Functional Effects of Low-Dose IL-2 in Patients with Chronic Graft Versus Host Disease

Abstract Introduction: CD4+CD25+FoxP3+ regulatory T cells (Treg) have broad suppressive activity and play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Interleukin-2 (IL-2) is a key growth factor for Treg and we previously reported that daily administration of low-dose IL-2 in patients with refractory cGVHD induces selective expansion of Treg and NK cells. No expansion of CD4 conventional T cells (Tcon) or CD8 T cells was noted. In a Phase 2 study, 64% of patients had clinical responses. Predictors of clinical response included younger age, early IL-2 initiation and a higher Treg:Tcon ratio at study baseline and at week 1 of IL-2 therapy. However, there were no significant differences in overall Treg expansion or plasma IL-2 levels between responders and non-responders during the initial 12 week treatment period. We therefore asked whether functional or gene expression differences in Treg or effector T cells could distinguish clinical response to IL-2. Methods: In vitro Treg suppression assays were performed using cryopreserved patient samples obtained at study baseline and week 6 of IL-2 therapy. CD4+CD25+CD127- Treg were cultured with CellTrace Violet-labeled CD4+CD25- Tcon in the presence of stimulating antibody-coated beads. Proliferation was measured as CellTrace Violet dilution by flow cytometry after 4 days of incubation. All patient samples were tested against the same healthy donor (HD) Treg and Tcon. Differential gene expression between clinical responders and non-responders was examined by RNA Seq analysis of sorted Treg, Tcon, CD8 and NK cells at study baseline and week 4 of IL-2 therapy. Results: To compare Treg cell function between response groups, we tested the ability of patient Treg to suppress the proliferation of HD Tcon. To determine whether there were qualitative differences in effector T cells between the response groups, we measured suppression of patient Tcon by HD Treg. At study baseline, clinical responders appear to have higher Treg suppressive function although the differences between the response groups were not significant due to small sample size (Figure 1A). Patient Tcon in responders and non-responders were similarly suppressed by HD Treg. At week 6 of IL-2 therapy, Treg from non-responders showed significant improvement in suppressive activity against HD Tcon, whereas there was no significant change in suppressive function of Treg from responders (Figure 1B). Thus, despite the lack of clinical improvement in cGVHD symptoms, non-responder Treg show both numeric increase and functional improvement in response to IL-2 therapy. To identify other cell-intrinsic differences that distinguish clinical response to IL-2, we performed RNA Seq on sorted Treg, Tcon, CD8 and NK cells in 3 responders and 3 non-responders. Differentially expressed genes were identified using the DEseq package. There were very few differentially expressed genes between response groups within the pre-treatment Treg, CD8 and NK cell samples (Figure 2). In contrast, 93 genes (92 upregulated, 1 downregulated) with greater than 4-fold difference in expression levels were identified in pre-treatment Tcon samples between responders and non-responders. The top 10 gene ontogeny terms associated with these differentially expressed genes include cell-cell adhesion and ion transport processes. By week 4 of IL-2 therapy, the gene expression profiles of responder and non-responder cells were very similar. There were no differentially expressed genes within Treg, and only 5 within Tcon. Conclusions: In addition to increasing absolute Treg numbers, daily low-dose IL-2 improves Treg suppressive function in patients who do not exhibit clinical cGVHD improvement. Interestingly, RNA Seq results suggest that the determinants of clinical response do not lie within the Treg. Instead, the greatest differences between response groups were detected in pre-treatment CD4 Tcon, implying that lack of clinical response reflects resistance of CD4 effector cells to suppression mediated by Treg in vivo. Further analysis of differentially expressed Tcon genes may help elucidate the mechanisms of resistance to IL-2 therapy in patients with cGVHD. Disclosures Koreth: LLS: Research Funding; prometheus labs inc: Research Funding; kadmon corp: Membership on an entity's Board of Directors or advisory committees; amgen inc: Consultancy; millennium pharmaceuticals: Research Funding; takeda pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Armand:Otsuka: Research Funding; Roche: Research Funding; BMS: Consultancy, Research Funding; Infinity: Consultancy; Merck & Co., Inc.: Consultancy, Research Funding; Sequenta: Research Funding; Sigma Tau: Research Funding; Tensha: Research Funding. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees.

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Indatuximab Ravtansine (BT062) in Combination with Low-Dose Dexamethasone and Lenalidomide or Pomalidomide: Clinical Activity in Patients with Relapsed / Refractory Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.4486.4486 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4486-4486 ◽

Cited By ~ 32

Author(s):

Kevin R. Kelly ◽

David S. Siegel ◽

Asher A. Chanan-Khan ◽

George Somlo ◽

Leonard T. Heffner ◽

...

Keyword(s):

