Deep Profiling of the Immune Microenvironment throughout Myeloma Disease Stages

Abstract Background The immune system is altered in multiple myeloma (MM) and contributes to therapy resistance. The availability of novel immunotherapies necessitates understanding the influence of the immune microenvironment on disease progression which may inform sensitivity to therapy. The objective of this study is to fully characterize the immune microenvironment in MM precursor diseases and MM and identify any immune contribution to progression. To accomplish this we used high-dimensional mass cytometry (CyTOF) to investigate immune alterations associated with progression in pre-malignant and malignant stages of MM. Methods Cryopreserved bone marrow mononuclear cells (BMMCs) from healthy donors (HD, n=13), MGUS (n=21), SMM (n=19), newly diagnosed MM (NDMM, n=17), and ~3 months post- first autologous stem cell transplant (ASCT, n=21) were assessed using a panel of 35 cell surface and 3 intracellular antibodies that includes cell lineage markers for identification of immune populations and functional markers indicative of positive or negative immune regulation. BMMCs were thawed, stained with antibodies, and analyzed on a Helios mass cytometer. Data were normalized using bead normalization, transformed using the inverse hyperbolic sine function with a cofactor of 5 and gated for 45+ live, intact, singlets for global analysis by gating in FCS express and clustering by viSNE for visualization. Differences in population abundance were identified in an unbiased manner by FlowSOM and in marker intensity by CITRUS. Marker intensity analysis was performed using the multiple testing permutation procedure (SAM), with an FDR of 1% and minimum population size of 0.5%. Results To identify changes in the immune microenvironment associated with progression we compared immune population abundance and marker intensity indicative of immune status including activation, exhaustion, or senescence. MGUS was distinguished from HD by increased abundance of CD4 central memory (CM, p<0.001), effector memory (EM, p<0.001) and plasmacytoid and monocyte-derived dendritic cells (DC, p< 0.01). In MGUS, TIM3 and CD57 were elevated on NK cells and NKT cells, respectively, compared to HD suggesting reduced activity. In SMM increased abundance of B regulatory cells (3.0 vs 5.9 %, p<0.01) but reduced inhibitory markers on T cells including PD1, CTLA4 CD55, FOXP3 and TIGIT was observed compared to MGUS. NDMM was distinguished from SMM by reduced abundance of CD4 EM (p<0.01), CD8 early EM (p< 0.001), and B regulatory cells (p<0.01) and increased abundance of active Tregs (CD38+, P<0.01) and total NK cells (p<0.01) which had increased CD55, a complement inhibitory protein. Post-ASCT changes in immune abundance include increased total CD8 and CD8 terminal effectors (CD57 +, p< 0.0001), B regulatory cells (p<0.0001), and reduced total CD4 and CD4 CM (p<0.0001), compared to NDMM. CD4 T cells post-ASCT were characterized by reduced CD127 and CCR7 and increased CD28, CTLA4, FOXP3 and TIGIT and CD8 T cells had reduced CD28, CD127 and CCR7 and increased CD57 and TIGIT compared to NDMM. Interestingly, significant difference in NK cells were not observed but post-ASCT NK cells may be active as suggested by reduced CD59 and TIM3 compared to NDMM. To determine whether the immune microenvironment had normalized by 3 months post-ASCT we compared population abundance to HD, MGUS, and SMM cases. Immune abundance post-ASCT revealed a significantly lower percentage of CD4 CM, 4 -8 - T cells, normal PCs, and post-switch B cells (25+) and elevated CD8 terminal effector (57+) and B regulatory cells than all 3 other groups. Overall major differences in abundance of total T and B cells and their subsets were observed with differences in NK cells between stages primarily reflected in marker expression (e.g. CD161+ subset) rather than abundance. Conclusions Early changes in the immune microenvironment observed in MGUS/SMM lead to immune suppression and eventually immune evasion allowing MM to emerge. In this study the immune ME did not appear to normalize 3 months post-therapy indicated by an increase in B regulatory cells and markers of inactive effector cells. Profiling of the immune microenvironment throughout MM treatment may allow us to identify novel therapeutic targets and optimal timing of administration of novel immunotherapies and patients that would most benefit from these therapies. Disclosures Walker: Sanofi: Speakers Bureau; Bristol Myers Squibb: Research Funding. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees.

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Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

Blood ◽

10.1182/blood-2021-148654 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 407-407

Author(s):

Frank Cichocki ◽

Jode P Goodridge ◽

Ryan Bjordahl ◽

Svetlana Gaidarova ◽

Sajid Mahmood ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Car T Cells ◽

Car T

Abstract Treatments for B-cell malignancies have improved over the past several decades with clinical application of the CD20-specific antibody rituximab and chimeric antigen receptor (CAR) T cells targeting CD19. Despite the success of these therapies, loss of CD20 after rituximab treatment has been reported in leukemia and lymphoma patients. Additionally, up to 50% of all patients receiving anti-CD19 CAR T-cell therapy relapse within the first year with many of those patients exhibiting CD19 loss. Thus, new therapeutic approaches are needed to address tumor antigen escape. Accordingly, we generated triple gene-modified iPSC-derived NK (iNK) cells, termed "iDuo" NK cells, tailored to facilitate multi-antigen targeting. The iPSC line was clonally engineered to express high-affinity, non-cleavable CD16a (hnCD16), an anti-CD19 CAR optimized for NK cell signaling, and a membrane-bound IL-15/IL-15R fusion (IL-15RF) molecule to enhance NK cell persistence (Fig. 1A). To model antigen escape, we generated CD19 knockout AHR77 lymphoma cells alongside wild type AHR77 cells (both CD20 +) as targets in cytotoxicity assays. Activated peripheral blood NK (PBNK) cells, non-transduced iNK cells, and iDuo NK cells were tested as effectors. Unlike PBNK cells or non-transduced iNK cells, iDuo NK cells efficiently eliminated wild type AHR77 cells with or without the addition of rituximab at all tested E:T ratios. Similarly, iDuo NK cells in combination with rituximab were uniquely able to efficiently eliminate CD19 KO AHR77 cells due to enhanced antibody-dependent cellular cytotoxicity (ADCC) driven by hnCD16 (Fig. 1B-E). Cytotoxicity mediated by iDuo NK cells was also evaluated using primary chronic lymphocytic leukemia (CLL) cells. Compared to expanded PBNK cells and non-transduced iNK cells, only iDuo NK cells (in the absence of rituximab) were able to kill primary CLL cells (Fig. 1F). Expression of IL-15RF by iDuo NK cells uniquely supports in vitro expansion without the need for cytokine supplementation. To determine whether IL-15RF supports in vivo persistence of iDuo NK cells, CD19 CAR iNK cells (lacking IL-15RF) and iDuo NK cells were injected into NSG mice without the addition of cytokines or CD19 antigen availability. iDuo NK cell numbers peaked within a week after injection and persisted at measurable levels for ~5 weeks, in marked contrast to CD19 CAR iNK cell numbers that were undetectable throughout (Fig. 1G). To evaluate the in vivo function of iDuo NK cells, NALM6 leukemia cells were engrafted into NSG mice. Groups of mice received tumor alone or were treated with 3 doses of thawed iDuo NK cells. iDuo NK cells alone were highly effective in this model as evidenced by complete survival of mice in the treatment group (Fig. 1H). To assess iDuo NK cells in a more aggressive model, Raji lymphoma cells were engrafted, and groups of mice received rituximab alone, iDuo NK cells alone, or iDuo NK cells plus rituximab. Mice given the combination of iDuo NK cells and rituximab provided extended survival compared to all other arms in the aggressive disseminated Raji lymphoma xenograft model (Fig. 1I). One disadvantage of anti-CD19 CAR T cells is their inability to discriminate between healthy and malignant B cells. Because NK cells express inhibitory receptors that enable "self" versus "non-self" discrimination, we reasoned that iDuo NK cells could have higher cytotoxicity against tumor cells relative to healthy B cells. To address this, we labeled Raji cells, CD19 + B cells from healthy donor peripheral blood mononuclear cells (PBMCs) and CD19 - PBMCs. Labeled populations of cells were co-cultured with iDuo NK cells, and specific killing was analyzed. As expected, iDuo NK cells did not target CD19 - PBMCs. Intriguingly, iDuo NK cells had much higher cytotoxic activity against Raji cells compared to primary CD19 + B cells, suggesting a preferential targeting of malignant B cells compared to healthy B cells. Together, these results demonstrate the potent multi-antigen targeting capability and in vivo antitumor function of iDuo NK cells. Further, these data suggest that iDuo NK cells may have an additional advantage over anti-CD19 CAR T cells by discriminating between healthy and malignant B cells. The first iDuo NK cell, FT596, is currently being tested in a Phase I clinical trial (NCT04245722) for the treatment of B-cell lymphoma. Figure 1 Figure 1. Disclosures Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Bjordahl: Fate Therapeutics: Current Employment. Gaidarova: Fate Therapeutics, Inc: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Rogers: Fate Therapeutics, Inc: Current Employment. Huffman: Fate Therapeutics, Inc: Current Employment. Lee: Fate Therapeutics, Inc: Current Employment. Szabo: Fate Therapeutics, Inc: Current Employment. Wong: BMS: Current equity holder in publicly-traded company; Fate Therapeutics, Inc: Current Employment. Cooley: Fate Therapeutics, Inc: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment. Miller: Magenta: Membership on an entity's Board of Directors or advisory committees; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees.

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Dissection of Bone Marrow Microenvironment By Single Cell RNA Sequencing in Warm AIHA Patients: A Proof-of-Concept Analysis

Blood ◽

10.1182/blood-2021-149546 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 931-931

Author(s):

Bruno Fattizzo ◽

Matteo Claudio Da Via' ◽

Juri Alessandro Giannotta ◽

Paola Bianchi ◽

Luca Baldini ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Complement Activation ◽

Advisory Committees ◽

Single Cell Rna Sequencing

Abstract Warm type autoimmune hemolytic anemia (wAIHA) is a rare disease characterized by variable severity and bone marrow (BM) compensation, unpredictable relapses and several complications (i.e. infections and thrombosis). Most therapies are aimed at restoring immune tolerance by targeting various immunologic mechanisms (autoantibody production, reticuloendothelial phagocytosis, and complement activation). BM composition during acute and chronic/relapsing disease phases is still under investigated, and preliminary studies showed that it may predict anemia severity and treatment response. We aimed at dissecting BM environment by single cell RNA sequencing (scRNA-seq) in patients with wAIHA. We selected 2 patients experiencing mild hemolytic reactivations handled with low dose steroids (M-AIHA) and 2 with severe relapses requiring high steroid doses and rituximab (S-AIHA). We focused on lymphoid/myeloid subpopulations and their gene expression and evaluated the association of output data with clinical features. We performed scRNA-seq for 17,989 single cells, detecting over 23,446 expressed genes per cell on average. Uniform manifold approximation and projection (UMAP), a method for nonlinear dimensionality reduction, showed the microenvironmental cell composition, including T-, B- and NK- lymphocytes and their subpopulations, plasma cells, CD14+ and CD16+ monocytes, hematopoietic stem cells, and myeloid precursors (Figure 1A). On the whole, BM microenvironment showed a high frequency of innate immunity effectors such as NK cells and monocytes (11% and 15% of total cells), likely reflecting the inflammatory state typical of autoimmune/autoinflammatory response. T-cell subpopulations were also highly represented. Specifically, more CD4+ memory than CD4+ naïve T-cells (58% vs 38%) were found, and T-regs represented a small fraction (4%). Also, CD8+ memory cells were more frequent than CD8+ naïve and CD8+ effectors (55% vs 24% vs 21%). Both CD8+ memory and effectors type 2 cells were higher than type 1 cells, indicating a likely participation of T cell compartment in disease phenotype. Finally, B cells were particularly underrepresented, probably due to recent therapy (steroids/rituximab). Figure 1B displays % of BM immune cells divided into M-AIHA and S-AIHA. S-AIHA patients showed higher CD14+ monocytes (57% vs 43%) and decreased NK cells (19% vs 81%) as compared to M-AIHA. Interestingly, within the latter compartment, the CD56 bright NKs were over-represented in S-AIHA (83% vs 17%), suggesting an attempt to negatively regulate activated lymphocytes. Moreover, S-AIHA showed a severe decrease of B cells as compared to M-AIHA, consistently with more recent rituximab treatment. Furthermore, we performed differential expression and gene set enrichment (GSEA) analysis within the different cell subset comparing M-AIHA and S-AIHA. Concerning T cells, we found differential expression of genes related to T-cell receptor, immunoglobulins and interferon alpha/gamma response. Regarding CD14+ monocytes, we observed a downregulation of pathways related to immunomodulatory/inflammatory cytokines, complement activation and apoptosis in S-AIHA versus M-AIHA. Finally, using the CopyKat tool, we found aneuploidies in myeloid cells, including stem cells, suggesting that the selective pressure from the immune environment may lead to accumulation of genetic lesions in chronic S-AIHA. This clonal evolution can possibly explain the clinical overlap with myeloid neoplasms. Overall, these preliminary data show for the first time that scRNA-seq technology is feasible in wAIHA patients and gives insights in the pathogenic role of bone marrow immunologic microenvironment. Additionally, BM composition appears to dynamically modify according to disease severity and treatment, potentially enabling tailored therapies. Figure 1 Figure 1. Disclosures Fattizzo: Kira: Speakers Bureau; Alexion: Speakers Bureau; Novartis: Speakers Bureau; Momenta: Honoraria, Speakers Bureau; Annexon: Consultancy; Apellis: Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bolli: Celgene/BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Honoraria; Amgen: Honoraria. Barcellini: Alexion Pharmaceuticals: Honoraria; Incyte: Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria.

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Rapid and Robust CD4+ and CD8+ T-, NK-, B- and Monocyte Cell Reconstitution after Nicotinamide-Expanded Cord Blood Transplantation

Blood ◽

10.1182/blood-2018-99-116700 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2123-2123

Author(s):

Jaap Jan Boelens ◽

Coco de Koning ◽

Mitchell E. Horwitz ◽

Guillermo Sanz ◽

Madan Jagasia ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Cord Blood ◽

B Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Immune Monitoring ◽

Cell Recovery ◽

Advisory Committees

Abstract Introduction Nicotinamide-expanded cord blood (NiCord) is a promising alternative source for allogeneic hematopoietic cell transplantation (HCT) when an HLA-matched donor is unavailable. A phase 1/2 trial with standalone NiCord HCT showed rapid neutrophil engraftment (median 11 days) and platelet engraftment (median 34 days). However, successful CD4+ immune reconstitution (IR) has shown to be crucial for infectious and relapse control associated with favorable survival (Admiraal JACI 2017). We performed unique in-depth immune monitoring to evaluate and compare the recovery of immune subsets after NiCord and conventional HCT. Methods In the phase1/2 multicenter trial, patients (n=36) with hematologic malignancies received NiCord-HCT after myeloablative (MA) conditioning without antithymocyte globulin (ATG). Immune monitoring was performed (harmonized sampling, handling and analyses in a central lab) in a random subgroup. The primary endpoint was probability of achieving CD4+ IR (>50*106/L within 100 days). Secondary endpoints were subset recovery over time of B-cells, CD4+ and CD8+ T-cells, natural killer (NK), monocytes, and dendritic cells (DC), during 7-365 days after HCT. In addition, TREC analyses are pending and will be available at the meeting. Data were compared with IR in cohorts of adolescent and young adult (AYA) patients at the UMC Utrecht receiving either unmanipulated cord blood transplantation (unCBT) or T-repleted unrelated bone marrow transplantation (BMT) for hematological malignancy after MA conditioning without ATG. Linear-mixed effects modelling in LOESS-regression curves and two-sided log-rank test for univariate comparisons in cumulative incidence plots were used. Results 24 NiCord recipients (median 41.5; 13.4-61.7 yrs) had blood samples available for in-depth early immune monitoring. NiCord cell dose consisted of median 6.4*106 CD34+/kg, and 2.3*106 CD3+T-cells/kg of the co-infused negative fraction (following CD133+-selection). 91% of patients achieved successful early CD4+ IR after NiCord (Fig 1). When comparing the NiCord with 27 unCBT (median age 15.4; range 12.2-22.1 yrs) and 20 BMT (median age 14.3; range 12.1-19.7 yrs), no difference in probability of early CD4+ IR was noted (p=0.76: Fig 1). Overall T-cell reconstitution was similar; CD3+ (p=0.99), CD4+ (p=0.71), CD8+ (p=0.08), although effector and central memory CD4+ and CD8+ T-cells, Tregs, gamma-delta T-cells, Th2, and Th17 recovered somewhat faster after NiCord. Recovery of conventional- (p=0.41) and plasmacytoid DCs (p=0.52) was similar as well. Overall reconstitution of NK-cells (p<0.001); especially naïve NK-cells, monocytes (p<0.001); mostly classical, and B-cells (p=0.026) was faster after HCT with NiCord, compared to unCBT and BMT cohorts (Fig 2). In B-cell recovery, strikingly faster early recovery of follicular B-cells (p=0.04), memory B-cells (p=0.003), and plasma cells (p=0.003) was observed in NiCord recipients. Conclusions In-depth immune monitoring reveals rapid and robust immune reconstitution in NiCord recipients, with high early CD4+ IR probability, and comparable recovery of T-cell-, NK-cell-, monocyte-, and DC-subsets to AYA controls receiving unCBT and BMT. Interestingly, B-cell recovery consisted of markedly higher follicular B-cell, memory B-cell, and plasma cell levels in NiCord recipients. Next to higher B-cell recovery, monocyte and NK-cells also recovered faster after NiCord transplantation, despite the younger age of the AYA cohort (expected to reconstituted faster). This may be explained by the higher stem cell dose and higher proliferative capacity of the NiCord- expanded product. Optimal comparison of IR in NiCord vs. unCBT in a randomized phase 3 trial is underway. Disclosures Horwitz: Gamida Cell: Research Funding. Jagasia:Incyte Corporation: Membership on an entity's Board of Directors or advisory committees. Wagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding. Cilloni:Janssen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees. Nierkens:Gamida: Research Funding.

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Favorable Progression-Free Survival Associated with Immune Bio-Markers Modulated By Pomalidomide in Relapsed/Refractory Multiple Myeloma: An Analysis of Phase III Study

Blood ◽

10.1182/blood-2019-131257 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1784-1784

Author(s):

Rao Prabhala ◽

Mehmet Kemal Samur ