoraria, Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding. Kay:Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding. Peng:Imanis: Other: Equity Ownership. Russell:Imanis: Other: Equity Ownership. Kenderian:Mettaforge: Patents & Royalties; Humanigen: Consultancy, Patents & Royalties, Research Funding; Lentigen: Research Funding; Torque: Consultancy; Novartis: Patents & Royalties, Research Funding; Kite: Research Funding; Gilead: Research Funding; Juno: Research Funding; BMS: Research Funding; Tolero: Research Funding; Sunesis: Research Funding; MorphoSys: Research Funding.

Introduction Despite the success of chimeric antigen receptor T (CART) cell therapy, it is limited by lower rates of durable responses related to inadequate CART cell expansion and trafficking and life-threatening complications such as cytokine release syndrome (CRS). Development of a strategy to efficiently track CART cells would allow the in vivo characterization of T cell expansion and trafficking to tumor sites as well as the development of strategies to potentially overcome these limitations. The sodium iodide symporter (NIS) is a sensitive reporter system that has been used for cell imaging in the clinic. Objectives NIS would be an efficient way to assess CART cell expansion, trafficking, and toxicity. Methods In vitro functions of CART cells were assessed by antigen specific proliferation, cytotoxicity, and cytokine production. NIS activity in vitro was evaluated by 125I uptake assay. In vivo imaging was performed using TFB PET/CT. CART cell trafficking assay was tested with OPM2 multiple myeloma (MM) xenografts and treated with either BCMA CART or NIS+BCMA CART cells. For CRS model, NSG mice were engrafted with patient-derived CD19+ acute lymphoblastic leukemia blasts. After the engraftment, mice were treated with either NIS+CART19 cells (5 × 106) or control. Results We generated NIS+CART19 and NIS+BCMA CART cells through dual transduction (Fig A) and revealed the exclusive 125I uptake in vitro (Fig B). In vitro functions of NIS+CART cells were equivalent to CART19 (Fig C). This indicates the incorporation of NIS into CART cells does not impair their antitumor activity. In vivo sensitivity assay revealed NIS+CART cells were detectable when cells were subcutaneously injected to NSG mice at a dose of 1.25 × 106 cells (Fig D). For in vivo trafficking assay, bioluminescence imaging (BLI) showed MM cells mainly engrafted in bones (Fig E). TFB PET confirmed the trafficking of NIS+BCMA CART cells to the bones, corresponding to BLI (Fig E). Both BCMA CART and NIS+BCMA CART cells exhibited similar antitumor activity. Finally, we explored whether TFB PET can detect CART massive expansion in vivo and predict CRS. One week after mice were treated with high dose NIS+CART19 cells, mice developed muscle weakness, hunched bodies, and weight loss (Fig F), which correlate with CRS. TFB PET revealed a significant uptake in the bone marrow, spleen, liver, and lungs (Fig G) of the diseased mice but not control mice. Flow cytometry revealed extensive infiltration of CART cells in the liver and spleen. This demonstrates the ability of TFB PET to detect NIS+CART cell expansion in vivo, correlating with the development of CRS. Conclusion Our results robustly show NIS+CART cells provides a sensitive, clinically applicable platform to image CART cells and to assess their expansion, trafficking to tumor sites, and the development of CRS. These studies illuminate a novel way to noninvasively assess CART cell functions in vivo.

Introduction Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, limitations include neurotoxicity (NT) and cytokine release syndrome (CRS). Emerging literature suggests that inhibitory myeloid cells and their cytokines induce CAR-T cell toxicities and inhibit effector functions. GM-CSF was specifically identified as a critical cytokine in the development of NT and CRS. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS, enhance CART anti-tumor activity, and reduce the presence of tumor associated macrophages. In addition, there appears to be a direct effect on CART19 cells. Objectives In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. Methods We used a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector to knock out GM-CSF during CART cell manufacturing. Edited and wildtype T cells and CART cells were sent for whole exome sequencing (WES) and RNA sequencing. We also used flow cytometry to assess the expression of GM-CSF receptors on CART cells, activated using CD3/CD28 beads or irradiated NALM6 cells. Results WES analysis revealed no significant difference in the single nucleotide variants or indel counts between GM-CSFk/o and GM-CSFwt CART19 (Fig 1A). WES was significant for only two alterations in the gene targeted by the gRNA (Fig 1B). This high efficiency and accuracy indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggests a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. Further investigation robustly indicates that while resting CART cells do not express any GM-CSF receptors, activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells were activated through their T cell receptor (Fig 1C) or CAR (Fig 1D). This was also seen in GM-CSFk/o CART19 cells, indicating this is specifically induced by T cell stimulation. These data suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Finally, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSFk/o CART19 revealed a distinct signature, including significant inhibition of the Fas death pathway (Fig 1E). This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion. Conclusion Our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSFk/o CART19 revealed a distinct transcriptome signature compared to GM-CSFwt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells.

Diarrhea after hematopoietic stem cell transplantation (HSCT) can be life-threatening, and its etiology includes conditioning regimens, graft-versus-host disease (GVHD), infections, and transplantation-associated microangiopathy (iTAM). Cord colitis syndrome (CCS) has been described as a syndrome of culture-negative and antibiotic-responsive persistent watery and non-bloody diarrhea of uncertain pathogenesis and occurs in umbilical cord blood transplantation (UCBT) recipients. We encountered a case similar to CCS that developed severe watery diarrhea after UCBT without any signs of GVHD or infection and responded well to metronidazole (MNZ) treatment. Since CCS is very rare, we herein describe a case of MNZ-effective diarrhea after UCBT.

Invasive fungal infection (IFI) is a major life-threatening problem encountered by patients with hematological malignancies receiving intensive chemotherapy. Empirical antifungal agents are therefore important. Despite the availability of antifungal agents for such situations, the optimal agents and administration methods remain unclear. We conducted a prospective phase 2 study of empirical 1 mg/kg/day liposomal amphotericin B (L-AMB) in 80 patients receiving intensive chemotherapy for hematological malignancies. All enrolled patients were high-risk and had recurrent prolonged febrile neutropenia despite having received broad-spectrum antibacterial therapy for at least 72 hours. Fifty-three patients (66.3 %) achieved the primary endpoint of successful treatment, thus exceeding the predefined threshold success rate. No patients developed IFI. The treatment completion rate was 73.8 %, and only two cases ceased treatment because of adverse events. The most frequent events were reversible electrolyte abnormalities. We consider low-dose L-AMB to provide comparable efficacy and improved safety and cost-effectiveness when compared with other empirical antifungal therapies. Additional large-scale randomized studies are needed to determine the clinical usefulness of L-AMB relative to other empirical antifungal therapies.

BACKGROUND Chimeric antigen receptor T cell (CART) therapy has revolutionized the treatment landscape for hematological malignancies, its efficacy remains limited in solid tumors. EGFRvIII is a truncated version of the wild type EGFR in which deletion of exons 2–7 of the extracellular domain leads the variant (EGFRvIII) that is strongly antigenic and is mostly expressed on tumor cells. EGFR amplification (tEGFR) almost uniformly precedes the presence of EGFRvIII on tumor cells. The heterogeneity of surface receptor expression and immunosuppressive stromal microenvironment underscore the need to develop CART strategies to target multiple tumor antigens simultaneously. METHODS we generated tEGFR/EGFRvIII directed CAR construct by cloning a clinically relevant tEGFR and EGFRvIII specific scFv into a second generation CAR construct (41BB stimulated) in a third generation lentivirus backbone. This was used to transfect 293T cells and the generated lentivirus particles were used to transduce T cells and generate EGFRvIII/tEGFR CART cells. GBM primary patient derived cell lines were used in these experiments. These cells were passaged and maintained in patient derived xenograft models. RESULTS EGFRvIII/tEGFR directed CART cells exhibited potent antitumor activity against EGFRvIII/tEGFR + GBM cell lines: with 100% killing at 1.25:1, 2.5:1, 5:1 and 10:1 E:T ratio on multiple PDX cell lines with EGFRvIII expression and EGFR over-expression (greater than five copies of EGFR gene) at 24 hours of incubation. Conclusion: We demonstrate that targeting EGFRvIII and over-expressed EGFR with CART cells is feasible, efficacious and represents a promising therapeutic strategy to target GMB. Data from in vivo and combinatorial CART experiments will be reported at the meeting.

Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell–associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF–deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

Introduction Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated a high response rate, the durable response rate is around 30%. Most patients lose their CART cells, and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in this field. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and immunosuppression. Objective CAFs from MM patients lead BCMA CART cell dysfunction and targeting both MM cells and CAFs can overcome this resistance. Methods CAFs were isolated from the bone marrow (BM) of MM patients and purified using anti fibroblast beads. MM-TME mouse model was established by injecting a 1:1 ratio of BCMA+ OPM2 (MM cell line) to CAFs. Five different CART cells were tested in this study. For single transduced CAR: BCMA, CS1, and fibroblast activation protein (FAP). For dual transduced CAR: BCMA-CS1 and BCMA-FAP CART. These were generated through the double transduction of two lentiviral vectors. Results BCMA CART cell proliferation assay showed inhibition of CART expansion in the presence of CAFs, and this was mediated by TGF-β (Fig A). MM-TME mouse model revealed a significant acceleration of MM progression (Fig B). To identify targets, we verified FAP expression on CAFs. Interestingly, there was a low level of CS1, but not BCMA expressed on CAFs (Fig C). Then, we evaluated the impact of CAFs on functions of BCMA CART cells compared to dual CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of CAFs, the proliferation of BCMA CART cells, but not the dual CART cells was significantly inhibited (Fig A). Similarly, in the presence of CAFs, the production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, we studied the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells was able to eradicate MM (Fig E). Then, the MM-TME model was used to assess the effect of targeting CAFs. Mice were treated with 1) control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A low dose (1 × 106 iv) of CART cell was used to induce relapse. BCMA CART cells led to a transient antitumor activity in this model (mice died within 2 weeks), while dual CART cells resulted in durable remissions, a higher number of circulating T cells, and long term survival (Fig F). Conclusion We demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in MM.

Objective Conditioning regimens for hematopoietic stem cell transplantation (HSCT) are well known to cause severe gastrointestinal toxicities that often disturb the oral intake of the patients followed by poor nutrition and life-threatening infection. An oral elemental diet (ED) is an easily consumed and assimilated form of liquid nutrients mainly composed of amino acids. It alleviates the digestive loading from the intestine and is mainly used for enteral nutritional support in patients with Crohn's disease. We herein report, for the first time, the efficacy of ED for patients undergoing HSCT. Methods We evaluated the efficacy of ED in a prospective cohort study. The primary endpoint for this study was the hospitalization period. The secondary endpoint was the occurrence of oral mucositis, nausea, diarrhea and fever. Patients A total of 73 patients were consecutively enrolled between March 2011 and March 2013. Twenty-three patients underwent autologous HSCT and 50 patients underwent allogeneic HSCT. The first 21 patients did not receive ED (non-ED group; NEG) while in the successive 52 patients (ED group; EG), oral ED was started before conditioning and was continued until 28 days after transplantation. Results The patient characteristics were similar between the two groups. The mean duration of ED administration for EG was 28.7 days (range, 3-37 days), and the mean total-dose of ED administration was 1904 g (range, 240-2,960 g). The median hospitalization period was significantly shorter in EG compared to NEG, (34 days vs. 50 days; p=0.007). Grade 3-4 oral mucositis occurred less in EG than NEG (25% vs. 48%; p=0.06). Conclusion Oral ED may promote an early mucosal recovery and thereby shorten the duration of hospitalization.

Little is known regarding the chimerism status after reduced-intensity conditioning transplantation when bone marrow is used as a stem cell source. We prospectively analyzed lineage-specific chimerism and retrospectively evaluated clinical outcomes in 80 adult patients who underwent unrelated donor bone marrow transplantation (URBMT) with fludarabine plus melphalan (FM) as the conditioning regimen. Mixed donor chimerism (MDC) was seen in 43 and 10 % of patients at days 14 and 28, respectively. Melphalan at ≤130 mg/m(2) was associated with an increased incidence of MDC at day 28 (P = 0.03). Patients with MDC at day 14 showed a marginally increased risk of primary graft failure and a marginally decreased risk of graft-versus-host disease. In multivariate analysis, MDC at day 14 was associated with higher overall mortality (hazard ratio (HR) = 2.1; 95 % confidence interval (CI), 1.1-4.2; P = 0.04) and relapse rate (HR = 3.0; 95 % CI, 1.2-7.5; P = 0.02), but not with non-relapse mortality (HR = 1.8; 95 % CI, 0.70-4.6; P = 0.23). Thus, the FM regimen yields prompt complete donor chimerism after URBMT, but the melphalan dose significantly impacts the kinetics of chimerism. Chimerism status evaluation at day 14 may be instrumental in predicting relapse after URBMT with the FM regimen.

Treatment with CD19-directed chimeric antigen receptor T cell (CART19) therapy has resulted in unprecedented clinical outcomes and was FDA-approved in acute lymphoblastic leukemia and non-Hodgkin B-cell lymphoma. However, its success in chronic lymphocytic leukemia (CLL) has been modest to date. An increasing body of evidence indicates that impaired CART cell fitness is the predominant mechanism of the relative dysfunction in CLL. The immunosuppressive microenvironment in CLL is well known and in part may be related to the abundance of circulating extracellular vesicles (EVs) bearing immunomodulatory properties. We hypothesized that CLL-derived EVs contribute to CART cell dysfunction. In this study, we aimed to investigate the interaction between circulating EVs isolated from CLL patient plasma (designated as CLL-derived EVs) and CART19 cells. We enumerated and immunophenotyped circulating EVs from platelet free plasma in untreated patients with CLL. We determined their interaction with CART19 cells using second generation, 41BB co-stimulated, lentiviral transduced CART19 cells generated in the laboratory from normal donors (FMC63-41BBζ CART cells). Our findings indicate that CLL-derived EVs impair normal donor CART19 antigen-specific proliferation against the CD19+ mantle cell lymphoma cell line Jeko-1 (Figure 1A). Next, we characterized CLL-derived EVs using nanoscale flow cytometric analysis of surface proteins and compared to healthy controls. Although the total EV particle count was not different between CLL and healthy controls (Figure 1B), there were significantly higher PD-L1+ EVs in patients with CLL (Figure 1C). Based on these results, we sought to assess the physical interaction between CLL-derived EVs and CART cells from normal individuals. When CLL-derived EVs were co-cultured with CART19 and CLL B cells and imaged with super-resolution microscopy, EVs were localized at the T cell-tumor junction (Figure 1D). Furthermore, CLL-derived EVs are captured by T cells as indicated by a significant reduction in the absolute count of EVs when co-cultured with resting T cells (Figure 1E). Having demonstrated that 1) there is an excess of PD-L1+ EVs in patients with CLL (Figure 1C) and 2) CLL-derived EVs physically interact with CART cells (Figures 1D-E), we sought to establish their functional impact on CART19 cells. Here, CART19 cells were stimulated with irradiated CD19+ JeKo-1 cells at a 1:1 ratio in the presence of increasing concentrations of CLL-derived EVs. There was a significant upregulation of inhibitory receptors such as PD-1 and CTLA-4 on the T cells (Figure 1F). This is associated with a reduction in CART effector cytokines (i.e., TNFβ) at higher concentrations of EVs (Figure 1G), suggesting a state of exhaustion in activated CART19 cells in the presence of CLL-derived EVs. This was further supported by transcriptome interrogation of CART19 cells. Here, CART19 cells were stimulated via 24-hour co-culture with the irradiated CD19+ cell line JeKo-1, in the presence of CLL-derived EVs at ratios of 10:1 and 1:1 EV:CART19 and then isolated by magnetic sorting. RNA sequencing of these activated CART19 cells indicated a significant upregulation of AP-1 (FOS-JUN) and YY1 (Figures 1H), known critical pathways in inducing T cell exhaustion. Finally, to confirm the impact of CLL-derived EVs on CART19 functions in vivo, we used our xenograft model for relapsed mantle cell lymphoma. Here, immunocompromised NOD-SCID-ɣ-/- mice were engrafted with the CD19+ luciferase+ cell line JeKo-1 (1x106 cells I.V. via tail vein injection). Engraftment was confirmed through bioluminescent imaging and mice were randomized to treatment with 1) untreated, 2) CART19 cells, or 3) CART19 cells co-cultured ex vivo with CLL-derived EVs for six hours prior to injection. A single low dose of CAR19 (2.5x105) was injected, to induce relapse. Treatment with CART19 cells that were co-cultured ex vivo with CLL-derived EVs resulted in reduced anti-tumor activity compared to treatment with CART19 alone (Figure 1I). Our results indicate that CLL-derived EVs induce significant CART19 cell dysfunction in vitro and in vivo, through a direct interaction with CART cells resulting in a downstream alteration of their exhaustion pathways. These studies illuminate a novel way through which circulating and potentially systemic EVs can lead to CART cell dysfunction in CLL patients. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Parikh:Ascentage Pharma: Research Funding; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board. Kenderian:Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.