Multiple Myeloma ◽

Phase I ◽

Clinical Response ◽

Board Of Directors ◽

Research Funding ◽

Low Dose ◽

Prior Exposure ◽

Tolerability Profile ◽

Advisory Committees ◽

Equity Ownership

Abstract Background: BT062 (Biotest AG, Dreieich, Germany) is an antibody-drug conjugate (ADC) comprising a CD138-binding chimerized antibody and the cytotoxic maytansinoid, DM4. It is designed to target and kill CD138-positive cancer cells. CD138 (Syndecan-1) is highly expressed on a number of solid tumors and hematological malignancies and is one of the most reliable markers for multiple myeloma (MM) cells. BT062 was previously evaluated as a monotherapy in patients with heavily pretreated relapsed/refractory MM and found to have an acceptable tolerability profile with preliminary evidence of activity (Heffner et al, Blood. 2012; 120: Abstract 4042). Phase I/IIa testing was initiated with BT062 in combination with lenalidomide (Len) and low-dose dexamethasone (dex). The combination was well tolerated at BT062 doses up to 100 mg/m², defined to be the recommended Phase 2 dose (RPTD), and induced meaningful responses, including in patients previously treated with both Len and bortezomib (Bort) (Kelly et al, Blood. 2014; 124: Abstract 4736). Based on these promising results, further investigation of BT062 in combination with pomalidomide (Pom) and dex was initiated in patients with prior Len and Bort exposure, a patient population known to have a poor outcome. Objectives: To evaluate the safety and activity of BT062 (on days 1, 8, and 15 in a 4-week cycle) used in combination with dex (20-40 mg on days 1, 8, 15, and 22) and Len (25 mg, daily on days 1-21) or Pom (4 mg, daily on days 1-21) in patients with relapsed/refractory MM. Methods: This is a prospective, open label, multicenter Phase I/IIa study. The RPTD of BT062 in combination with Len/dex was defined to be 100 mg/m², and 38 patients were treated with BT062/Len/dex at the BT062 RPTD. An additional 17 patients were treated with BT062/Pom/dex at the BT062 RPTD. Patients aged ≥18 years with relapsed/refractory MM were eligible to participate. Prior treatment with Len, Pom, and/or dexamethasone (any dose) was allowed. To qualify for treatment with BT062/Len/dex at the BT062 RPTD, patients must have received at least one but no more than six prior therapies.To qualify for treatment with BT062/Pom/dex, patients must have received at least two prior therapies, including both Len and Bort, and progressed on or within 60 days of completion of their last therapy, with no limit on number of prior therapies. Patients with clinical response (or no evidence of disease progression) without unacceptable toxicities were eligible to receive additional treatment cycles. Toxicities were assessed by CTCAE v4. Clinical response was assessed by the investigator according to International Myeloma Working Group criteria. Results: Sixty-four patients have received BT062 in combination with dex and Len or Pom in this ongoing study. The combinations have been generally well tolerated, with approximately 90% of adverse events (AEs) reported CTC grade 1 or 2. The most common AEs reported are diarrhea, fatigue, and nausea. Forty-seven patients have received BT062 with Len/Dex (3 at 80 mg/m², 38 at 100 mg/m², 6 at 120 mg/m²), with 8 patients still on treatment. Among these 47 patients, median progression-free survival (PFS) was 16.4 months. Forty-three patients completed at least two treatment cycles and were evaluable for response. Of these patients 33 achieved a partial response (PR) or better, with an overall response rate (ORR) of 77% and a median duration of response (DOR) of 21.0 months. Thirteen of the evaluable BT062/Len/dex-treated patients had prior exposure to both Len and Bort and progressed on or within 60 days of their last therapy. ORR was 54% among these patients, including 1 complete response (CR), 4 very good partial responses (VGPR) and 2 PRs. Seventeen patients were treated with BT062/Pom/dex, all had prior exposure to both Len and Bort and progressed on or within 60 days of their last therapy. ORR was 79%, with 4 VGPR and 7 PR among the 14 patients evaluable for efficacy. Median PFS has not been reached after 7.5 months median follow up, with 7 patients still on treatment. Updated safety and activity data will be presented. Conclusion: BT062 has been found to be well tolerated when used in combination with Len/dex or Pom/dex, with encouraging activity even in patients with Len- and Bort-pretreated disease progressing on or within 60 days of completion of their last therapy. Disclosures Kelly: Novartis: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Siegel:Novartis: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Merck: Honoraria. Somlo:Millennium: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Heffner:Millennium: Research Funding; AbbVie: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding. Madan:Onyx: Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Celgene: Speakers Bureau. Lonial:Celgene: Consultancy; Celgene: Consultancy; BMS: Consultancy; Onyx: Consultancy; Millenium: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Merck: Consultancy; BMS: Consultancy. Barmaki-Rad:Biotest AG: Employment. Rühle:Biotest AG: Employment. Herrmann:Biotest AG: Employment. Wartenberg-Demand:Biotest AG: Employment. Haeder:Biotest AG: Employment. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Acetylon: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

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Therapeutic and Immunologic Responses Elicited By in Situ Vaccination with CpG, Ibrutinib, and Low-Dose Radiation

Blood ◽

10.1182/blood-2021-154017 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3539-3539

Author(s):

Tanaya Shree ◽

Sarah Haebe ◽

Debra K. Czerwinski ◽

Grady Day ◽

Anuja Sathe ◽

...

Keyword(s):

