Systematic STAT3 Mutation Testing Identifies Patients with Unsuspected T-Cell Large Granular Lymphocytic Leukemia

Abstract The initial clinical presentation of T-cell large granular lymphocytic leukemia (T-LGL) and myelodysplastic syndromes (MDS) can be similar, each characterized by unexplained peripheral cytopenias. However, these diseases are pathobiologically distinct and associated with stark differences in prognosis and therapy. T-LGL is a clonal lymphoid disorder defined by phenotypically abnormal cytotoxic T cells and an indolent clinical course, while MDS is a clonal disorder of hematopoietic stem cells defined by ineffective hematopoiesis, morphologic dysplasia, and an elevated risk of acute leukemia. Despite these differences, distinction between T-LGL and MDS can be challenging and misdiagnosis can significantly delay initiation of appropriate therapy. The recent identification of STAT3 mutations in LGL may facilitate this distinction: activating STAT3 mutations occur in 40-70% of T-LGL cases, primarily within the SH2 domain, but have not been reported in patients with MDS without concomitant T-LGL. STAT3 is included within our clinical next generation sequencing (NGS) panel, which is used to evaluate patients with known or suspected hematologic malignancies, primarily acute myeloid leukemia, MDS and myeloproliferative neoplasms, as well as various lymphocytic leukemias. We report the frequency and type of STAT3 mutations within our patient population and assess the impact of this information on diagnosis. Between 1/1/2015 and 6/30/2016, 3414 samples (primarily peripheral blood (PB) or bone marrow (BM)) from 2530 unique patients evaluated at Brigham and Women's Hospital and/or Dana-Farber Cancer Institute underwent clinical NGS with a custom, 95-gene, amplicon-based panel (PMID: 27339098). Exons 2-17 and 21-23 of the STAT3 gene were analyzed in each sample using reference transcript 1 (NM_139276). We identified 40 patients with 40 candidate STAT3 mutations (Figure 1). Based on domain localization, variant allele fraction, and population allele frequency, we classified these sequence variants as somatic SH2 domain mutations (n = 21), somatic non-SH2 domain mutations of unknown significance (n = 5) or germline variants (n = 14). Of the 21 patients with somatic SH2 domain mutations, 9 carried a prior diagnosis of T-LGL and 8 were concurrently diagnosed with T-LGL by conventional diagnostic criteria (clonal aberrant T cells in the setting of neutropenia, anemia, or lymphocytosis). The final 4 patients with STAT3 SH2 domain mutations were unexpected diagnoses of T-LGL. These 4 patients were initially referred from outside institutions for MDS based on the reported presence of unilineage erythroid dysplasia (n=2), unquantified ring sideroblasts (n=1), or pancytopenia with unspecified marrow findings (n=1). In 3 of these cases, the STAT3 mutation discovery prompted T-cell flow cytometric analysis of peripheral blood, which revealed an aberrant immunophenotype, and T-cell receptor gamma gene rearrangement studies, which were clonal; these tests are pending in the 4th case. BM evaluation was performed in 12 of 21 patients, including the 4 with suspected MDS; in all cases, the findings did not meet diagnostic criteria for MDS by expert hematopathology review and all showed a normal karyotype. Five additional cases demonstrated somatic non-SH2 domain STAT3 mutations of unknown pathobiologic significance: 3 myeloid neoplasms, 1 chronic lymphocytic leukemia, and 1 autoimmune hemolytic anemia. Additional non-STAT3 mutations were also frequently identified in tumors other than T-LGL. Our experience demonstrates that STAT3 sequencing is a critical component of the evaluation of unexplained cytopenias, and identification of a mutation can clarify ambiguous phenotypes thus averting the consequences of misdiagnosis or diagnostic delay. Notably, several series have reported that MDS and T-LGL can infrequently occur concurrently and thus identification of a STAT3 mutation and a clonal T-LGL population does not exclude the possibility of concomitant MDS. In our cohort, however, no T-LGL patient with a STAT3 mutation demonstrated pathologic evidence of MDS and the majority (19 of 21) showed no other myeloid-associated somatic mutations. In addition, 5 cases in our cohort had likely somatic, non-SH2 domain STAT3 mutations in the context of disparate clinical scenarios, suggesting that these mutations may have a pathogenic role in other hematologic malignancies, a subject of future study. Disclosures DeAngelo: Ariad: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Incyte: Consultancy; Celgene: Consultancy. Stone:Celator: Consultancy; Jansen: Consultancy; ONO: Consultancy; Agios: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Merck: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy.

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Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Blood ◽

10.1182/blood-2020-138601 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 42-43

Author(s):

Prajish Iyer ◽

Lu Yang ◽

Zhi-Zhang Yang ◽

Charla R. Secreto ◽

Sutapa Sinha ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Gene Signature ◽

T Cell Subsets ◽

Rna Seq ◽

Advisory Committees

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: 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; 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; 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; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding: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; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

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IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽

10.1182/blood.v116.21.2431.2431 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2431-2431

Author(s):

Marta Coscia ◽

Francesca Pantaleoni ◽

Chiara Riganti ◽

Candida Vitale ◽

Micol Rigoni ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Board Of Directors ◽

Stromal Cells ◽

Peripheral Blood ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Spontaneous Apoptosis ◽

Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

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Cellular Immune Responses To Vector In a Gene Therapy Trial For Hemophilia B Using An AAV8 Self-Complementary Factor IX Vector