Despite the success of chimeric antigen receptor T (CART) cell therapy, it is limited by 1) lower rates of durable responses related to inadequate CART cell expansion and trafficking to tumor sites and 2) development of life-threatening complication such as cytokine release syndrome (CRS). Development of a strategy to efficiently and robustly image and track CART cells in the clinic would allow the in vivo characterization of T cell expansion and trafficking to tumor sites as well as the development of strategies to potentially overcome these limitations. The sodium iodide symporter (NIS) is a characterized and sensitive reporter system that has been used for cell imaging in the clinic. We hypothesized that the incorporation of NIS into CART cells would be a sensitive and efficient way to assess CART cell expansion, trafficking, and toxicity. To test our hypothesis, we used two CART cell constructs that are characterized in preclinical models and studied extensively in the clinic: CART19 (41BB costimulated) and BCMA-CART cells (41BB costimulated). First, we generated NIS+CART19 and NIS+BCMA-CART cells through dual transduction of lentiviral vectors (Fig A) and revealed the exclusive 125I uptake by these NIS+CARTs and its inhibition by the NIS inhibitor KClO4in vitro (Fig B). We then analyzed T cell functions of NIS+CART cells. Here, NIS+CART19 or CART19 cells were cultured with the CD19+ acute lymphoblastic leukemia (ALL) cell line NALM6. There was no difference in CART cell cytotoxicity (Fig C), proliferation, or cytokine production (not shown) between NIS+CART19 and CART19. This indicates that the incorporation of NIS into CART cells does not impair their antitumor activity. Next, we evaluated the sensitivity of NIS+CART19 cell detection by TFB-PET in vivo; imaging was performed using an Inveon TFB-PET/CT scanner. Mice received 250 μCi 18F-TFB 45 minutes prior to image acquisition. NIS+CART cells were detectable with TFB-PET when cells were subcutaneously injected at a dose of 1.25x106 cells (Fig D). Having demonstrated that the incorporation of NIS in CART cells provides a sensitive way of their detection by TFB-PET and does not interfere with their effector functions, we tested its efficiency to assess CART cell trafficking in vivo, using multiple myeloma (MM) xenografts. Here, immunocompromised NOD-SCID-ɣ-/- (NSG) mice were engrafted with the BCMA+OPM2 MM cell line (1x106 IV). After engraftment, the tumor burden was assessed by bioluminescence imaging (BLI) and mice were randomized to receive 1) BCMA-CART or 2) NIS+BCMA-CART cells (5x106 IV). Mice were then serially imaged for 1) bioluminescence as a measure of disease burden, and 2) with TFB-PET to assess CART cell expansion and trafficking. As expected, BLI demonstrated that MM predominantly engrafts in bones (Fig E). TFB-PET confirmed trafficking of the NIS+BCMA-CART cells to the bones, corresponding to the areas involved by MM based on BLI (Fig E, right). Both BCMA-CART and NIS+BCMA-CART cells exhibited similarly potent antitumor activity in this model (not shown). Finally, we aimed to explore whether TFB-PET can detect CART massive expansion in vivo and predict the development of CRS. Here, we used an established CRS model in our laboratory. NSG mice were engrafted with patient derived relapsed ALL blasts (5x106 IV). Engraftment was confirmed by peripheral blood sampling. When the leukemic burden is >10 CD19+ cell/µl, mice were treated with either high dose NIS+CART19 cells (5x106 IV) or PBS control. One week after NIS+CART19 cell treatment, mice developed muscle weakness, hunched bodies, and weight loss (Fig F), which correlate with an extreme elevation of cytokines (Sterner et al. Blood 2018). TFB-PET revealed a significant uptake in the bone marrow, spleen, liver, and lungs (Fig G) of the diseased mice but not control mice. Mice were then euthanized, and tissues were harvested. Flow cytometric analysis confirmed an extensive infiltration of CART cells in the liver and spleen. This demonstrates the ability of TFB-PET to detect NIS+CART cell expansion in vivo, correlating with the development of CRS. In summary, our results robustly show that the incorporation of NIS into CART cells provides a sensitive, clinically applicable platform to image CART cells using TFB-PET and to assess their expansion, trafficking to tumor sites, and the development of CRS. These studies illuminate a novel way to noninvasively assess CART cell functions in vivo. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Suksanpaisan:Imanis: Employment. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Kay:Agios: Other: DSMB; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; MorphoSys: Other: Data Safety Monitoring Board. Peng:Imanis: Equity Ownership. Russell:Imanis: Equity Ownership. Kenderian:Kite/Gilead: Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Novartis: Patents & Royalties, Research Funding.

BACKGROUND Pulmonary infiltrates are common after allogeneic hematopoietic cell transplantation (HCT) and can have infectious or non-infectious etiologies. Fiberoptic Bronchoscopy (FOB) with bronchoalveolar lavage (BAL) is frequently used to evaluate these patients. However, the diagnostic yield can be highly variable according to patient populations, and the impact on management is not well defined, especially in the era of modern antimicrobial therapies. The aim of this study was to investigate the diagnostic and therapeutic yield of FOB-BAL in adult patients with pulmonary infiltrates during the first 100 days following HCT. METHODS We retrospectively reviewed the medical records of adult HCT patients who underwent FOB-BAL during the first 100 days post HCT between January 2005 and December 2015 at Mayo Clinic, Rochester, MN. The interval from HCT to FOB was stratified into early (days 1-30) vs. late (days 31-100). The finding of progressively more hemorrhagic effluent and/or ≥ 20% hemosiderin-laden macrophages in BAL were considered diagnostic for diffuse alveolar hemorrhage (DAH). Univariate and multivariate logistic regression models were fit to identify clinical and transplant characteristics that may impact the diagnostic yield of FOB-BAL. RESULTS A total of 114 patients (median age 55 years), representing 12% of all HCT patients underwent FOB-BAL. Underlying diagnoses were AML in 36%, MDS in 16%, ALL in 16%, MPN in 12% , CLL in 7%, and other in 13%. Transplants were from matched related donor in 43%, matched unrelated donor in 40%, and other donor/matching status in 17%. FOB-BAL was performed during the early period in 61% and during the late period in 39% of patients. In the early period, FOB-BAL provided a specific diagnosis in 49% of patients (30% DAH and 19% infection). In the late period, FOB-BAL had a diagnostic yield of 55% (14% DAH and 36% infection) (Figure 1). The distribution of bacterial and viral infections identified via BAL were similar during the early period (39% and 38%, respectively), whereas more bacterial than viral infections were identified in the later period (56% vs. 19%, respectively) (Figure 2). Non-invasive testing for respiratory tract infection was performed in 34% of patients. These included nasopharyngeal swab for RSV/Influenza PCR in 16% (all negative), multiplex respiratory pathogen PCR in 5% (1 positive for coronavirus), and sputum culture in 21% (positive in 8 patients). This compares with the identification of an infectious pathogen via BAL in 36% of patients; bacterial in 16%, viral in 11%, and fungal in 10%. Transbronchoscopic lung biopsy biopsies were performed in 8 patients and provided diagnostic information in 3 patients (eosinophilic pneumonia, organizing pneumonia and diffuse alveolar damage each in 1 patient). Procedural complications occurred in 3 patients; 2 after transbronchoscopic lung biopsy (1 bleeding and 1 pneumothorax) and 1 during FOB-BAL (atrial fibrillation with rapid ventricular rate). In a multivariable logistic regression model, the underlying diagnosis, presence of acute respiratory failure, and early vs. later time period after HCT were independently affecting the diagnostic yield of FOB-BAL, with higher likelihood of DAH being diagnosed in patients with AML/MDS and during the early post-HCT period, and higher likelihood of DAH being diagnosed in patients with acute respiratory failure (Table 1). In 40% of patients, FOB-BAL findings lead to treatment changes. These included addition of antimicrobials in 24% and addition of corticosteroids in 22% (17% DAH, 2% idiopathic pneumonia syndrome, and 1% organizing pneumonia) (Figure 3). FOB findings lead to more antimicrobial modifications in the early than in the later period (32% vs. 18%, respectively). Overall survival was poor in all the three groups, although significantly better in patients with non-diagnostic FOB findings (median of 2, 3, and 11 months in patients with DAH, infection, or non-diagnostic FOB finings, respectively) (p = 0.03) (Figure 4). CONCLUSION FOB-BAL provides clinically useful information in the post-transplant period. BAL findings were diagnostic in 51% of patients, and led to management changes in 40% of patients. Transbronchoscopic lung biopsy is associated with higher rates of procedural complications. Further characterization of the underlying diagnosis in patients with non-diagnostic FOB-BAL findings remains an unmet clinical goal. Disclosures Sakemura: Humanigen: Patents & Royalties. Kenderian:Morphosys: Research Funding; Kite/Gilead: Research Funding; Lentigen: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Tolero: Research Funding; Novartis: Patents & Royalties, Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated an overall response rate of 70-90% in patients with relapsed/refractory MM, the durable response rate is around 30%. Most patients lose their CART cells and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in CART cell therapy for MM. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and in the generation of an immunosuppressive microenvironment. We hypothesized that CAFs from bone marrows of patients with MM (MM-CAFs) inhibit BCMA CART cells and contribute to their failure and that targeting both the malignant plasma cells and CAFs can overcome this resistance. To test this hypothesis, we isolated MM-CAFs and studied their interaction with BCMA CART cells generated from normal donors (41BB costimulated, lentivirally transduced). Our initial findings suggest that MM-CAFs inhibit BCMA CART cell antigen specific proliferation in the presence of the BCMA+ MM cell line OPM2, and this inhibition is predominantly mediated through the secretion of TGF-β (Fig A). MM-CAFs also promoted MM tumor growth in an MM-TME xenograft model established in the laboratory (Fig B). Here, immunocompromised NOD-SCID-γ-/- (NSG) mice were engrafted with 1x106 luciferase+ BCMA+ OPM2, in combination with either 1x106 CAFs or vehicle control intraveneously (IV). Subsequent tumor burden was monitored by bioluminescent imaging of these mice. The presence of CAFs in this model significantly accelerated MM progression (Fig B). Based on these findings, we aimed to develop CART cell therapy targeting both malignant MM cells and their CAFs and to determine whether this strategy can reverse MM-CAF induced CART cell inhibition. To identify targets for these CART cells, we first verified the expression of Fibroblast Associated Protein (FAP), an established CAF target, on MM-CAFs. Flow cytometric analysis of MM-CAFs showed significantly higher expression of FAP, compared to fibroblasts derived from normal bone marrow (Fig C). In addition, our screening flow cytometric analysis identified CS1 as another protein overexpressed by MM-CAFs (Fig C). We therefore designed and generated FAP CART cells (41BB costimulated, lentivirally transduced) and CS1 CART cells (CD28 costimulated, lentivirally transduced). We also generated dual CART cells for both BCMA-FAP CART cells and BCMA-CS1 CART cells. These dual CART cells were generated through the dual transduction of two lentiviral vectors during CART manufacturing. Next, we evaluated the impact of CAFs on effector functions of BCMA CART cells compared to dual targeting CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of MM-CAFs, the antigen specific proliferation of BCMA CART cells, but not the dual targeting CART cells was significantly inhibited (Fig A). Similarly, in the presence of MM-CAFs, production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, to verify the significance of our laboratory findings, we investigated the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells were able to completely eradicate MM (Fig E). However, to determine the effect of targeting CAFs, we used our MM-TME model. Here, NSG mice were engrafted with the luciferase+ MM cell line OPM2, along with MM-CAFs, as described in Fig 1B. Mice were then imaged for engraftment and randomized to treatment with 1) untransduced control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A lower dose (1x106 IV) of CART cell was used to induce relapse post BCMA CART cells. Treatment with BCMA CART cells led to a transient antitumor activity in this MM-TME model (mice died within 2 weeks), while dual targeting CART cells resulted in durable remissions and long term survival of these mice (Fig F). In summary, we demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in multiple myeloma. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Lentigen: Research Funding; Kite/Gilead: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Tolero: Research Funding; Novartis: Patents & Royalties, Research Funding; Morphosys: Research Funding.