T Cells ◽

Immune Responses ◽

Board Of Directors ◽

Research Funding ◽

Low Dose ◽

Low Dose Radiation ◽

Advisory Committees ◽

Tumor Reduction ◽

Dose Radiation

Abstract Introduction: In situ vaccination aims to induce an immune response locally at one tumor site that propagates systemically to all tumor sites. This approach can be effective in indolent lymphoma (Brody et al., JCO 2010, Frank et al., Cancer Discov 2018, Hammerich et al., Nat Med 2019). We designed a novel clinical strategy combining in situ vaccination with systemic ibrutinib, a kinase inhibitor that modulates B and T cells. Our preclinical work had shown that combined intratumoral CpG injection and systemic ibrutinib administration was curative of systemic disease in a mouse lymphoma model, an effect that was T cell dependent (Sagiv-Barfi, Blood, 2015). Here we report the results and correlative data from the Phase I/II clinical trial testing this combination along with local low-dose radiation in adults with recurrent low-grade B cell lymphoma (NCT02927964). Methods: Enrolled patients received intratumoral injections of CpG (SD-101, 3 mg) weekly for 5 doses and local radiation (4Gy in two fractions) to the same site. Daily oral ibrutinib (560mg) began after the second intratumoral injection. Revised Lugano criteria (Cheson et al., JCO, 2014) were used to assess overall radiographic responses to therapy. Distal responses were assessed by excluding the injected site and measuring only non-injected sites. Fine needle aspirates (FNAs) were obtained from CpG-injected and non-injected nodal tumor sites pre- and post-treatment and analyzed by flow cytometry and droplet-based single-cell RNA sequencing (scRNAseq). Results: Among the twenty patients treated on study, median age was 64, 55% were male, and all but one had a diagnosis of follicular lymphoma. All patients were previously treated with an average of 2 lines of therapy, and half had previously received chemotherapy. Adverse events (AEs) were consistent with known effects of ibrutinib (including diarrhea and rash) and of CpG (including fever and flu-like reactions). No drug-related grade 4, serious, or unexpected AEs were observed. As anticipated, all patients experienced tumor reduction at the locally treated site (median 84% reduction). Remarkably, all patients experienced some tumor reduction at non-injected non-irradiated index lesions (median 45%, range 13-100%), suggesting the generation of systemic immune responses (Figure 1A). By Cheson criteria, ten patients achieved an objective response, including one complete response (ORR 50%). Despite an overall improvement in tumor burden, three patients had new or progressing non-index lesions and scored as progressive disease. Treatment induced an expansion of naïve and effector memory T cells and reductions in T follicular helper (Tfh) and activated regulatory T cells (Tregs) at the injected site. T cells with high expression of transcripts related to oxidative phosphorylation (Toxphos) increased preferentially in patients with subsequent clinical tumor reduction (Figure 1B), implicating T cell metabolism in successful generation of immune responses. Analysis of single cell T cell receptor (TCR) sequencing data revealed>300 clones that were comprised of at least 2 cells at each timepoint and which expanded or contracted at least two-fold during treatment. Expanding clones were more likely than contracting clones to be activated or memory T cells and less likely than contracting clones to be Tfh or Tregs (Figure 1C-D). Clone dynamics were often similar at the two sampled tumor sites, reflecting systemic immune responses. Finally, in vitro assays showed treatment-induced expansion of tumor-specific T cells in the peripheral blood of all 6 evaluable patients. Conclusion: The combination of oral ibrutinib, intratumoral CpG, and local low-dose radiation is safe and can generate systemic antitumor immune responses and systemic tumor shrinkage in low-grade B cell lymphoma. Figure 1 Figure 1. Disclosures Shree: Gilead: Other: Spouse's employment. Khodadoust: CRISPR Therapeutics, Nutcracker Therapeutics: Research Funding; Myeloid Therapeutics: Membership on an entity's Board of Directors or advisory committees; Alexion, AstraZeneca Rare Disease: Other: Study investigator. Frank: Kite-Gilead: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Research Funding; Allogene Therapeutics: Research Funding. Beygi: Kite/Gilead: Current Employment. Levy: GigaGen: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Kira: Membership on an entity's Board of Directors or advisory committees; Abintus Bio: Membership on an entity's Board of Directors or advisory committees; Khloris: Membership on an entity's Board of Directors or advisory committees; Virsti: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees.

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Transcriptomic Systems Biology Analyses with Buparlisib (BKM120) Pan PI3K Inhibition in Canine B Cell Lymphoma (BCL): Leveraging Comparative Oncology for Cancer Therapeutics and Biomarker Discovery

Blood ◽

10.1182/blood-2018-99-118548 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4114-4114

Author(s):

Ravi Dashnamoorthy ◽

Afshin Beheshti ◽

Sarah Cass ◽

Athena Kritharis ◽

Kristine Burgess ◽

...

Keyword(s):