Blood ◽

10.1182/blood.v122.21.717.717 ◽

2013 ◽

Vol 122 (21) ◽

pp. 717-717

Author(s):

Etiena Basner-Tschakarjan ◽

Federico Mingozzi ◽

Yifeng Chen ◽

Amit Nathwani ◽

Edward Tuddenham ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Factor Ix ◽

High Dose ◽

Advisory Committees ◽

Ifn Γ ◽

Cellular Immune

Abstract In a clinical study of gene transfer for hemophilia B an adeno-associated virus vector serotype 8 (AAV8) expressing a self-complementary liver-specific expression cassette for the factor IX (FIX) transgene was administered intravenously in ten affected subjects. The results of the first part of the study have been published (NEJM 365:2357-65, 2011). In this abstract we present the immunomonitoring data, using Interferon-gamma (IFN-γ) ELISpot and polyfunctional T cell analysis of peripheral blood mononuclear cells (PBMCs) to monitor cellular immune responses to vector capsid and to Factor IX. We have previously shown that the cellular immune response was directed solely towards AAV capsid epitopes, not FIX, and that the response was dose-dependent. Out of six subjects infused in the high dose cohort (2x1012vg/kg), 4/6 manifested a minor rise in liver enzyme levels and detection of capsid-specific T cell reactivitiy in the ELISpot assay at ∼7-10 weeks post vector infusion. Maximum results on IFN- γ ELISpots ranged from 200-500 sfu/million cells. In two of these cases a modest decline in FIX level also occurred. Prompt initiation of prednisolone reversed these effects and rescued FIX levels. The remaining two subjects infused at the high dose, showed no rise in liver enzyme levels at any time point. However capsid reactive T cells were detectable in one subject as early as one to two weeks after vector infusion in peripheral blood by IFN-γ ELISpot assay, while no activation at all was detected in the other subject, possibly due to low cell recovery and viability of the cells. A similar immune response profile, with early detection of activated T cells but no rise in liver enzymes, was also observed in both subjects in the intermediate dose cohort in the first part of this study. Polyfunctional T cell analysis revealed concurrent Interleukin-2, Tumor necrosis factor-alpha and CD107a positivity in activated T cells at the peak of activation. Furthermore it showed that capsid-specific early T cell responses were detectable in the CD4+ T cell and later in the CD8+T cell compartment. Long-term immune monitoring of all subjects is ongoing. Importantly in one of the first two subjects treated at the high dose, capsid reactive T cells were detected by ELISpot 1.5 years after gene transfer; these cells were not detected in the other subject in whom long-term follow-up samples are available. Of note, capsid-reactive T cells were also seen at late time points (>1 year after infusion) in a middle dose subject and a low dose subject. Despite detectable T cell reactivity towards the AAV capsid in the peripheral blood FIX expression remained stable, suggesting that there is a short window of time during which transduced hepatocytes present a target for cytotoxic T cells, and that T cell positivity after this window is without any clinical consequences. In conclusion, for this scAAV8 vector there appears to be a critical threshold vector dose for a clinically detectable immune response, starting at 2x1012 vg/kg. The clinically detectable response occurred in four out of six subjects so far, and was manifest within a critical time interval of 7-10 weeks post infusion. The capsid-specific response was polyfunctional and detected in CD4+ and CD8+T cells in peripheral blood. It is important to note that not all subjects treated at the high dose developed an immune response. However, given the limited dataset, it is not yet possible to define predictive parameters, e.g. HLA type of a subject, for an immune response. Continued monitoring and future studies with more subjects will be necessary to confirm the presented findings, in particular time and rate of occurrence of a cellular response as well as successful treatment with a short course of Prednisolon. Disclosures: Tuddenham: Pfizer: Consultancy. Reiss:Hemophilia of Georgia: Honoraria. High:BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

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Characterization of Allogeneic T Cells Expressing Inducible Caspase-9 Following Adoptive Transfer in Children Receiving an HLA-Haploidentical Hematopoietic Stem Cell Transplant for the Treatment of Myeloid Malignancies

Blood ◽

10.1182/blood-2018-99-119779 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4534-4534

Author(s):

Joanne Shaw ◽

Xiaoou Zhou ◽

An Lu ◽

Valentina Bertaina ◽

Daria Pagliara ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Adoptive Transfer ◽