Background: Unprecedented clinical outcomes were reported after CD19 chimeric antigen receptor T-cell (CART19) therapy. However, the complete response rate in chronic lymphocytic leukemia (CLL) is much lower, at approximately 20%–30%. Several immune defects have been identified in CLL that result from the complex interaction between CLL cells and the microenvironment. This leukemic microenvironment is rich with extracellular vesicles (EVs) secreted by B-CLL cells. Here, we aimed to investigate the role of EVs play in the diminished CART response seen in some CLL patients. Methods: EVs were isolated from peripheral blood of 16 patients with untreated CLL at different Rai stages and risk profile by FISH. Cytometry was used to determine size, number of particles per μl, and CD19 expression. To investigate the impact of EVs on CAR T-cell functions, CART19 cells were stimulated with either CLL B cells or the CD19-positive cell line JEKO and different effector functions were analyzed. Results: Two patterns of EVs in CLL patients were identified; a single versus 2 distinct EV populations, characterized by size (small [EVssmall] and large [EVslarge], Fig 1A). In 25% of patients, EVs were CD19 positive (EVCD19+). CD19 positivity was detected only in patients with the EVslarge (Fig 1B). The EVs concentration, CD19 expression (EVsCD19+ vs EVsCD19-), or the size (EVssmall vs EVslarge) did not correlate with disease stage (early vs advanced Rai stage) or risk profile of CLL (low vs high risk). To investigate our hypothesis that EVs could modulate CART19 function, CART19 cell effector functions were examined in the presence of EVsCD19+, EVsCD19-, EVssmall, or EVslarge. EVs alone were insufficient to stimulate CART19 cells. However when CART19 cells were stimulated with the CD19-positive cell line JEKO, their effector functions were reduced only in the presence of EVsCD19+ but not EVsCD19-. This included a significant reduction in CART-specific killing (Fig 1C) and a reduction in cytokine production. The impairment of CART cell functions was independent of the size of EVs, ie, there was no impairment of CART functions with large or small size EVCD19- in co-culture. Conclusion: We identify CD19-positive large size EVs from patients with CLL and demonstrate that these EVs play a role in the leukemic microenvironment by reducing CAR T-cell activity. Studies are ongoing to define the mechanism(s).

Despite the remarkable outcomes of CD19-directed chimeric antigen receptor T (CART19) cell therapy in B-cell malignancies, the durable responses in diffuse large B-cell lymphoma are less than 40%, and strategies to enhance this response are desperately needed. Inhibition of AXL RTK with TP0903, a high-affinity AXL inhibitor has been found to induce robust apoptosis of malignant B cells. Here, we aimed to examine the role of AXL RTK inhibition with TP0903 on T-cell function in B-cell malignancies. First, we investigated the influence of TP0903 on CART19 cell phenotype and functions. Here, we used 41BB costimulated, lentiviral-transduced CART cells. AXL inhibition led to polarization of CART cells into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line Jeko or with leukemic B cells isolated from patients with chronic lymphocytic leukemia (CLL), in the presence of TP0903 (Fig 1A). Exposure of activated CART cells to TP0903 also resulted in significant downregulation of inhibitory receptors on activated CART cells (Fig 1B), a reduction of conical cytokines known to be associated with the development of cytokine release syndrome (CRS) (Fig 1C). To investigate the effect of AXL RTK inhibition of CART cells with TP0903 in vivo, we established MCL xenografts through the injection of 1.0x106 of Jeko into NSG mice. A week later, mice were treated with either vehicle alone, TP0903 (20 mg/kg/day) alone, 0.5x106 of CART19 alone, or TP0903 (20mg/kg/day)+0.5x106 of CART19. Three weeks after the treatment, mice were rechallenged with 1.0x106 of Jeko. Mice treated with CART19 and TP0903 rejected the tumor challenge while mice previously treated with CART19 alone redeveloped MCL, suggesting that AXL inhibition enhanced CART cell persistence (Fig 1D). Finally, we validated our preclinical findings in correlative analyses of phase 1 clinical trial of TP0903 in patients with solid tumors (NCT02729298). Similar to our findings, there was a significant reduction in Tregs, reduction of inhibitory receptors and polarization to a Th1 phenotype. These findings will be further investigated in a planned phase 1 clinical trial of TP0903 in relapsed/refractory CLL (NCT03572634) In summary, we demonstrated for the first time that AXL inhibition polarizes T cells into a Th1 phenotype, downregulates inhibitory receptors, and synergizes with CART cells in B-cell malignancies. These findings encourage further study of TP0903 as an enhancer of T-cell immunotherapies.

Chimeric antigen receptor T-cell therapy (CART) is limited by the development of cytokine release syndrome (CRS) and neurotoxicity (NT). CRS is related to extreme elevation of cytokines and massive T cell expansion. Preliminary studies suggest that NT might be mediated by myeloid cells that cross the blood brain barrier. This is supported by correlative analysis from CART19 pivotal trials where CD14+ cell numbers were increased in the cerebrospinal fluid of patients that developed severe NT (Locke et al, ASH 2017). Thus, we aimed to investigate the role of GM-CSF neutralization in preventing CRS and NT after CART cell therapy via monocyte control. The human GM-CSF neutralizing antibody lenzilumab (Humanigen, Burlingame, California), which has been shown to be safe in phase II clinical trials, does not impair CART cell function in vitro (10 ug/mL) when CART19 cells are stimulated with the CD19+ Luciferase+ acute lymphoblastic leukemia (ALL) cell line NALM6. Lenzilumab results in enhanced CART cell antigen specific proliferation in the presence of monocytes. In vivo, NOD-SCID-g−/− mice were engrafted with high disease burdens of NALM6 and treated with low doses of CART19 or control T cells, in combination with lenzilumab or isotype control antibody. The combination of CART19 and lenzilumab does not impair CART cell activity in vivo (Fig 1A). The impact of GM-CSF neutralization on CART toxicities in a novel patient derived xenograft model was explored. NOD-SCID-g−/− mice were engrafted with primary ALL blasts (1-3 × 106 cells). Mice were treated with high doses of CART19 cells (2-5 × 106 iv) and developed CART cell related toxicities. The combination of CART19, lenzilumab (to neutralize human GM-CSF), and murine GM-CSF blocking antibody (to neutralize mouse GM-CSF) resulted in prevention of weight loss (Fig 1B), decrease in critical myeloid cytokines (Fig 1C-D), reduction of cerebral edema (Fig 1E), enhanced leukemic disease control in the brain (Fig 1F), and reduction in brain macrophages (Fig 1G). As CRISPR/Cas9 gene editing of the GM-CSF gene during the process of CART cell manufacturing would result in CART cells with innately reduced secretion of GM-CSF, we designed guide RNA targeting exon 3 of the GM-CSF gene to generate GM-CSFk/o CART19 cells. These CARTs produce significantly less GM-CSF upon activation but other T cell cytokine production is not inhibited (Fig 1H). Using the NALM6 high tumor burden relapse xenograft model as described above, GM-CSFk/o CART19 cells resulted in slightly enhanced disease control compared to CART19 cells (Fig 1I). These studies illuminate a novel approach to abrogate NT and CRS through GM-CSF blockade that also potentially enhances CART cell functions. Based on these results, we have designed a phase II clinical trial using lenzilumab as a modality to prevent CART related toxicities in patients with diffuse large B cell lymphoma.

Despite the remarkable results of CD19 directed chimeric antigen receptor T cell (CART19) therapy, the durable responses in B cell lymphoma and chronic lymphocytic leukemia (CLL) is limited. This may be due to the loss of CART persistence, poor trafficking and inhibition by the tumor microenvironment. Strategies to enhance CART function are thus needed. Recent data have shown that abnormal expression of receptor tyrosine kinase (RTK) AXL portends poor prognosis. Inhibition of AXL with TP0903, a high affinity AXL inhibitor, has been found to induce robust apoptosis of CLL B cells. We therefore examined the role of AXL inhibition with TP0903 on T cell and CART19 function. First, we investigated the effect of AXL inhibition on naive T cells. Cells stimulated with PMA/Ionomycin and cultured with TP0903 showed significant reduction of Th2 cytokines and inhibitory receptors (Fig 1a). Effector and regulatory T cells (Treg) treated with TP0903 showed a preferential reduction of Tregs (Fig 1b). Next, we investigated the influence of TP0903 on 41BB costimulated CART19. TP0903 led to polarization of CARTs into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP0903 treatment also significantly downregulated inhibitory receptors on activated CARTs, including cytokine release syndrome (CRS) related cytokines (Fig 1c). The combination of CART19 and TP0903 yielded a synergistic cytotoxicity against JeKo in vitro (Fig 1d). We compared the transcriptome of activated CARTs treated with TP0903, and noted >100 genes that were differentially expressed compared to non-treated cells. Among these, cell junction, cell migration, and immune synapse related genes were significantly increased in activated CARTs treated with TP0903. To investigate the effect of AXL inhibition of CARTs with TP0903 in vivo, we established MCL xenografts via the injection of JeKo into NSG mice. The mice were then treated with vehicle alone, TP0903 alone, CART19 alone, or TP0903 + CART19. Three weeks after the treatment, mice were rechallenged with JeKo. Mice treated with CART19 and TP0903 rejected the JeKo challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART persistence (Fig 1e). Finally, we validated our findings in a phase I clinical trial of TP0903 (NCT02729298). T cells isolated from 3 cancer patients showed significant reduction of Tregs. This will be further investigated in a planned phase I clinical trial of TP0903 in relapsed/refractory CLL (NCT03572634). In summary, we showed for the first time that AXL inhibitior polarizes T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes CARTs in B cell malignancies. These findings encourage further study of TP0903 as an enhancer of T cell immunotherapies.

Impact of T-cell chimerism on relapse after cord blood transplantation for hematological malignancies: Nagoya Blood and Marrow Transplantation Group study

BACKGROUND Respiratory failure syndromes are common after allogeneic hematopoietic cell transplantation (allo-HCT), and are associated with substantial morbidity and mortality. These syndromes are incompletely understood, and may be associated with histopathologic evidence of acute lung injury (ALI). The role of biopsy in this setting is controversial, with uncertain benefits and considerable procedural risk. We report the clinical, radiologic and histopathologic features of patients with biopsy-proven ALI following allo-HCT in a large multi-site retrospective series. METHODS The medical records of 1,864 consecutive adults who underwent allo-HCT at Mayo Clinic, Minnesota (n = 916), Arizona (n = 694), and Florida (n = 254) (Jan 2005-Dec 2017) were reviewed. A total of 152 patients underwent a lung biopsy: 126 bronchoscopic, 22 surgical, and 4 CT-guided biopsies (Figure 1). Patients with a histopathologic diagnosis of ALI were included in the study, and were classified into clinicopathologic subgroups per published American Thoracic Society guidelines (Am J Resp Crit Care Med, 2011). Patients whose diagnosis remained uncertain after a detailed review of the medical records were included in a separate subgroup as undifferentiated ALI. Transplant characteristics, radiologic features, pulmonary function test (PFT) results, treatments, and outcomes were compared between groups. RESULTS Clinicopathologic diagnoses in 62 patients with biopsy-proven ALI consisted of an infectious etiology in 15 (24%), cryptogenic organizing pneumonia (COP) in 15 (24%), idiopathic pneumonia syndrome (IPS) in 9 (15%), bronchiolitis obliterans syndrome (BOS) in 9 (15%), diffuse alveolar hemorrhage in 3 (5%), periengraftment respiratory distress syndrome in 2 (3%), and undifferentiated ALI in 9 (15%) patients (Figure 2). Transplant characteristics were comparable between groups. Infectious pneumonia and IPS were the most common diagnoses during the first 100 days post HCT (50% and 36%, respectively). Between 100 and 365 days, a range of diagnoses were seen including infectious pneumonia (20%), COP (23%), IPS (14%), BOS (23%), and undifferentiated ALI (20%) (Table 1). In 14/15 (93%) infectious pneumonia cases, biopsy did not change management and diagnosis was concurrently established by bronchoalveolar lavage (BAL) or non-invasive testing. Patients with IPS, BOS, and undifferentiated ALI more commonly had a pleural effusion (66%, 44%, and 44%, respectively). Nodular infiltrates were most commonly seen in patients with infectious pneumonia (55%). Other radiographic findings were not clearly discriminative. BOS and infectious pneumonia were more commonly associated with an obstructive PFT (78% and 33%, respectively), whereas restrictive pattern was more frequently seen in COP and IPS (50% and 57%, respectively) (Table 2). Despite a histopathologic diagnosis of ALI, 9 (15%) patients had clear clinical and radiographic evidence of BOS. Steroids were used commonly in noninfectious syndromes: 93% of patients with COP and in 100% of IPS patients. Of 4 patients with IPS who received Etanercept, 2 (50%) showed complete recovery, whereas the remaining 5 patients who did not receive Etanercept died from fulminant respiratory failure. Most common treatment response was complete recovery in COP (73%), fulminant respiratory failure leading to death in IPS (78%), and chronic respiratory failure in BOS (78%). Estimated OS was shortest in the group with IPS and undifferentiated ALI (median 5 and 17 mons, respectively), whereas patients with COP had the best prognosis (mOS not reached) followed by the infectious pneumonia and the BOS groups (mOS of 59 and 55 mons, respectively). Pulmonary complications were the most common cause of death in patients with non-infectious ALI. Lung biopsy was associated with post-procedural pneumothorax in 17 (27%) patients; 12 of whom required a chest tube. CONCLUSIONS In this largest case series to date of histopathologic ALI after allo-HCT, biopsy does not appear to meaningfully change management in the majority of patients, and is associated with substantial procedural risk. Histopathologic findings do not clearly correlate with the clinical and radiographic picture in a sizeable proportion of the patients. PFT results, BAL findings, the timing of the onset of respiratory failure after HCT, and the response to steroids seem to be of greater diagnostic and/or prognostic value. Disclosures No relevant conflicts of interest to declare.

Despite its efficacy, chimeric antigen receptor T-cell therapy (CART) is limited by the development of cytokine release syndrome (CRS) and neurotoxicity (NT). While CRS is related to extreme elevation of cytokines and massive T cell expansion, the exact mechanisms for NT have not yet been elucidated. Preliminary studies suggest that NT might be mediated by myeloid cells that cross the blood brain barrier. This is supported by correlative analysis from CART19 pivotal trials where CD14+ cell numbers were increased in the cerebrospinal fluid of patients that developed severe NT (Locke et al, ASH 2017). Therefore, we aimed to investigate the role of GM-CSF neutralization in preventing CRS and NT after CART cell therapy via monocyte control. First, we investigated the effect of GM-CSF blockade on CART cell effector functions. Here, we used the human GM-CSF neutralizing antibody (lenzilumab, Humanigen, Burlingame, California) that has been shown to be safe in phase II clinical trials. Lenzilumab (10 ug/kg) neutralizes GM-CSF when CART19 cells are stimulated with the CD19+ Luciferase+ acute lymphoblastic leukemia (ALL) cell line NALM6, but does not impair CART cell function in vitro. We have found that malignancy associated macrophages reduce CART proliferation. GM-CSF neutralization with lenzilumab results in enhanced CART cell antigen specific proliferation in the presence of monocytes. To confirm this in vivo, NOD-SCID-g-/- mice were engrafted with high disease burdens of NALM6 and treated with low doses of CART19 or control T cells (to induce tumor relapse), in combination with lenzilumab or isotype control antibody. The combination of CART19 and lenzilumab resulted in significant anti-tumor activity and overall survival benefit compared to control T cells (Fig 1A), similar to mice treated with CART19 combined with isotype control antibody, indicating that GM-CSF neutralization does not impair CART cell activity in vivo. This anti-tumor activity was validated in an ALL patient derived xenograft model. Next, we explored the impact of GM-CSF neutralization on CART cell related toxicities in a novel patient derived xenograft model. Here, NOD-SCID-g-/- mice were engrafted with leukemic blasts (1-3x106 cells) derived from patients with high risk ALL. Mice were then treated with high doses of CART19 cells (2-5x106 intravenously). Five days after CART19 treatment, mice began to develop progressive motor weakness, hunched bodies, and weight loss that correlated with massive elevation of circulating human cytokine levels. Magnetic Resonance Imaging (MRI) of the brain during this syndrome showed diffuse enhancement and edema, associated with central nervous system (CNS) infiltration of CART cells and murine activated myeloid cells. This is similar to what has been reported in CART19 clinical trials in patients with severe NT. The combination of CART19, lenzilumab (to neutralize human GM-CSF) and murine GM-CSF blocking antibody (to neutralize mouse GM-CSF) resulted in prevention of weight loss (Fig 1B), decrease in critical myeloid cytokines (Fig 1C-D), reduction of cerebral edema (Fig 1E), enhanced leukemic disease control in the brain (Fig 1F), and reduction in brain macrophages (Fig 1G). Finally, we hypothesized that disrupting GM-CSF through CRISPR/Cas9 gene editing during the process of CART cell manufacturing would result in functional CART cells with reduced secretion of GM-CSF. We designed guide RNA targeting exon 3 of the GM-CSF gene and generated GM-CSFk/o CART19 cells. Our preliminary data suggest that these CARTs produce significantly less GM-CSF upon activation but continue to exhibit similar production of other cytokines and exhibit normal effector functions in vitro (Fig 1H). Using the NALM6 high tumor burden relapse xenograft model as described above, GM-CSFk/o CART19 cells resulted in slightly enhanced disease control compared to CART19 cells (Fig 1I). Thus, modulating myeloid cell behavior through GM-CSF blockade can help control CART mediated toxicities and may reduce their immunosuppressive features to improve leukemic control. These studies illuminate a novel approach to abrogate NT and CRS through GM-CSF neutralization that also potentially enhances CART cell functions. Based on these results, we have designed a phase II clinical trial using lenzilumab as a modality to prevent CART related toxicities in patients with diffuse large B cell lymphoma. Disclosures Ahmed: Humanigen: Employment. Sahmoud:Humanigen: Employment. Durrant:Humanigen: Employment. Russell:Vyriad: Equity Ownership. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding.