Clinical Study ◽

Systems Biology ◽

Cancer Risk ◽

Board Of Directors ◽

Differentially Expressed Genes ◽

Research Funding ◽

Biological Pathways ◽

Differentially Expressed ◽

Advisory Committees ◽

Key Genes

Abstract Background: The canine is a highly appealing model for cancer research and discovery in part due to comparable histopathological features with humans, a fully intact immune system, similar clinicopathologic features, a more comparable body size and pharmacokinetic properties than the mouse, varied breed-specific incidence rates as well as a shared environment with humans. We and others have shown prominent transcriptomic overlap of human and canine NHL (cNHL) (McDonald T et al. Onctogarget, 2018). PI3K/Akt signaling plays an important role in lymphomagenesis, which is also a promising therapeutic target. However, identification of predictive genetic aberrations of therapeutic efficacy remains elusive. We evaluated the clinical activity of the pan-PI3K inhibitor, buparlisib, in a pilot clinical study in cNHL. Methods :We enrolled and treated 10 dogs with buparlisibwho were diagnosed with BCL in an IRB and IACUC approved clinical study. Cases included 2 treatment naïve and 8 dogs with relapsed disease that had relapsed s/p CHOP (6), L' asparaginase (1) and VELCAP (1) treatment. Pet owners were consented and the study subjects received buparlisib9mg/kg orally for 28 consecutive days. Analysis for tumor response were evaluated on weekly basis through direct tumor measurement or use of x-rays. Post-therapy fine needle aspirates (FNA) were collected on Days 0, 7 and 21 to examine predictors of response to BKM120. RNA from fine need aspirate cells were isolated and the transcriptomic changes were evaluated using Canine Genome 2.0 Affymetrix Array, followed by unbiased systems biology assessment for biological pathways using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). We performed unbiased assessment to determine pertinent biological pathways associated with treatment response. The overall impact was used to determine the global effect on tumor progression and cancer risk based on the specific regulation of each gene. A Carcinogenic Risk Score (CRS) was calculated based on these values to determine if there is a promoted risk for cancer (positive value) or inhibitory risk for cancer (negative value) by summing the log2 fold-change values of key genes and subtracting this from the sum of log2 fold-change values of the tumor suppressors when comparing pre-treated to BKM120 treated dogs. Results: Following four weeks of BKM120 treatment, the overall response rate was 30% with 1 complete response lasting 42 days; 2 partial responses lasting 55 and 72 days; 3 stable disease; and 4 progressive disease. Mild treatment related toxicities such elevated blood glucose, thrombocytopenia and anemia, fever, nausea and lethargic symptoms, with no treatment related toxicities in 2 cases were noted. Principal Component Analysis (PCA) and hierrachical clustering analysis of differentially expressed genes show that differentially expressed genes to cluster together in all dogs during post 2 week, indicating a consistent biological activity by BKM120 in all dogs regardless of breed, prior treatment or disease status. Pathway network analysis based on differentially expressed genes predicted activation of upstream regulators associated with tumor suppression including SOX1, SOX3 and GMNN (Week 1) and CEBPA (Week 2). Analysis of "key genes" involved in multiple biological processes appeared to be associated with response of PI3K inhibitortreatment. This included down regulation of CREBBP with a Cancer Risk Score (CRS) of -0.97 and downregulation of VIM, CDH3, WNT3, WNT5B and FGFR2 with a CRS of -2.98 (Fig 1). Conclusion: Results from our pilot study in cNHL showed encouraging clinical responses with a pan-PI3K inhibitor in 3 of 10 dogs. Furthermore, our unbiased characterization of biological pathways revealed that the observed GEP changes associated with tumor suppression and they reduced the risk for cancer progression. Overall, the canine model appears to be particularly attractive model that may be leveraged for the study of clinical and biological responses to novel therapeutic oncologic agents. Disclosures Evens: Bayer: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Novartis: Consultancy; Acerta: Consultancy; Seattle Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics International DMC: Membership on an entity's Board of Directors or advisory committees; Tesaro: Research Funding; Janssen: Consultancy; Affimed: Consultancy.

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Post-Treatment Clone Size Predicts Survival Independently of IPSS-R and Response after Azacitidine Therapy for MDS.

Blood ◽

10.1182/blood-2020-139082 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-13

Author(s):

Yasuhito Nannya ◽

Magnus Tobiasson ◽

Shinya Sato ◽

Elsa Bernard ◽

Maria Creignou ◽

...

Keyword(s):

Clinical Response ◽

Board Of Directors ◽

Research Funding ◽

Post Treatment ◽

Private Company ◽

Advisory Committees ◽

Clone Size ◽

Otsuka Pharmaceutical ◽

Pre Treatment

Background DNA hypomethylating agents (HMAs), including azacytidine (AZA) have been established as key drugs for higher-risk myelodysplastic syndromes (MDS). We and others have explored the role of mutation profile before AZA administration on predicting outcomes. Actually, we have previously identified mutated-TP53 as a marker associated with higher rate of achieving complete remission (CR). In addition, mutations in TP53 and DDX41 predicted reduced and prolonged survival after treatment, respectively. However, the clinical significance of evaluating clone size changes early after treatment has not been determined. In this study, we explored the role of post-treatment clone size in predicting outcomes of AZA treatment for MDS and related diseases. Methods We enrolled 290 AZA-treated cases, including 88 from a Japanese prospective study (JALSG MDS-212 trial), 149 from Karolinska Institute, and 53 from a retrospectively collected Japanese cases. The diagnoses were MDS (n=242), MDS/MPN (n=25), and AML-MRC (n=23). For all patients, tumor samples were collected both before and after AZA administration and were analyzed for mutations in 66 genes implicated in myeloid neoplasms using targeted-capture sequencing. The median cycles of AZA treatment before sampling was 4 (range 1-7). Clone size was calculated from variant allele frequency adjusted for ploidy or allelic imbalances.Survival was calculated with a Cox regression model. Results In post-treatment samples, we identified 870 mutations in 51 genes in 255 (88%) patients with a median of 3 mutations per sample, while 943 mutations were seen in 279 (96%) patients in the pre-treatment samples. Most frequently detected mutations in post-treatment samples were seen in TET2, TP53, RUNX1, and ASXL1. Germline DDX41 mutations were excluded from clone size evaluation. Median clone sizes were 0.63 and 0.54 for pre-treatment and post-treatment samples (P=.011), respectively. The largest clone sizes (max(VAF)) in post-treatment samples had a strong negative correlation with hematological response according to IWG criteria (P < .0001). We next explored whether max(VAF) in post-treatment samples provides a more precise estimation of long-term survival than IPSS-R. Max(VAF) further stratified each IPSS-R risk group in subgroups with discrete OS (P < .0001 for IPSS-R very high and P = .0004 for high risk group). Incorporating pre-treatment mutation data (mutations in TP53 and DDX41) and max(VAF) values in addition to IPSS-R scores and clinical response, we constructed a multivariate model and found that all these factors had an independent and significant impact on OS (Figure 1A). Next, we examined whether max(VAF) combined with IPSS-R and clinical response can improve the model. For this purpose, we randomly split the cohort into 75% training and 25% validation subsets and for each split, we constructed different models using the training set, performance of which was evaluated by calculating the concordance index (c-index) using the validation set. The mean c-index in 10,000 simulation sets increased by 0.025 by adding response data to IPSS-R score (I versus IR in Fig 1B). Further improvements were obtained by adding gene mutation and max(VAF), in which the c-index increased by 0.034 (IR versus IGR in Fig 1B) and 0.010 (IGR versus IGRP in Fig 1B), respectively. For the 53 patients who received allogeneic stem cell transplantation, the median post-transplant OS was 82.6 months (range, 36.3 to not reached). Notably, max(VAF) significantly stratified OS after allo-SCT (HR, 3.3; 95%CI, 1.3 to 8.3; P = .014). Conclusions Our study revealed that post-treatment clone size significantly correlated with clinical response and the evaluation of post-treatment clone size allows for more precise prognostication after AZA treatment compared with IPSS-R and clinical response alone. Table Disclosures Naoe: NIPPON SHINYAKU CO.,LTD.: Speakers Bureau; Sysmex co.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Astellas Pharma Inc.: Speakers Bureau; Bristol-Myers Squibb Company: Speakers Bureau. Miyazaki:Celgene: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Otsuka Pharmaceutical: Honoraria; Astellas Pharma Inc.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Papaemmanuil:Kyowa Hakko Kirin: Consultancy, Honoraria; Prime Oncology: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; MSKCC: Patents & Royalties; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Ogawa:Eisai Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding.