Peripheral Blood ◽

Employment Equity ◽

Advisory Committees ◽

Myeloid Malignancies ◽

Donor T Cells ◽

Equity Ownership

Abstract Background: Adoptive transfer of allogeneic donor T cells can be an effective treatment for hematological malignancies through recognition of leukemia-associated antigens (LAAs) on tumor cells or through alloreactivity. However, alloreactive T cells can also cause graft-versus-host disease (GvHD) limiting their use as an immunotherapy. To leverage the anti-tumor effects of allogeneic polyclonal T cells while minimizing GvHD, we have genetically modified donor T cells with the inducible caspase-9 (iC9) safety switch, which induces apoptosis following exposure to the small molecule ligand rimiducid. Here we show that iC9-modified allogeneic T cells (BPX-501) persist, expand and contain functional LAA-specific T cells in children receiving an alpha/beta TCR and CD19-depleted HLA-haploidentical hematopoietic stem cell transplant (haplo-HSCT) for the treatment of myeloid malignancies. Methods: Pre-infusion products (BPX-501: donor T cells modified with the bicistronic retroviral vector encoding iC9 and truncated CD19 (ΔCD19)) and patient peripheral blood mononuclear cells (PBMCs) were analyzed from twelve patients (AML (10), MDS (1), JMML (1)) receiving BPX-501 (1x106 cells/kg) following an alpha/beta T cell and CD19 B cell-depleted haplo-HSCT (BP-004U: NCT03301168). Engraftment and persistence were measured by coexpression of CD3 and CD19 by flow-cytometry. Endogenous and gene-modified T cells were also phenotyped for CD4:CD8 ratios, memory cell composition (TN, TCM, TEM, TEMRA; CD45RA and CD62L) and T cell receptor Vβ diversity. BPX-501 products and post-treatment samples were characterized for LAA-specific T cells using IFN-γ ELISpot against peptide pools (15 aa overlapping by 5 aa) derived from WT1, PRAME, MAGE (A1, C1, C3), NE and PR3, with and without exposure to 10 nM rimiducid to determine the anti-leukemic contribution of BPX-501. Results: BPX-501 was infused at a median time of 22.5 days after HSCT (range 12-34, one patient was infused at day 89 and one patient was infused at day 147). BPX-501 cells (CD3+CD19+) were detectable in the peripheral blood at 1-2 weeks after infusion in all 12 patients, reaching a peak expansion frequency of a median of 24% ± 17% of total CD3+ T cells, and an absolute cell number of 66.9 ± 112 cells/µl at 2 months post-infusion and could be detected for up to 24 months. BPX-501 T cells showed a CD8-skewed phenotype whereas endogenous T cells exhibited a more balanced CD4:CD8 ratio. BPX-501 were predominantly CD45RA-CD62L+ and CD45RA-CD62L- central and effector memory T cells, respectively. In BPX-501 products, we detected LAA-specific T cells by ELISpot using overlapping peptide pools to WT1, PRAME, MAGE, NE and PR3, and in peripheral blood samples obtained 2 to 5 months post-T cell infusion. Importantly, LAA-reactivity was greatly diminished with exposure to iC9-activating rimiducid. Further, we measured the TCR Vβ usage and observed highly-skewed TCR repertoire in BPX-501 T cells compared to endogenous T cells in 6 months after HSCT indicating selection and expansion of TCR clones. Three patients engrafted BPX-501 were treated with rimiducid to control GvHD resulting in a rapid decrease (62% ± 12%) of CD3+CD19+ T cells in the peripheral blood. In patients treated with rimiducid, CD3+CD19+ T cells recover without further instances of GvHD suggestive of in vivo depletion of alloreactive T cell clones using iC9. Summary: Allogeneic T cells engineered with the iC9 safety switch engraft, expand and demonstrate long-term persistence following adoptive transfer into patients receiving a haplo-HSCT. LAA-specific T cells and alloreactive T cells within the BPX-501 product are detectable in the peripheral blood following infusion and likely contribute to elimination of myeloid malignancies. Disclosures Shaw: Bellicum Pharmaceuticals: Employment, Equity Ownership. Zhou:Bellicum Pharmaceuticals: Employment, Equity Ownership. Lu:Bellicum Pharmaceuticals: Employment, Equity Ownership. Aldinger:Bellicum Pharmaceuticals, Inc.: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Foster:Bellicum: Employment, Equity Ownership.

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Synergistic Role of Leukemic and Non-Leukemic Immune Repertoires in CD8+ T-Cell Large Granular Lymphocytic Leukemia As Identified By Single-Cell Transcriptomics

Blood ◽

10.1182/blood-2021-146539 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1318-1318

Author(s):

Dipabarna Bhattacharya ◽

Jani Huuhtanen ◽

Matti Kankainen ◽

Tapio Lönnberg ◽

Cassandra M Kerr ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Healthy Controls ◽