Immunosuppressive treatment using antithymocytic globulin (ATG) is the treatment of choice for severe aplastic anemia patients [1, 2]. Iron chelation therapy is also effective for patients with frequent blood transfusions [3–5]. However, the effects of these treatments on hemodialysis patients have not been reported. Here, we present a case of severe aplastic anemia on hemodialysis, treated with immunosuppressive and iron chelation therapy. A 49-year-old male presented in December 2007 with fever and pancytopenia. He required regular hemodialysis following history of chronic renal failure 25 years earlier, and had also been treated with recombinant epoetin beta. Laboratory studies revealed a white blood cell count of 1.2 9 10/ll, neutrophil cell count of 468/ll, platelet count of 0.9 9 10/ll, hemoglobin concentration of 5.4 g/dl, and reticulocytes at only 0.9 9 10/ll. Bone marrow biopsy revealed severe hypoplasia. He was diagnosed with severe aplastic anemia, and required frequent transfusions of PC and RBC to maintain a 2 9 10/ll platelet level and 8.0 g/dl hemoglobin concentration. He was given the antibiotic, meropenem, for febrile neutropenia, which rapidly controlled his fever. The same drug, epoetin beta, was continued for treatment. Cyclosporin A and granulocyte colony-stimulating factor (G-CSF) were given as the first therapy for severe aplastic anemia, but his bone marrow remained hypoplastic. Horse ATG was added at a dose of 15 mg/kg for 5 days. Methylprednisolone was used at a dose of 2 mg/kg/day for 5 days, and then tapered off. The only adverse event was a grade 2 allergic reaction. His hematopoietic system recovered slightly, and G-CSF was lowered, but transfusion frequency could not be reduced. An anabolic steroid was added, but yielded no improvement. Then, 8 months after the first ATG, a second rabbit ATG was given at a dose of 5 mg/kg for 5 days. Methylprednisolone was given concomitantly, and no significant adverse event was seen, except an acute allergic reaction. Total red-blood cell transfusion volume was over 80 Japanese units, so deferasirox was given. Bone marrow biopsy revealed a moderate number of positive granules on iron staining, which were not observed at the initial examination. The initial dose was 10 mg/kg/day, due to renal failure, but the serum ferritin level remained above 3000 ng/ml. The dose was increased to 20 mg/kg/day. Six months later, the serum ferritin level was below 1000 ng/ml. Transfusion frequency was reduced. One year later after the second ATG therapy, he became transfusionfree (Fig. 1). Bone marrow biopsy revealed the recovery of hematopoiesis, but on iron staining, a moderate number of positive granules were still observed. During this course of treatments, he continued dialysis three times per week in an open space, and bacterial infection was not a threat. J. Hiraga (&) R. Sakemura S. Mizuno Department of Hematology, Toyota Memorial Hospital, 1-1 Heiwa-cho, Toyota 471-8513, Japan e-mail: junji_hiraga@mail.toyota.co.jp

In allo-stem cell transplantation (SCT), it is unclear whether donor-specific anti-HLA Abs (DSAs) can actually mediate graft rejection or if they are simply surrogate markers for the cellular immunity that causes graft rejection. Here, we first analyzed a case of cord blood allograft rejection in which DSA and cytotoxic T lymphocyte (CTL) specific for donor HLA-B*54:01 were detected at the time of graft rejection. Both the DSA and CTL inhibited colony formation by unrelated bone marrow mononuclear cells sharing HLA-B*54:01, suggesting that the humoral and cellular immune responses were involved in the graft rejection. Interestingly, the DSA and CTL were also detected in cryopreserved pre-transplant patient blood, raising a hypothesis that the presence of anti-HLA Abs could be an indicator for corresponding HLA-specific T cells. We then evaluated the existence of HLA-specific CD8(+) T cells in other patient blood specimens having anti-HLA class I Abs. Interferon-γ enzyme-linked immunospot assays clearly confirmed the existence of corresponding HLA-specific T-cell precursors in three of seven patients with anti-HLA Abs. In conclusion, our data demonstrate that integrated humoral and cellular immunity recognizing the same alloantigen of the donor can mediate graft rejection in DSA-positive patients undergoing HLA-mismatched allo-SCT. Further studies generalizing our observation are warranted.

Treatment of primary central nervous system lymphoma (PCNSL) improved in recent years. However, the high neurotoxicity and low survival rates associated with this condition remain unresolved. We report 13 consecutive patients with PCNSL for whom upfront melphalan, cyclophosphamide, etoposide, and dexamethasone (known as LEED) followed by autologous stem-cell transplantation (ASCT) was planned at the Anjo Kosei Hospital. All patients were pathologically diagnosed with diffuse large B-cell lymphoma and were negative for human immunodeficiency virus. All patients were to receive three cycles of high-dose methotrexate-based induction chemotherapy, two cycles of high-dose AraC-based chemotherapy, and LEED followed by ASCT. All 13 patients achieved a partial response, and the 3-year overall survival (OS) rate was 76.2 %. Seven of the 13 patients were alive at the last follow-up, without any adverse events, including neurotoxicity. Six of the 13 (46.2 %) patients underwent ASCT and the 3-year OS rate was 80.0 %. Although this study included only a limited number of patients, these preliminary signs of efficacy and tolerability merit further consideration. To make further improvements in survival, the rate of patients undergoing ASCT should be increased. Other prospective studies involving greater numbers of patients are required to confirm these findings.

[Introduction] Regenerating islet-derived protein 3 alpha (REG3A), a protein produced by Paneth cells, has been reported as a biomarker of lower gastrointestinal graft-versus-host disease (GVHD). The single nucleotide polymorphism (SNP) rs7588571 is located at the upstream of REG3A gene within 2kb, suggesting that the expression of REG3A might be affected by the SNP. In this study, we analyzed the association between the rs7588571 genotype in recipients and transplant outcomes. [Patients and methods] The study population included 118 adult Japanese patients who had undergone T cell replete allogeneic bone marrow transplantation from an HLA-identical sibling donor with GVHD prophylaxis consisting of short-term methotrexate and cyclosporine between 1987 and 2006. Sixty-eight (58%) male and 50 (42%) female patients, with a median age of 35 years (range 15-56), were included. Eighty-eight (75%) patients were classified as having standard risk disease defined as acute myeloid leukemia in first or second complete remission, acute lymphoblastic leukemia without Philadelphia chromosome in first complete remission, chronic myeloid leukemia in chronic phase, myelodysplastic syndrome without excess of blasts, aplastic anemia and paroxysmal nocturnal hemoglobinuria, while other patients were classified as having high risk disease. Conditioning regimen was myeloablative in 113 (96%) patients and reduced-intensity in other patients. Genomic DNA was extracted from patient's peripheral blood or bone marrow by standard methods. Genotyping was performed using TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, California, USA) on ABI 7300 Real-Time PCR systems (Applied Biosystems). Multiple logistic regression model was used to identify risk factors associated with acute and chronic GVHD, and multivariate Cox model was created for overall survival (OS), non-relapse mortality (NRM), and relapse. Variables with a P-value < 0.1 in the univariate analysis were entered into the stepwise selection method. All tests were two-sided, and P [Results] The frequencies of the GG, AG, and AA genotypes were 41%, 43%, and 16%, respectively, which were compatible with those reported in the HapMap-JPT database. There was no significant association between the distribution pattern of the rs7588571 genotypes and each patient characteristic. Grade II-IV acute GVHD developed in 12% of all patients, 14% in those with GG genotype, 14% with AG genotype and 11% with AA genotype, respectively. In univariate analysis, the higher patient age (≥35) was the only significant factor for higher incidence of grade II-IV acute GVHD (OR: 5.4, 95% CI: 1.3-32, P=0.011). Extensive chronic GVHD developed in 38% of all patients, 27% in those with GG genotype, 45% with AG genotype and 50% with AA genotype, respectively. The incidence of extensive chronic GVHD in non-GG genotype (46%) was significantly higher than that in GG genotype (27%) (P=0.05). In multivariate analysis, non-GG genotype (OR: 2.5, 95% CI: 1.0-5.8, P=0.04), female to male transplant (2.8, 1.2-7.0, 0.023), and high risk disease (2.8, 1.1-7.2, 0.038) were significant factors for higher incidence of extensive chronic GVHD. The relapse rates at 4-year were 26% in non-GG and 17% in GG genotype (HR: 1.16, 95% CI: 0.49-2.7, P=0.73). High risk disease was the only significant factor for higher relapse rate. The OS and NRM at 4-year were 57% and 20% in non-GG, and 65% and 17% in GG genotype, respectively. The rs7588571 genotypes affected neither OS nor NRM. [Discussion] REG3A polymorphism was associated with the incidence of extensive chronic GVHD. Given its capacity to control the tissue regeneration and intestinal microbiota, REG3A may not only reflect the intestinal damage caused by GVHD but also have a certain role in the pathogenesis of GVHD. Our findings need to be confirmed in larger cohort, and further investigation is required in order to clarify the biological effect of rs7588571 genotypes and the exact role of REG3A in the pathophysiology of GVHD. Disclosures Kiyoi: MSD K.K.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Eisai Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Alexion Pharmaceuticals: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Celgene Corporation: Consultancy; Chugai Pharmaceutical Co. LTD.: Research Funding.

Introduction: Endogenous or gene-modified T cells with T cell receptors (TCRs) are major designs of cytotoxic T lymphocytes (CTL) therapies. However, gene-modified TCR-CTL therapies targeting on malignant tumors have been generally unsuccessful in previous clinical trials. The major reasons were poor expansion and short persistence of CTL. To overcome these problems, TCR-affinity have been genetically enhanced, but often resulted in life-threatening toxicities. We aimed to modify TCR-CTL in order to improve the function by enhancing intracellular signaling after TCR ligation without affinity modulation. Method: We generated three artificial T cell activating adapter molecules (ATAM), which are CD3z, CD3z/CD28, and CD3z/4-1BB. CD28 or 4-1BB intracellular domain was inserted in the middle of CD3z intracellular domain, so that they can assemble with internal TCR complex when stimulated with corresponding antigen. ATAM-T2A-tEGFR was packaged into retrovirus vector to transduce the T cells. In order to evaluate the functional alteration, we established ATAM-CMV CTL model. We stimulated T cells with CMV pp65 peptide to induce endogenous CMV CTL. On day 4 after stimulation, ATAMs were retrovirally transduced into CMV CTL. After a selection with anti-EGFR mAb, we achieved more than 90% purity of ATAM positive CMV CTL and used in 51Cr release assay, cytokine release assay, and the growth assay. In the growth assay, we stimulated the ATAM CMV-CTL weekly and assayed the viable cell number, tetramer positivity, phenotype, and apoptosis. Regarding these assay, we used HLA transduced K562 (HLA-K562) as APC. Results: First, we confirmed the expression of each ATAM on CD8+ T cells, which showed similar expression among each ATAM. To determine the cytotoxicity of CMV CTL according to each ATAM or control tEGFR transduction, we performed 51Cr release assay against HLA-K562 pulsed with or without CMV peptide. In the presence of CMV peptide, each ATAM-CMV CTL showed similar cytolytic activities to control CMV CTL. [specific lysis (%): tEGFR 32.0+-6.0, CD3z 32.4+-2.6, CD3z/CD28 31.1+-8.8, and CD3z/4-1BB 37.4+-1.5: at an E:T ratio = 3:1, respectively}. In the intracellular cytokine assay against HLA-K562 pulsed with CMV peptide, similar proportions of responder were positive for IFN-g and IL-2 in control and ATAM-CMV CTL. [IFN-g (%): tEGFR, 67.3+-9.4, CD3z, 69.0+-7.4, CD3z/CD28, 66.5+-8.0, and CD3z/4-1BB, 69.6+-7.9; IL-2 (%): tEGFR, 17.5+-5.0, CD3z, 17.7+-4.3, CD3z/CD28, 15.9+-4.3, and CD3z/4-1BB, 18.9+-4.8, respectively]. The cytotoxicity and cytokine secretion upon irrelevant stimulation were completely negative among each ATAM-CMV CTL. Although the surface phenotype was not changed in control CMV-CTL, that of ATAMs-CMV CTL was significantly expanded after antigen stimulation [central memory (CD45RA-CD62L+) (%): tEGFR, 6.0+-2.9, CD3z, 26.8+-11.1, CD3z/CD28, 19.4+-9.5, and CD3z/4-1BB, 35.8+-26.8: at 7 days after stimulation]. Regarding the cell growth, CD3z/CD28 and CD3z/4-1BB showed greater CTL expansion upon weekly antigen stimulation. The fold expansion of tEGFR, CD3z, CD3z/CD28, and CD3z/4-1BB-CMV CTL were 4.9+-0.7, 8.2+-2.4, 12.3+-1.1, and 13.0+-1.6 at day7, 1.6+-0.4, 8.0+-2.5, 18.6+-3.9, and 21.3+-7.7 at day14, and 0.4+-0.2, 1.4+-0.7, 5.1+-3.0, and 30.4+-22.9 at day21, respectively (Fig). Comparing with control, CD3z/4-1BB-CMV CTL showed continuous greater expansion after several stimulations (p˂0.01: during day0 to day7, p˂0.05: during day7 to day14). To examine the reason for the growth advantage, we investigated the apoptosis after the stimulation with DAPI/Annexin V staining. It revealed that the live cells after specific stimulation elevated in ATAMs-CMV CTL compared with control CMV CTL. Conclusion: We generated three ATAMs and successfully transduced into CMV CTL. Particularly, the whole cell growth and continuance of growth after weekly stimulation were significantly improved in CD3z/4-1BB CTL. This greater cell expansion may improve in vivo persistence and further effectiveness of TCR-CTL therapy. ATAM-CMV CTL did not recognize any APC without corresponding peptide, indicating that ATAM transduction could improve the CTL effectiveness without altering CTL specificity. Thus we believe that we could potentially apply ATAM transduction to other TCR-CTLs targeting malignant tumors safely. Figure Figure. Disclosures Kiyoi: Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding; Celgene Corporation: Consultancy.

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on T cells and contributes to T cell exhaustion during chronic infections. Moreover, it has been shown that engagement of PD-1 on T cells by PD-L1 on tumor cells is associated with the immune escape of tumors, and recent clinical trials have highlighted the anti-tumor efficacy of blockade of the PD-1/PD-L1 pathway. Along with costimulatory molecules such as CD80 and CD86, PD-L1 is also highly expressed on dendritic cells (DCs), which are professional antigen-presenting cells (APCs) that are widely used to generate antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy. However, the roles of PD-L1 on APCs have not been well examined. Thus, we evaluated the roles of PD-L1 on APCs in the induction of cytomegalovirus (CMV)-specific CTLs (CMV-CTLs). First, peripheral blood mononuclear cells (PBMCs) were obtained from five CMV-seropositive healthy donors (HLA-A*02:01, n = 3; A*24:02, n = 1; B*07:02, n = 1). Mature DCs were generated from adherent PBMCs by stimulation with GM-CSF/IL-4 and maturational cytokines (TNF-a, IL-1b, IL-6, and PGE2), and freshly isolated CD3+ T cells were stimulated with mature DCs pulsed with CMV pp65-derived HLA-restricted peptides (NLVPMVATV for HLA-A*02:01, QYDPVAALF for A*24:02, and TPRVTGGGAM for B*07:02) in the presence or absence of anti-PD-L1 blocking antibody. After 14 days of culture, the presence of anti-PD-L1 antibody resulted in a less efficient induction of CMV-CTLs (Figure 1), suggesting that PD-L1 might play a positive role in the induction of antigen-specific CTLs. For further evaluation, we generated K562-based artificial APCs (aAPCs), which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1 (named K562, K562+CD80/86, K562+PD-L1, and K562+CD80/86+PD-L1). CD3+ T cells isolated from nine CMV-seropositive healthy donors (HLA-A*02:01, n = 4; A*24:02, n = 4; B*07:02, n = 1) were stimulated weekly for 28 days using HLA-restricted CMV peptide-pulsed K562-based aAPCs. K562+CD80/86+PD-L1 led to significantly higher induction of CMV-CTLs than K562 or K562+CD80/86 (Figure 2). Since the original K562 cells slightly express CD80, we completely knocked out the CD80 expression in the cells using the CRISPR/Cas9 system, and then we transduced the cells with or without PD-L1 (named CD80KO K562+PD-L1 and CD80KO K562, respectively) to determine whether PD-L1 itself has a stimulatory effect on the induction of CMV-CTLs. CD80KO K562 and CD80KO K562+PD-L1 induced the same amount of CMV-CTLs, suggesting that PD-L1 itself does not have a stimulatory effect for the induction of CMV-CTLs. Phenotypic analysis showed that CMV-CTLs induced by K562+CD80/86+PD-L1 contained more CD45RA- CD62L+ central memory T cells, which persist longer and are more effective in vivo after adoptive transfer in CTL therapy, than CMV-CTLs induced by other K562-based aAPCs. In addition, when compared with CMV-CTLs induced by other K562-based aAPCs, CMV-CTLs induced by K562+CD80/86+PD-L1 did not express higher levels of exhaustion markers such as PD-1 and TIM-3, and exhibited similar levels of IFN-gamma and IL-2 production and CD107a expression in response to the specific peptide stimulation. Furthermore, to evaluate the effect of K562-based aAPCs on the induction of tumor antigen-specific CTLs, CD3+ T cells were isolated from three HLA-A*24:02-positive healthy donors and stimulated with HLA-A*24:02-restricted WT1 peptide-pulsed K562-based aAPCs weekly. After four stimulations, only K562+CD80/86+PD-L1 could clearly expand the WT1-specific CTLs from all of the three donors (Figure 3). In conclusion, our findings demonstrate that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of antigen-specific CTLs without causing excessive differentiation or functional exhaustion of the induced CTLs. These results suggest that stimulation with K562+CD80/86 might result in the activation-induced cell death of antigen-specific CTLs and that PD-L1 might be involved in fine-tuning excessive stimulation of CD80/86. Further analyses to determine the mechanisms of our findings are warranted. Our results also highlight the possibility that K562+CD80/86+PD-L1 has therapeutic potential as novel aAPCs for the generation of CTLs for adoptive immunotherapy. Disclosures Kiyoi: Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Teijin Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding.

Dear Editor, A 64-year-old man was diagnosed with symptomatic multiple myeloma (MM) in June 2009. No extramedullary region including the testes was detected. Serum protein electrophoresis showed IgA-κmonoclonal protein. Durie-Salmon stagewas IIIA and International Staging System stage was II. Treatment was initiated with pulsed dexamethasone and bortezomib (1.3 mg/m) twice weekly with dexamethasone (20 mg on the day of bortezomib administration and the next day).Without any adverse events, he achieved very good partial response (VGPR) after three cycles of bortezomib therapy [1]. Subsequently, he received high-dose melphalan (200 mg/m) followed by autologous stem cell transplantation (ASCT) in November 2009. After ASCT, he was followed up without any therapeutic agents. However, in September 2010, he complained of hip pain, and serum IgA level was elevated to 2,014 mg/dl. As recurrence of MM was diagnosed, we started weekly bortezomib with dexamethasone. The patient soon experienced improvement of symptoms, and serum IgA decreased to normal levels. In July 2011, during weekly bortezomib and dexamethasone, the patient noticed enlargement of his scrotum. Magnetic resonance image revealed enlarged bilateral testes. Serum IgA levels and all other laboratory data were normal. Bilateral orchidectomy was performed in October 2011. Microscopic examination showed positive results for CD56, CD138, and IgA and κ light chain (Fig. 1a, b). The pathological diagnosis wasmetastasis of plasma cell myeloma to the bilateral testes, and the right spermatic cord had positive margins. In November 2011, serum IgA was elevated to 2,304 mg/dl. At relapse of systemicMM including spermatic margins after orchidectomy, a 28-day cycle of lenalidomide (25 mg on days 1–21) and dexamethasone (40 mg on days 1, 8, 15, and 22) was initiated [2]. Serum IgA levels decreased to 1,026 mg/dl and reached a plateau without stump recurrence of the spermatic cord. After 6 cycles of lenalidomide, the patient was treated with cyclophosphamide, vincristine, adriamycin, and dexamethasone (C-VAD) and achieved VGPR after two cycles of C-VAD therapy. In August 2012, he successfully underwent a second ASCT. Two months after ASCT, he started a 28-day cycle of lenalidomide maintenance therapy (10mg on days 1–21) [3]. As of the time of writing, the patient remains alive without recurrence.

Abstract Background: Dasatinib is a novel kinase inhibitor of BCR-ABL and SRC family kinases that has shown promising anti-leukemic activity against chronic myeloid leukemia (CML). However, some patients experience treatment-related toxicity shortly after initiating dasatinib administration and are managed by drug interruption and/or dose reduction (altered treatment) which can limit the potential benefits of treatment. Methods: To determine the factors that influence compliance and the efficacy of dasatinib therapy, we conducted a phase 2 study of mid-term compliance and the effectiveness of dasatinib therapy in patients with chronic phase CML (CML-CP) (DARIA-01 Study). Treatment was administered until disease progression or intolerable toxicity, as determined by the treating physician. Dasatinib at 100 mg once daily was initially administrated. Dose interruption or reduction to 50 mg once daily was allowed after grade 2 or worse nonhematologic toxicity or grade 3 or worse hematologic toxicity. Trough plasma concentrations of dasatinib (Cmin) at steady state were assessed at 28 days of therapy. Results: In April 2014, 32 CML-CP patients were enrolled in the study, which included at least 6 months of follow-up. The median age was 53 years (range, 20–86). Twenty-four (75.0%) were previously untreated patients with CML-CP, and the remaining eight (25.0%) were switched from other tyrosine kinase inhibitors (imatinib, five patients; nilotinib, three patients) because of intolerance or resistance to these drugs. During the initial 28 days, 28 patients continued with 100 mg/day treatment, and four received doses of 50 mg/day. The incidence of altered treatment was 25% during the initial 3 months and 34% during the initial 6 months from dasatinib administration. Four patients who experienced dose reduction during the initial 3 months continued with 50 mg/day treatment thereafter at their request. Except one who experienced gastro-intestinal bleeding, all patients could continue dasatinib therapy. Age (>60 years) and Cmin divided by the given dasatinib dose (Cmin/D) were significantly correlated with a higher incidence of altered treatment during the initial 3 months (p = 0.0095) (p = 0.0083). Twenty-five (78%) patients achieved complete cytogenetic response (CCyR), and 13 (40%) achieved major molecular response at 6 months. The incidence of pleural effusion during the initial 28 days was significantly correlated with Cmin/D (p = 0.032) but was not related to the molecular response rate at 6 months (40% vs. 41%, p = 0.98). Conclusion: Current data demonstrate that patients treated with dasatinib who displayed a high Cmin/D (>1.0) value at day 28 and/or who were aged 60 and over were at a high risk for altered treatment during the early periods of therapy. Patients who experienced pleural effusion could be well managed by dose reduction and/or temporal interruption of dasatinib and could still achieve the preferable molecular response rate. Individualizing therapy by incorporating a pharmacokinetic assay would improve compliance and the efficacy of dasatinib therapy, particularly for elderly patients. Disclosures Morita: Bristol: Honoraria.

Background: Oral elemental diet (ED) is easily assimilated form of liquid nutrients composed of amino acids, sugars, vitamins and minerals. It alleviates the digestive loading from the intestine and mainly used for enteral nutritional support in the treatment of patients with Crohn's disease. High dose alkylating agents and total body irradiation (TBI) commonly used in conditioning regimens for hematopoietic stem cell transplantation (HSCT) are well known to cause severe gastro intestinal toxicities including mucositis associated with oral pain which often disturb oral intake of the patient. Inadequate nutrition requiring total parenteral nutrition (TPN) prolongs hospitalization and life-threatening infection may result from bacterial translocation through damaged mucosal membranes. Objectives: To our best knowledge, there are no previous reports of the efficacy of ED in patients with HSCT. We conducted a prospective cohort study to determine whether ED improved patients' outcome. Patients: We enrolled 73 consecutive patients who underwent HSCT between Mar. 2011 and Mar. 2013 in Anjo Kosei Hospital. Fifty patients undergone allogeneic HSCT, and 23 patients undergone autologous HSCT. The sources of allogeneic HSCT were unrelated bone marrow (n=16), related peripheral blood stem cell (n=16), unrelated cord blood (n=18). Median age was 47 years old (range, 17-72 years). There were 41 male and 32 female patients. Patients had leukemia (n = 38), malignant lymphoma (n = 24), multiple myeloma (n = 7), and others (n = 3). At transplant, 52 patients were in complete remission (CR), 21 patients were in non-CR. Twenty-one patients who were transplanted before November 2011 did not receive any ED (non-ED group; NEG), and 52 patients transplanted afterward November 2011 received an ED as standard care (ED-group; EG). Methods: ED administration was initiated from day -8 to day -4 to all patients when conditioning regimens for HSCT began, and ended on day +28 after HSCT. The recommended dose was 80g (1255 kJ) daily for total oral ED intake. We didn’t exclude the intake after failure of ED administration, additional food intake, and total parenteral nutrition (TPN). Our standard practice for allogeneic HSCT has been to start TPN on day -1 and continue until patients achieve a documented caloric intake of 6279 kJ per day after engraftment. For autologous HSCT, our protocol has been to start TPN when patients fail to reach a documented caloric intake of 6279 kJ per day by food intake and continue until this level is reached. This study was approved by our institutional review board. Results: Patient characteristics were not significantly different between the two groups. The median days of ED administration was 30 days (range, 3–37 days). The median number of days of oral food intake for EG and NEG were 22 days (range, 2–37) and 25 days (range, 3–35 days) days, respectively (P = 0.90). There were no adverse events reported due to ED administration. Grade 3/4 oral mucositis occurred in 13 (25%) EG patients and 10 (48%) NEG patients (P=0.06). Mean duration of mucositis in EG and NEG were 2.8 days and 5.6 days (P=0.07), respectively. Grade 2 to 4 fever occurred in 43 (83%) EG patients and 14 (67%) NEG patients (P=0.13), and the mean duration of fever were 3.9 days and 5.0 days, respectively (P=0.95). Acute GVHD occurred in 8 (19%) EG patients and 7 (46%) NEG patients (P=0.21). The mean duration of patient hospitalization was 36.6 days in EG patients and 57.3 days in NEG patients (P=0.003). Day100 non-relapse mortalities were 4 (8%) in EG patients and 3 (14%) in NEG patients (P=0.40). Conclusions: Oral intake of ED in patients with HSCT significantly reduced the duration of hospitalization and showed the possibility to improve the occurrence of grade 3/4 oral mucositis without adverse events. Disclosures No relevant conflicts of interest to declare.

Method Fourteen cases of young (under 66 years old) PCNSL who were treated at Anjo-Kosei Hospital between May 2002 and December 2011 enrolled in this study. Result Among 14 cases, 35–65 (median 56) years old, the pathorogical diagnosis are: 13 cases of diffuse large B cell lymphoma (DLBCL), 1 case of intravascular large B cell lymphoma (IVL), and 6 cases are treated by upfront auto PBSCT. The median follow-up was 38.7 months. Between auto-PBSCT and no-auto-PBSCT arms, there are no significant differences in the patient's background such as age (53.5 years old versus 58 years old), performance status, serum LDH (205 versus 198), serum sIL2R (436 versus 680). We checked the prognostic scoring system for PCNSL, but there are no differences between the two arms. In auto-PBSCT arm, they were treated by high-dose MTX-based therapy and high-dose AraC therapy, when they achieved CR/ near CR, they were carried out auto PBSCH. The conditioning regimen for auto PBSCT was LEED therapy in all cases. The overall survival at 4 years was superior in the auto-PBSCT arm (75.0% versus 41.7% P = 0.262). The relapse rate at 4 years was superior in the auto-PBSCT arm (25.0% versus 61.9% P = 0.273). In auto PBSCT arm, five cases maintain complete remissions and no one has severe complications such as leukoencephalopathy. In no-auto PBSCT arm, four cases are died from the relapse and only three cases maintain complete remission. Conclusion Our results emphasize the effectiveness and safety of upfront auto PBSCT to PCNSL.

Introduction Hematology malignancy is often complicated by many critical diseases including DIC. The resolution of DIC safely is one of the most important aspects in getting over hematological malignancy. We would like to report our comparative review of the administration of Recombinant Thrombomodulin (Recomodulin) versus Dalteparin Na (Fragmin) and this time, we would also report the possibility of reducing blood products such as fresh frozen plasma (FFP). Patient Thirteen patients (mean age 57.0 years, men/women 5/8, AML 7, APL 4, ALL 2) were administered recombinant thrombomodulin and 13 patients (mean age 50.5 years, men/women 8/5, AML 6, APL 4, ALL 3) were administered Dalteparin Na. Result The mean duration of therapies was 3.8 and 11.3 days, respectively. All patients has achieved DIC resolution, and the mean resolution time was 5.5 and 11.6 days (P = 0.020). The mean administration of FFP was 7.5 and 16.9 units (P = 0.091). The average of FDP, Fib, and JMHW DIC score has improved in both therapies. On the other hand, there were no changes in PT-INR and AT3. No bleeding and other adverse events were reported during both administration. Conclusion Recombinant thrombomodulin adminisitration is effective therapy rather than Dalteparin Na administration for patients with hematological malignancy and also tends to reduce the use of FFP.