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Integrated Transcriptomic and Proteomic Analyses of Inflammasome in Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2019-128094 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2991-2991

Author(s):

María Zurdo ◽

Ana M Hurtado López ◽

Tzu Hua Chen-Liang ◽

Helios Martínez-Banaclocha ◽

Laura Palomo ◽

...

Keyword(s):

Board Of Directors ◽

Differentially Expressed Genes ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Myelomonocytic Leukemia ◽

Differentially Expressed ◽

Advisory Committees ◽

Myelomonocytic Leukemia

Background and aim: Inflammasome and pyroptosis overactivation have recently been associated as fundamental mechanisms in the ineffective hematopoiesis of myelodysplastic syndromes (MDS). Chronic myelomonocytic leukemia (CMML) shares histological and clinical characteristics with MDS but, within clinical differences, It stands out a high association with inflammatory/autoimmune diseases in which a disproportionate activation of inflamasome has been implicated. Our hypothesis is that CMML cases show a higher inflammasome activation with respect to the MDS subset, a relevant difference both in terms of potential therapeutic targets and pathogenic clues. The main objective is to confirm, describe and quantify these differences using high-performance and multi-gene/protein methods. Methods: We performed enhanced RNA-seq in bone marrow mononucleated cells of 27 CMML at diagnosis, 10 MDS and 9 controls (103 million average readings). We selected 116 genes related to the inflammasome and reviewed the differential expression between cases and controls. We evaluated by multiplex immunoassay the profile of 28 cytokines in peripheral blood in 35 CMML patients, 37 MDS and 8 controls. Subsequently, we studied whether these differentially expressed genes / cytokines showed differences in CMML depending on the mutational state of TET2, SRSF2 and ASXL1. Finally, we compared in vitro the degree of activation of inflamasome in the monocytoid component of 8 CMML patients versus 7 controls. Results: In the transcriptomic analysis of the inflamasome genes in patients with CMML, we found 30 of 116 differentially expressed genes compared with healthy controls. Of those 30 genes, 26 showed a pro-inflammatory function and, of them, 18 were up-regulated. Of the 4 differentially expressed genes with an anti-inflammatory function, 3 were significantly under-expressed in CMML patients. We highlight, due to the quantitative difference, the overexpression of two genes coding for monocyte chemotactic proteins, CCL7 and CCL2 (FC = 269.21, p = 0.032; FC = 11.79, p = 0.03) That pro-inflammatory transcriptional profile was not so evident in the cases of MDS: of the 29 differentially expressed genes with pro-inflammatory function, 18 were down-regulated. Subsequently, we designed a customized panel for proteomic analysis including 9 of the 30 differentially expressed genes in CMML. We found that, in a relevant percentage of cases, also proinflammatory cytokines derived from these differentially expressed genes were elevated (62.5%) in peripheral blood of patients, compared to healthy donors; pointing towards the key role of gene transcription in the definition of the pro-inflammatory sense of the proteomic dimension of inflammasome in CMML. Next, we found that those patients with CMML and somatic mutations of TET2 had a higher expression of CCL7 and CCL2 compared to patients with CMML wild type, with a tendency to significance in the first case and significant in the second (FC 11.9, p = 0.15; FC 7.8, p = 0.03). Finally, we conducted in vitro stimulation studies at diagnosis in patients with CMML confirming that the canonical activation of the NLRP3 inflammasome (increased production of IL-1β) is significantly enhanced with respect to control individuals. Conclusion: We describe for the first time, a hyperactivation in CMML, compared with MDS, of the components of the inflammasome. Hyperactivation associated in CMML to a gene transcriptional mechanism and related, in the case of the two most over-expressed genes, CCL2 and CCL7, to the presence of mutations in TET2. Our findings point to new therapeutic targets whose modulation could restore inefficient hemopoiesis and potential diagnostic and prognostic biomarkers in CMML. Disclosures Díez-Campelo: Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerez:Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Longitudinal T Cell Immunoprofiling of Patients with Relapsed and/or Refractory Myeloma Who Receive Daratumumab Monotherapy: A Subanalysis of a Phase 2 Study (the REBUILD Study)

Blood ◽

10.1182/blood-2019-124655 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3167-3167 ◽

Cited By ~ 1

Author(s):

Elisavet Vlachonikola ◽

Anna Vardi ◽

Eftathios Kastritis ◽

Evdoxia Hatjiharissi ◽

Eirini Katodritou ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Clinical Response ◽

Board Of Directors ◽

Research Funding ◽

Post Treatment ◽

Secondary Endpoint ◽

Median Frequency ◽

Advisory Committees ◽

Pre Treatment

Recent evidence suggests immunomodulatory effects of daratumumab in heavily pre-treated Multiple Myeloma (MM) patients (pts); however, the precise effects remain under-characterized, particularly at the molecular level. REBUILD is an ongoing prospective, multicenter, non-comparative, open-label, phase II study that evaluates the effects of daratumumab monotherapy on bone metabolism of pts with relapsed and/or refractory MM (RRMM) who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Secondary endpoint of the study included the evaluation of T cell dynamics by comprehensive analysis of the T cell receptor (TR) repertoire employing next generation sequencing (NGS) and multi-color flow cytometry. Herein we report the results of this secondary endpoint for the first 14 pts who completed 3 cycles of daratumumab monotherapy. In total, we analyzed 28 paired samples collected at screening (n=14) and on Day 1 of Cycle 4 (C4D1, n=14) of treatment in order to assess potential changes in relation to treatment and clinical response. Patients were grouped based on best responses into responders (i.e. patients with partial response [PR, n=1] and very good PR [VGPR, n=6]), and non-responders (i.e. patients with minimal response [MR, n=2], stable disease [SD, n=4], or progressive disease [PD, n=1]). Starting material was peripheral blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw NGS reads (n=6,715,406 | median 221,145/sample) were processed through a previously published, purpose-built bioinformatics pipeline. Only productive TRBV-TRBD-TRBJ rearrangements were taken into consideration (n=3,097,565 | median 101,670/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 151,153 distinct clonotypes (median 5,084 clonotypes/sample) were assessed. Both groups (responders/non-responders) displayed clonal T cell expansions both pre- and post-treatment. Clonality was found to be increased after treatment for both responders and non-responders, with statistical significance in the former (median cumulative frequency of the 10 most expanded T cell clonotypes/sample: 31% pre-treatment versus 40% post-treatment, respectively | p=0.04). In both groups, the clonotype repertoire appeared to be renewed with only a small fraction of pre-treatment clonotypes remaining after treatment (1% for non-responders; 0.6% for responders). Interestingly, in the responders' group we noticed a significant shift in the major clonotype repertoire at screening vs C4D1. In particular, in the responders' group the 10 most expanded clonotypes/sample at C4D1 represented expansions of clonotypes present at very low frequency at screening, whereas the most expanded clonotypes at screening decreased or even diminished post-treatment, suggesting that daratumumab treatment led to the emergence of anti-myeloma T cell clones which contributed to clinical response. On the contrary, the 10 most expanded pre-treatment clonotypes in the non-responders' group tended to dominate also the post-treatment repertoire. Of note, 13 shared clonotypes were identified amongst the post-treatment repertoires of different patients (responders/non-responders); shared clonotypes were not found in other entities in public databases, raising the possibility that they may be "disease-specific" and selected by common tumor-associated antigens. With a single exception, shared clonotypes were detected in cases with relevant HLA restrictions, which is noteworthy given the random HLA background of our cohort. Finally, flow cytometry analysis revealed a significant increase post treatment in the percentage of CD3+ T cells (median frequency at screening 60% versus 83% at C4D1 | p=0.003), driven mostly by the expansion of the CD8+ T cell compartment (median frequency at screening 30.8% versus 48.9% at C4D1 | p=0.03) in both groups. In conclusion, TR clonality increases post-treatment through a renewal mechanism; however, pre-treatment clones significantly expanded post-treatment in responders, alluding to the existence of clonotypes with anti-MM properties that may be activated after treatment with daratumumab, arguably contributing to clinical response. Disclosures Kastritis: Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria; Amgen: Honoraria, Research Funding. Hatjiharissi:Janssen: Honoraria. Katodritou:Genesis: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Gavriatopoulou:Genesis: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses; Amgen: Honoraria. Delimpasi:Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Genesis: Honoraria, Other: Travel grant. Symeonidis:Sanofi: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: 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, Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Janssen: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Genesis: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria; Takeda: Honoraria, Other: Travel expenses, Research Funding. Chatzidimitriou:Janssen: Honoraria.

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Automated Manufacture of Matched Donor-Derived Allogeneic CD19 CAR T-Cells for Relapsed/Refractory B-ALL Following Allogeneic Stem Cell Transplantation: Toxicity, Efficacy and the Important Role of Lymphodepletion

Blood ◽

10.1182/blood-2019-129640 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 776-776

Author(s):

Claire Roddie ◽

Maeve A O'Reilly ◽

Maria A V Marzolini ◽

Leigh Wood ◽

Juliana Dias Alves Pinto ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Peak Car ◽

Matched Donor

Introduction: 2nd generation CD19 CAR T cells show unprecedented efficacy in B-ALL, but several challenges remain: (1) scaling manufacture to meet patient need and (2) feasibility of generating products from lymphopenic patients post allogeneic stem cell transplant (allo-SCT). To overcome these issues we propose: (1) use of the CliniMACS Prodigy (Miltenyi Biotec), a semi-automated cGMP platform that simplifies CAR T cell manufacture and (2) the use of matched donor T cells to overcome the challenge posed by patient lymphopenia, albeit this may come with a heightened risk of graft versus host disease (GvHD). CARD (NCT02893189) is a Phase I study of matched donor derived CD19 CAR T cells generated on the CliniMACS Prodigy in 14 adult patients with relapsed/refractory (r/r) B ALL following allo-SCT. We additionally explore the requirement for lymphodepletion (LD) in the allogeneic CAR T cell setting and report on the incidence of GvHD with this therapy. Methods: Manufacturing: CARD utilises non-mobilised matched donor leucapheresate to manufacture 2nd generation CD19CAR T cells using a closed CliniMACS® Prodigy/ TransACTTM process. Study design: Eligible subjects are aged 16-70y with r/r B ALL following allo SCT. Study endpoints include feasibility of CD19CAR T cell manufacture from allo-SCT donors on the CliniMACS Prodigy and assessments of engraftment and safety including GvHD. To assess the requirement for LD prior to CD19CAR T cells in lymphopenic post-allo-SCT patients, the study is split into Cohort 1 (no LD) and Cohort 2 (fludarabine (30 mg/m2 x3) and cyclophosphamide (300mg/m2 x3)). To mitigate for the potential GvHD risk, cell dosing on study mirrors conventional donor lymphocyte infusion (DLI) schedules and is based on total CD3+ (not CAR T) cell numbers: Dose 1=1x106/kg CD3+ T cells; Dose 2= 3x106/kg CD3+ T cells; Dose 3= 1x107/kg CD3+ T cells. Results: As of 26 July 2019, 17 matched allo SCT donors were leukapheresed and 16 products were successfully manufactured and QP released. Patient demographics are as follows: (1) median patient age was 43y (range 19-64y); (2) 4/17 had prior blinatumomab and 5/17 prior inotuzumab ozogamicin; (3) 7/17 had myeloablative allo SCT and 10/17 reduced intensity allo SCT of which 6/17 were sibling donors and 12/17 were matched unrelated donors. No patients with haploidentical transplant were enrolled. To date, 12/16 patients have received at least 1 dose of CD19CAR T cells: 7/16 on Cohort 1 and 5/16 on Cohort 2 (2/16 are pending infusion on Cohort 2 and 2/16 died of fungal infection prior to infusion). Median follow-up for all 12 patients is 22.9 months (IQR 2.9-25.9; range 0.7 - 25.9). At the time of CAR T cell infusion, 7/12 patients were in morphological relapse with >5% leukemic blasts. Despite this, CD19CAR T cells were administered safely: only 2/12 patients experienced Grade 3 CRS (UPenn criteria), both in Cohort 1, which fully resolved with Tocilizumab and corticosteroids. No patients experienced ≥Grade 3 neurotoxicity and importantly, no patients experienced clinically significant GvHD. In Cohort 1 (7 patients), median peak CAR expansion by flow was 87 CD19CAR/uL blood whereas in Cohort 2 (5 patients to date), median peak CAR expansion was 1309 CD19CAR/uL blood. This difference is likely to reflect the use of LD in Cohort 2. CAR T cell persistence by qPCR in Cohort 1 is short, with demonstrable CAR in only 2/7 treated patients at Month 2. Data for Cohort 2 is immature, but this will also be reported at the meeting in addition to potential mechanisms underlying the short persistence observed in Cohort 1. Of the 10 response evaluable patients (2/12 pending marrow assessment), 9/10 (90%) achieved flow/molecular MRD negative CR at 6 weeks. 2/9 responders experienced CD19 negative relapse (one at M3, one at M5) and 3/9 responders experienced CD19+ relapse (one at M3, one at M9, one at M12). 4/10 (40%) response evaluable patients remain on study and continue in flow/molecular MRD negative remission at a median follow up of 11.9 months (range 2.9-25.9). Conclusions: Donor-derived matched allogeneic CD19 CAR T cells are straightforward to manufacture using the CliniMACS Prodigy and deliver excellent early remission rates, with 90% MRD negative CR observed at Week 6 in the absence of severe CAR associated toxicity or GvHD. Peak CAR expansion appears to be compromised by the absence of LD and this may lead to a higher relapse rate. Updated results from Cohorts 1 and 2 will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Qasim:Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

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Identification of Two CAR T-Cell Populations Associated with Complete Response or Progressive Disease in Adult Lymphoma Patients Treated with Axi-Cel

Blood ◽

10.1182/blood-2019-130815 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 779-779 ◽

Cited By ~ 2

Author(s):

Zinaida Good ◽

Jay Y. Spiegel ◽

Bita Sahaf ◽

Meena B. Malipatlolla ◽

Matthew J. Frank ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Advisory Committees ◽

Car T Cells ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory

Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

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Human CD83 Targeted Chimeric Antigen Receptor T Cell for the Prevention of Graft Versus Host Disease and Treatment of Myeloid Leukemia

Blood ◽

10.1182/blood-2019-124031 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 196-196

Author(s):

Bishwas Shrestha ◽

Kelly Walton ◽

Jordan Reff ◽

Elizabeth M. Sagatys ◽

Nhan Tu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Graft Versus Host ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Fund Research

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.

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BCX9930, a Potent, Selective, Oral Factor D Inhibitor, Demonstrates Proof-of-Concept As Monotherapy in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Blood ◽

10.1182/blood-2020-138838 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-15

Author(s):

Austin Kulasekararaj ◽

Jacques Le Roux Malherbe ◽

Andrew McDonald ◽

Melanie Cornpropst ◽

Phil Collis ◽

...

Keyword(s):

South Africa ◽

Board Of Directors ◽

Research Funding ◽

Total Bilirubin ◽

Proof Of Concept ◽

Advisory Committees ◽

Clone Size ◽

The Past ◽

History Of ◽

Pre Treatment

INTRODUCTION: PNH, a rare, chronic, life-threatening disease, is characterized by hemolytic anemia due to uncontrolled activity of the complement alternative pathway (AP), bone marrow failure, and thrombosis. Inhibition of C5 by intravenously administered eculizumab and ravulizumab reduces intravascular hemolysis, but PNH red blood cells (RBCs) become opsonized and susceptible to extravascular hemolysis (Risitano et al, Blood 2009). Only approximately half of PNH patients become transfusion independent with eculizumab treatment (Hillmen et al, NEJM 2006). BCX9930 is a potent, selective, orally administered inhibitor of complement factor D. Inhibition of factor D may prevent both intravascular and extravascular hemolysis in PNH. In healthy subjects, BCX9930 showed linear pharmacokinetics and dose-related AP suppression, and was safe and generally well-tolerated over a wide dose range. Here we describe safety and laboratory data establishing proof-of-concept for BCX9930 monotherapy in PNH patients in Study BCX9930-101 (NCT04330534). METHODS: Ongoing Study BCX9930-101 includes an open-label, dose-ranging evaluation of BCX9930 in PNH subjects who may either be naïve to C5 inhibitors (and receive BCX9930 as monotherapy) or have an incomplete treatment response to eculizumab or ravulizumab (with BCX9930 added to existing treatment). Up to 4 sequential cohorts each use a forced titration design for the first 28 days (Figure 1). Subjects enrolled in South Africa can participate in an individualized 48-week extension if they derive benefit at Day 28. Clinical benefit from BCX9930 is evaluated using laboratory monitoring and symptom assessment. Safety and tolerability are evaluated via clinical and laboratory monitoring, causality of adverse events is assessed by investigators, and the study is overseen by an independent Data Monitoring Committee. Data from Cohort 1 through 28 days is reported; data from the extension and subsequent cohorts will be subsequently summarized as available. RESULTS: To date, four C5 inhibitor naïve PNH subjects in South Africa have enrolled in Cohort 1. These subjects had PNH for a median of 4.5 years; 2 subjects had a history of transfusions in the past year; 1 subject each had a history of aplastic anemia or major thrombosis. Pre-treatment lactate dehydrogenase (LDH), total bilirubin, hemoglobin (Hb), reticulocyte count, and RBC PNH Type III clone size ranged from 3.7-11.1 × ULN, 0.61-3.3 mg/dL, 6.1-11.6 g/dL, 0.13-0.29 × 106/µL, and 41.4%-88.6% respectively. Treatment over 28 days with 50 mg twice daily (BID; Days 1-14) and 100 mg BID (Days 15-28) of BCX9930 produced dose-dependent, clinically meaningful improvements across hemolysis biomarkers (Figure 2). Decreases were observed in LDH (4/4), reticulocytes (4/4), and total bilirubin (2/2 subjects with elevated pre-treatment values). Increases were observed in Hb (3/4) and PNH RBC clone size (4/4). One subject showed an initial response to BCX9930 50 mg BID, followed by worsening indicators of hemolysis temporally associated with an upper respiratory tract infection (URTI; onset on Day 7). With an increase in dose to 100 mg BID and resolution of the URTI, LDH and reticulocytes fell and Hb rose. All four subjects reported one or more PNH-associated symptoms, including hemoglobinuria, jaundice, fatigue, erectile dysfunction, headache and abdominal pain, prior to enrollment. With the exception of one subject with persistent hemoglobinuria, all symptoms resolved by Day 28 on BCX9930. Three subjects experienced moderate headache that resolved in < 3 days after initiating BCX9930. One subject developed a rash during treatment with amoxicillin for an URTI; the rash resolved while continuing BCX9930 dosing. One subject on concomitant chronic corticosteroids and azathioprine had an unrelated fatal serious adverse event of disseminated varicella during the study extension. Based on review of safety data, Cohort 2 opened at doses of 200 mg BID and 400 mg BID and, in the 3 subjects who continued into the extension, the dose was titrated to ≥ 200 mg BID. CONCLUSIONS: Oral BCX9930 elicited rapid changes in laboratory parameters indicative of reduced hemolysis and clinical benefit and was safe and generally well-tolerated over a 28-day dosing interval. These interim results establish proof of concept for monotherapy with BCX9930 in the treatment of C5-inhibitor naïve PNH patients and support evaluation of higher doses. Disclosures Kulasekararaj: Alexion:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau;Ra Pharma:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau;BioCryst Pharmaceuticals, Inc.:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Apellis:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau;Roche:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Novartis:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau;Celgene:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau.Malherbe:Key Oncologics:Honoraria, Other: Conference sponsor;Novartis:Other: Conference sponsor;Astellas:Honoraria, Other: Conference sponsor;Takeda:Consultancy;Acino:Honoraria;Shire:Other: Conference sponsor;BioCryst Pharmaceuticals, Inc.:Consultancy;Janssen:Consultancy, Honoraria, Other: Conference sponsor;Roche:Honoraria, Other: Conference sponsor.McDonald:venetoclax advisory board in South Africa (in CLL context):Consultancy;Alberts Cellular Therapy:Current Employment.Cornpropst:BioCryst Pharmaceuticals, Inc.:Current Employment.Collis:BioCryst Pharmaceuticals, Inc.:Current Employment.Davidson:BioCryst Pharmaceuticals, Inc.:Current Employment.Chen:BioCryst Pharmaceuticals, Inc.:Current Employment.Tower:BioCryst Pharmaceuticals, Inc.:Current Employment.Gesty-Palmer:BioCryst Pharmaceuticals, Inc.:Current equity holder in publicly-traded company, Ended employment in the past 24 months.Sheridan:BioCryst Pharmaceuticals, Inc.:Current Employment.Risitano:Alexion:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Alnylam:Research Funding;Novartis:Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Pfizer:Speakers Bureau;Achillion:Membership on an entity's Board of Directors or advisory committees;Apellis:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Biocryst:Membership on an entity's Board of Directors or advisory committees;RA pharma:Research Funding;Amyndas:Consultancy;Samsung:Membership on an entity's Board of Directors or advisory committees;Roche:Membership on an entity's Board of Directors or advisory committees;Jazz:Speakers Bureau.

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