Advisory Committees ◽

Cell Clones ◽

Tcr Repertoire

Abstract Background: T-cell large granular lymphocytic leukemia (T-LGLL), a rare lymphoproliferative disorder of mature T cells, is characterized by the accumulation of activated effector T cells leading to a clonally restricted T-cell receptor (TCR) repertoire. Chronic antigen stimulation together with activating somatic STAT3 mutations have been proposed to lead to clonal expansion of leukemic cells. However, no holistic research has been done to show how leukemic and non-leukemic cells liaise to sustain abnormal immune reactivity in T-LGLL. Methods: We investigated the transcriptome and TCR repertoire in T-LGLL using: 1) single-cell RNA and TCR (scRNA+TCRαβ) sequencing from CD45+ sorted blood cells (T-LGLL n=11, healthy n=6), 2) TCRβ sequencing from blood mononuclear cells (T-LGLL n=48, healthy n=823), 3) bulk RNA sequencing (T-LGLL n=15, healthy n=5), 4) plasma cytokine profiling (T-LGLL n=9, healthy n=9), and 5) flow cytometry validations (T-LGLL n=6, healthy n=6) (Figure) Results: ScRNA+TCRαβ-seq data revealed that in healthy controls, hyperexpanded CD8+ T-cell clones (at least 10 cells with identical TCRs) preferentially had an effector memory phenotype, whereas in T-LGLL, the hyperexpanded clonotypes represented a more cytotoxic (increased expression of GZMB, PRF1, KLRB1) and exhausted (LAG3 and TIGIT) phenotype. Using flow cytometry, we confirmed that upon anti-CD3/CD28/CD49 antibody stimulation, T-LGLL clones (CD8+CD57+) expressed higher levels of cytotoxic proteins (GZMA /GZMB , PRF1) but were deficient in degranulation responses and cytokine secretion as measured by expression of CD107a/b and TNFα/IFNγ, respectively. Focused re-clustering of the extracted T-LGLL clones from the scRNA+TCRαβ-seq data revealed considerable heterogeneity among the T-LGLL clones and partly separated the mutated (mt) STAT3 and wild type (wt) STAT3 clones. STAT3wt clones upregulated T-cell activation and TCR signaling pathways, with a higher cytotoxicity and lower exhaustion score as compared to STAT3mt clones. This was validated with bulk RNA-seq data. To understand the antigen specificities of the T-LGLL clones, we combined previously profiled T-LGLL TCRs with our data to form the largest described dataset of 200 T-LGLL clones from 170 patients. Notably, T-LGLL clones were found to be private to each patient. Furthermore, the analysis by GLIPH2 algorithm grouping TCRs did not reveal detectable structural similarities, suggesting the absence of a unifying antigen in T-LGLL. However, in 67% of T-LGLL patients, the TCRs of leukemic clones shared amino acid level similarities with the rest of the non-leukemic TCR repertoire suggesting that the clonal and non-clonal immune repertoires are connected via common target antigens. To analyze the non-clonal immune repertoire in T-LGLL in detail, we compared our data to other published scRNAseq data from solid tumors (n=4) and hematologic cancers (n=8) and healthy controls (n=6). The analysis revealed that in T-LGLL also the non-leukemic CD8+ and CD4+ T cells were more mature, cytotoxic, and clonally restricted. When compared to healthy controls and other cancer patients, in non-leukemic T-LGLL the most upregulated pathway was IFNγ response. Finally, most of the upregulated cytokines in T-LGLL (e.g., CCL2/3/7, CXCL10/11, IL15RA) were secreted predominantly by monocytes and dendritic cells, which also had upregulated HLA class II expression and enhanced scavenging potential in T-LGLL patients. Ligand-receptor analysis with CellPhoneDB revealed that the number of predicted cell-cell interactions was significantly higher in T-LGLL as compared to reactive T-cell clones in healthy controls. The most co-stimulatory interactions (e.g., CD2-CD58, TNFSF14-TNFRSF14) occurred between the IFNγ secreting T-LGLL clones and the pro-inflammatory cytokine secreting monocytes. Conclusions: Our study shows a synergistic interplay between the leukemic and non-leukemic immune cell repertoires in T-LGLL, where an aberrant antigen-driven immune response including hyperexpanded CD8+ T-LGLL cells, non-leukemic CD8+ cells, CD4+ cells, and monocytes contribute to the persistence of the T-LGLL clones. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clones in patients with T-LGLL. Figure 1 Figure 1. Disclosures Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy. Mustjoki: Novartis: Research Funding; BMS: Research Funding; Janpix: Research Funding; Pfizer: Research Funding.

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Pluripotent Cell-Derived Off-the-Shelf TCR-Less CAR-Targeted Cytotoxic T Cell Therapeutic for the Allogeneic Treatment of B Cell Malignancies

Blood ◽

10.1182/blood-2018-99-116843 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4546-4546 ◽

Cited By ~ 1

Author(s):

Raedun Clarke ◽

Sjoukje Van Der Stegen ◽

Chia-Wei Chang ◽

Mushtaq Husain ◽

Yi-Shin Lai ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

B Cell Malignancies ◽

Advisory Committees ◽

Car T Cells ◽

Car T

Abstract The advent of off-the-shelf chimeric antigen receptor (CAR) T cell therapeutics is widely recognized to be a major potential advancement for the treatment of cancer. Several obstacles currently hamper the broad use of CAR T cells, including the inherent variability and cost of manufacturing of autologous cellular populations, the absolute requirement for precise genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity. Here we present pre-clinical data for FT819, a first-of-kind off-the-shelf human induced pluripotent stem cell (hiPSC)-derived CAR T cell product. FT819 is defined by the precise genetic engineering of multiple targeting events at the single cell level to create a clonal master iPSC line. The engineered features include the targeted integration of a novel, modified CD19 CAR into the T cell receptor α (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease (GvHD), and the expression of a high-affinity, non-cleavable form of CD16 (hnCD16) to deliver an adjustable system to address tumor antigen escape. Through a proprietary cellular reprogramming platform, peripheral blood derived T cells are converted to hiPSCs, engineered to contain the modified CD19 CAR targeted into the TRAC locus and hnCD16, and clonally selected to create a master hiPSC line (TRAC-TiPSC, FT819). Molecular characterization of the TRAC-TiPSC master cell line by 5' junction, 3' junction and internal sequence PCR confirmed homology directed repair and bi-allelic targeting of the CD19 CAR into the TRAC locus. The origin of the clonal master cell bank was confirmed to be a TCRαβ T cell by PCR-mediated detection of TCRδ locus deletion and methyl-seq analysis of the TCRα locus. Flow cytometric analysis demonstrated the maintenance of a uniform population of hiPSCs (>95% SSEA4/TRA-1-81/OCT4/NANOG) and expression of hnCD16 transgene (>95% CD16). Utilizing our stage-specific T cell differentiation protocol, we demonstrate that the TRAC-TiPSCs yield TRAC-iT cells with uniform expression of the CAR (>95%), complete elimination of TCR surface expression and clinically enabling expansion through the manufacturing process (>50,000 fold). To confirm the lack of alloreactivity conferred by the deletion of endogenous TCR expression, mixed lymphocyte reactions were performed using TRAC-iT, primary TCR+ T cells and primary TCR+CAR+ T cells as responders and HLA-mismatched peripheral blood mononuclear cells (PBMCs) as targets. In comparison to primary T cells and primary CAR-T cells, TRAC-iT did not respond and proliferate in response to TCR stimulation or HLA-mismatched PBMCs indicating that the risk of GvHD was alleviated. In vitro functional studies established that TRAC-iT possess a potent cytotoxic T lymphocyte response to CD19 antigen challenge in a similar manner to peripheral blood CAR T cells as demonstrated by expression of markers indicative of degranulation (CD107a/b, Granzyme B), T cell activation (CD69, CD25), and production of INFγ, TNFα and IL2. Importantly, TRAC-iT targeted tumor in an antigen specific manner as verified by lysis of CD19+, but not CD19-, tumor cell lines as seen by in vitro cytolytic assays (50% killing E:T; TRAC-iT = 1:8, primary CAR-T = 1:4). In vivo studies demonstrated that TRAC-iT cells effectively control tumor progression in a mouse model of acute lymphoblastic leukemia Nalm6 (TRAC-iT versus no treatment, p<0.0001). To validate the capability of TRAC-iT to simultaneously target multiple antigens, TRAC-iT was co-cultured with mixtures of CD19+CD20+ and CD19-CD20+ tumor cells in the presence of anti-CD20 monoclonal antibody, Rituxan. In vitro cytolytic assays demonstrate that only TRAC-iT cells can effectively identify and eliminate CD19-CD20+ tumor cells when combined with Rituxan. Importantly, the antibody-dependent cellular-cytotoxicity did not appear to interfere with CAR function as TRAC-iT maintained its directed cytotoxic capacity. Collectively, these preclinical studies suggest that FT819 is a consistent and uniform off-the-shelf product than can be effectively and safely used in the treatment of B cell malignancies in the allogeneic setting. Disclosures Clarke: Fate Therapeutics Inc.: Employment. Chang:Fate Therapeutics Inc.: Employment. Husain:Fate Therapeutics Inc.: Employment. Lai:Fate Therapeutics Inc.: Employment. Peralta:Fate Therapeutics Inc.: Employment. Stokely:Fate Therapeutics Inc.: Employment. Abujarour:Fate Therapeutics Inc.: Employment. Dinella:Fate Therapeutics Inc.: Employment. Lee:Fate Therapeutics Inc.: Employment. Pribadi:Fate Therapeutics Inc.: Employment. Chu:Fate Therapeutics Inc.: Employment. Truong:Fate Therapeutics Inc.: Employment. Sabouri-Ghomi:Fate Therapeutics Inc.: Employment. Meza:Fate Therapeutics Inc.: Employment. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Sadelain:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

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Clonally Expanded Bone Marrow T Cells Show Effector Differentiation and Rarely Recognize Disease-Associated Antigens in Multiple Myeloma

Blood ◽

10.1182/blood-2020-136991 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-7

Author(s):

Carlotta Welters ◽

Meng-Tung Hsu ◽

Christian Alexander Stein ◽

Livius Penter ◽

María Fernanda Lammoglia Cobo ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Cell Expansion ◽

Advisory Committees ◽

Myeloma Cells ◽

T Cell Expansion

Multiple myeloma is a malignancy of monoclonal plasma cells accumulating in the bone marrow. The critical influence of tumor-infiltrating T cells on disease control and therapeutic responses has been shown in a variety of malignancies, however, the role of multiple myeloma bone marrow-infiltrating T cells is incompletely understood. Although it has been shown that multiple myeloma neo-antigen-specific T cells can be expanded in vitro, little is known about functions and specificities of clonally expanded multiple myeloma-infiltrating bone marrow T cells. Here we asked at the single cell level whether clonally expanded T cells i) were detectable in multiple myeloma bone marrow and peripheral blood, ii) showed characteristic immune phenotypes, and iii) recognized antigens selectively presented on multiple myeloma cells. A total of 6,744 single bone marrow T cells from 13 treatment-naïve patients were index-sorted and sequenced using our methodologies for determination of paired T cell receptor (TCR) αβ sequences along with immune phenotype, transcription factor and cytokine expression. Clonal T cell expansion occurred predominantly within the CD8+ compartment. Phenotypes of clonally expanded T cells were distinctive of cytolytic effector differentiation and significantly different from non-expanded CD8+ T cells. Less than 25% of expanded CD8+ T cell clones expressed the immune checkpoint molecules programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), or T cell immunoglobulin and mucin-domain containing-3 (TIM-3), while B and T lymphocyte attenuator (BTLA) was expressed on more than half of the expanded clones. Clonal T cell expansion did not correlate with neo-antigen load as determined by whole exome and RNA sequencing of purified multiple myeloma cells. Furthermore, peripheral blood TCRβ repertoire sequencing from five selected patients with substantial bone marrow T cell expansion identified 90% of expanded bone marrow T cell clones overlapping with peripheral blood. To determine whether clonally expanded bone marrow T cells recognized antigens selectively presented on multiple myeloma cells, 71 dominant TCRs from five selected patients with substantial clonal T cell expansion were re-expressed in 58α-β- T-hybridoma reporter T cells and co-incubated with CD38-enriched multiple myeloma cells from the same patients. Only one of these TCRs recognized antigens selectively presented on multiple myeloma cells and this TCR was not neo-antigen-specific. Hypothesizing that the target antigen was a non-mutated self-antigen, we could show that this TCR also recognized the plasma cell leukemia cell line U-266 in an HLA-A*02:01-restricted manner. In summary, clonally expanded T cells in multiple myeloma bone marrow of newly diagnosed patients show cytolytic effector differentiation. In the majority of patients, clonally expanded bone marrow T cells do not recognize antigens presented on multiple myeloma cells and are not neo-antigen-specific. Our findings are relevant for the design of future therapeutics and clinical trials. The identified TCR, which recognizes a multiple myeloma antigen shared with U-266 in an HLA-A*02:01-restricted manner, could be a promising candidate for T cell therapy. Disclosures Bullinger: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Abbvie: 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; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

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Safety Data from a First-in-Human Phase 1 Trial of NKG2D Chimeric Antigen Receptor-T Cells in AML/MDS and Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.4052.4052 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4052-4052 ◽

Cited By ~ 16

Author(s):

Sarah Nikiforow ◽

Lillian Werner ◽

Joana Murad ◽

Matthew Jacobs ◽

Lauren Johnston ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Dose Escalation ◽

Phase 1 ◽

Hematologic Malignancies ◽

Advisory Committees ◽

Cd8 Cells ◽

Car T Cells ◽

Car T

Abstract Introduction: Conventional CAR-T cells express a single chain antibody variable fragment that restricts recognition to one tumor antigen and a limited set of cancers. This study employs a novel CAR fusing full-length human NKG2D with the CD3z signaling domain. In autologous transduced CM-CS1 T cells, NKG2D CAR receives endogenous costimulation via DAP10 to target multiple NKG2D-ligands that are upregulated in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. Methods: A phase 1 dose-escalation study to establish safety and feasibility of a single infusion of CM-CS1 T cells without lymphodepleting conditioning enrolled subjects with AML/MDS-RAEB or relapsed/refractory progressive multiple myeloma (MM) without standard therapy options (NCT02203825). Eligibility criteria included suitable organ function, no CNS disease, no prior allogeneic SCT or adoptive T-cell therapy, no therapy within 3 weeks prior to infusion, no immune suppression, and no uncontrolled infection. Dose-escalation spanned 4 cohorts [half-log increments from 1x106 to 3x107 CM-CS1 T cells] according to a 3+3 design. DLTs included ≥ Grade 3 non-hematologic toxicity or ≥ Grade 2 autoimmune toxicity related to CAR T cells. Initial assessment was at 28 days. At least 1 AML/MDS and 1 MM subject were mandated in each dose level. Manufacturing included PBMC stimulation with OKT3 and IL-2 followed by 2 rounds of retroviral transduction at DFCI's Cell Manipulation Core Facility. Vector copy number (VCN) and replication-competent retrovirus (RCR) testing were performed on whole blood and PBMCs, respectively, using quantitative PCR. Results: From April 2015 to July 2016, 11 subjects were infused, and 10 completed the DLT period. Eight of 11 were male, 6 had AML/MDS, and median age was 70 (range 44 to 79) (Panel A). Median WBC was 2.3 (range 0.7 to 7.2 K/uL); median ALC was 0.74 (range 0.09-2.37 K/uL). Five had cells manufactured from peripheral blood; 6 underwent apheresis. Median percentage of blasts in bone marrow for AML/MDS patients was 50% (range 4-68%). All myeloma patients had undergone ≥ 5 therapies including ≥1 autologous SCT. Four of the 6 AML/MDS patients had secondary disease, 3 had complex cytogenetics, 3 had p53 mutations, and 1 had a FLT3-ITD mutation. Dose-escalation proceeded from 1x106 to 3x107 CM-CS1 T cells. All 11 products passed release criteria, and there were no infusion reactions. Products consisted of median 97.2% CD3+ cells and 31.0% CD8+ cells, with vector-specific NKG2D expression on median 74.6% of CD3+ and 66.3% of CD8+ cells (Panel B). The first 10 subjects completed their 28 day evaluation period without DLTs. There were no cases of cytokine release syndrome, cell-related neurotoxicity, auto-immunity, or CAR T-related death. SAEs included a Grade 4 intracochlear bleed and an episode each of grade 4 neutropenia and thrombocytopenia deemed related to disease progression. Forty percent of patients experienced some Grade 3 toxicity, all related to underlying disease or a complication thereof (Panel C). At these initial cell doses, no patient to date has had objective tumor response at the 28 day evaluation mark. Nine initiated subsequent therapies; there have been 4 deaths secondary to disease or complications of subsequent therapies. However, cases of unexpected survival without further therapy and responses to subsequent treatments were noted. For example a patient with p53-mutated AML survived 4 months despite 50% blasts at infusion, and another entered PR at 6+months after cells on an IDH-1 inhibitor with <5% IDH and 54% p53 mutation burden at initiation. RCR testing at 3 (n=6) and 6 months (n=1) was negative. As anticipated, no CAR T cell persistence has been detected at or beyond 2 weeks, with 1 exception. CAR T cell DNA has been detected sporadically from 1 hour to 1 week after infusion. Conclusion: In the first 3+ dose-escalation cohorts of patients with AML/MDS and myeloma, a single dose of CM-CS1 T cells without lymphodepletion was feasible and well-tolerated, with no DLTs. CAR T cells generally have not persisted beyond 1 week, consistent with pre-clinical models. Correlative analyses including post-infusion immunophenotyping are in process. Future studies of multiple infusions of NKG2D CAR T cells in both hematologic malignancies and solid tumors at the higher cell doses associated with efficacy in pre-clinical models are in planning. Table Table. Disclosures Murad: Celdara Medical, LLC: Employment. Reder:Celdara Medical, LLC: Employment. Sentman:Celdara Medical, LLC: Membership on an entity's Board of Directors or advisory committees, Other: Holds patents on this technology. Wade:Celdara Medical, LLC: Employment. Schmucker:Celdara Medical, LLC: Employment. Lehmann:Celyad, SA: Employment. Snykers:Celyad, SA: Employment. Allen:Celyad, SA: Employment. Stone:Celator: Consultancy; Jansen: Consultancy; Novartis: Consultancy; Merck: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Roche: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Xenetic Biosciences: Consultancy; Agios: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Pfizer: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Consultancy. Dranoff:Novartis: Employment. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees.

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Safety and Efficacy of Multiantigen-Targeted T Cells for Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-113118 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1014-1014 ◽

Cited By ~ 2

Author(s):

Premal Lulla ◽

Ifigeneia Tzannou ◽

George Carrum ◽

Carlos A. Ramos ◽

Rammurti Kamble ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Advisory Committees ◽

T Cell Clones ◽

Cell Clones ◽

Equity Ownership ◽

T Cell Lines

Abstract Despite an array of approved agents for the treatment of multiple myeloma (MM), most patients eventually relapse after conventional treatments. The adoptive transfer of tumor-targeted T cells has demonstrated efficacy in the treatment of patients with chemo-refractory hematological malignancies including MM. While the majority of T cell-based immunotherapeutic studies in the clinic explore genetically modified T cells that target a single tumor-expressed antigen, we have developed a strategy to generate non-engineered T cell lines that simultaneously target multiple MM-expressed antigens, thereby reducing the risk of tumor immune evasion. We manufacture multiTAA-specific T cells targeting the tumor-associated antigens PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin by culturing patient-derived PBMCs with autologous DCs loaded with pepmixes (15mer peptides overlapping by 11 aminos acids) spanning all 5 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail. In our current clinical trial (NCT02291848), we have successfully generated multi-antigen-targeted lines from 18/ of 19 patients thus far, with one in production. The T cell lines comprise of CD3+ T cells (mean 95.6±2.2%) with a mixture of CD4+ (28.9±7.2%) and CD8+ (56.6±7.2%) T cells, which express central and effector memory markers (CD45RO+/CD62L+/CCR7+ -- 1.21±0.2%; CD45RO+/CD62L+/CCR7- -- 15.16±2.5%; CD45RO+/CD62L-/CCR7- -- 56.9±6.3%). All the expanded lines were specific for two to five target antigens with the majority of lines (13 of 18) specific for ≥3, (PRAME: Mean 45, range: 0 to 205 spot forming units (SFU)/2x105 input cells ; SSX2 mean: 57, 0 to 583, NYESO1: mean: 51, 0 to 125 , MAGE-A4 Mean: 67, 0 to 377 and Survivin mean: 53, 0 to 51), and did not react against non-malignant autologous recipient cells (2±3% specific lysis; E:T 20:1). We assessed the clonal diversity of the clinical product using TCR vβ deep sequencing analysis. We found both polyclonality and that the majority (mean 79%; range: 59 to 95%) represented rare T cell clones that were unique to the ex vivo expanded cell line and below levels of detection in the patients peripheral blood prior to infusion, thereby enabling in vivo tracking studies.. To date we have infused 18 patients with at least 2 infusions, 2 weeks apart of doses ranging from 0.5 to 2x107/m2. These patients had received a median of 4 lines of prior therapy including high dose chemotherapy with autologous stem cell rescue. Ten patients were refractory to their latest therapy and had active MM, while 8 were in remission at the time of infusion. At the 6 week evaluation period, of the 10 patients receiving multiTAA-specific T cells to treat active disease, 1 had a complete remission (CR) by the international myeloma working group (IMWG) response criteria, 1 had a partial remission (PR) and 8 others had stable disease (SD). Seven of these 10 patients were infused more than 1 year ago. Although 2 of the 7 subsequently had disease progression, the remaining 5 continue to respond, with sustained CR (1), PR (2) or SD (2). Of the 8 patients in CR at the time of T cell infusion, all remained in CR at the week 6 disease assessment and of the 6 evaluable patients who are >1 year post T cells, only one patient has relapsed, at 7 months after T cell infusion. These clinical responses correlated with the emergence and persistence (>6 months) of "line-exclusive" tumor-reactive T cells in patient peripheral blood, as assessed by longitudinal tracking of infused T cell clones using TCR deep sequencing. These infused product-derived T cells were detected in both peripheral blood (mean 0.43% ±SD of 0.3 of the total repertoire) and the marrow (mean 0.61%±0.24% of total repertoire). The expansion of product-derived T cell clones was higher among patients with active MM than in patients treated in remission (active: 0.60±0.39%, remission: 0.2±0.08%, p=0.048). Notably, no patient, including the complete responder, had infusion-related systemic- or neuro-toxicity. Thus, autologous multiTAA-targeted T cells directed to PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin can be safely administered to patients with MM, in whom they can subsequently be detected long-term in peripheral blood and marrow, and where they produce sustained tumor responses including CR. It will be of interest to discover whether larger or more frequent doses of these T cells can produce further benefit with maintained safety. Disclosures Brenner: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Viracyte: Equity Ownership; Cell Medica: Research Funding; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Cytosen: Membership on an entity's Board of Directors or advisory committees. Vera:Marker: Equity Ownership. Leen:Marker: Equity Ownership.

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Richter's Transformation after CD-19 Directed CAR-T Cells for Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood-2021-149815 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1430-1430

Author(s):

Amanda Blackmon ◽

Alexey V. Danilov ◽

Lili Wang ◽

Raju Pillai ◽

Hormoz Babaei Mirshkarlo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

T Cell Therapy ◽

Car T Cells ◽

Car T

Abstract Introduction Approximately 5-10% of patients with chronic lymphocytic lymphoma (CLL) will develop transformation to a more aggressive lymphoma, usually diffuse large B-cell lymphoma (Richter's transformation, RT). The median overall survival after transformation is less than one year. It remains difficult to predict which patients will transform although there is a correlation with poor risk features of CLL, like del17p/TP53 mutation and Notch1 mutations among others. While data emerging from trials of CD19-directed CAR-T cells (CD19CART) in CLL are showing promising results in the relapsed/refractory setting, there appears to be an emergence of RT in some cases even when there is no measurable residual CLL. For instance, in the phase 1 portion of the TRANSCEND CLL 004 trial, in the monotherapy arm with lisocabtagene maraleucel (n=23), 5 RT cases emerged subsequently and 3 of these had no recurrent CLL or MRD conversion to positive [Siddiqi T, et al. ASH 2020]. Four of these RT events were in patients who had progressed on both ibrutinib and venetoclax. Here we describe patients who developed RT after receiving CD19CART for CLL at City of Hope. Methods A retrospective chart review was performed to identify RT emergence and to analyze key factors surrounding the development of RT after CD19CART for CLL at City of Hope. Patient characteristics were assessed including age, sex, prior number of treatments, CLL FISH panel, mutational analysis, time on BTK inhibitor therapy, response to CAR T cell therapy, time to RT after CD19CAR T cell therapy, and outcomes after RT. Pathology samples from RT were assessed for CD19 expression and will be assessed for PDL-1, MYC, SYK, ZAP70, AKT, ERK expression by IHC or flow cytometry. Results A total of 7 out of 27 patients have been identified who received CD19CART for CLL at City of Hope and subsequently relapsed with RT [Table 1]. The median age at the time of CD19CART was 66 years (range, 54-68) and median number of prior therapies was 5 (range 4-7). All patients had features associated with high risk CLL prior to CD19CART: 5/7 had del17p; 3/7 had TP53 mutations, 2/7 had NOTCH1 mutations, and 1/7 had SF3B1 mutations. Most patients, 6/7, achieved an objective response to CD19CART with 4/7 undetectable minimal residual disease to a level of <10 -4 cells (uMRD4) CRs on imaging and bone marrow examination, and 1 uMRD4 PR. The median time to transformation after administration of CD19CART was 9.5 months (range 3.5-40 months). All patients had received BTK inhibitor therapy prior to CAR T cells, with the median length of treatment being 1 year (5 months - 4 years) and 6/7 had received prior venetoclax as well. Biopsy material at the time of RT indicated 6/7 were positive for CD19 expression by immunohistochemistry or flow cytometry (1 was only weakly positive). PD-L1, MYC, SYK, ZAP70, AKT, ERK expression will be analyzed, and results presented at the meeting. Of these patients, 3/7 were unable to be treated for RT and died shortly after diagnosis of RT due to frailty, sepsis/respiratory failure/compartment syndrome, and CNS involvement/altered mental status/hypercalcemia/tumor lysis. Two patients achieved CR (one with R-CHOP, one with O-CHOP/pembrolizumab/acalabrutinib) and underwent allogeneic hematopoietic stem cell transplantation - one of which now has relapsed SLL 2.5 years later. Two patients are on clinical trials and are pending response evaluation. Conclusions Given the expression of CD19 in the RT pathology of most cases in this series, it appears that a different mechanism of escape or resistance is occurring in these cases. All 7 pts had poor risk features of their CLL before CD19CART like del17p/TP53 mutation, Notch1 mutation and SF3B1 mutation. We are investigating the RT pathology specimens further and will compare these RT cases with other CLL patients we have treated with CD19CART thus far and who have not relapsed/progressed with RT in order to examine the differences in treatment history, cytogenetic features, proliferative/accelerated nature of CLL at baseline, and PDL1 expression before and after CAR T cell therapy. Improved treatment combinations are needed in high risk, multiply relapsed CLL patients to prevent emergence of RT despite excellent responses of the CLL itself. Figure 1 Figure 1. Disclosures Danilov: Gilead Sciences: Research Funding; Pharmacyclics: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; TG Therapeutics: Consultancy, Research Funding; Takeda Oncology: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria. Siddiqi: Janssen: Speakers Bureau; Oncternal: Research Funding; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: CD19 CAR T products used in clinical trials for relapsed/refractory chronic lymphocytic leukemia

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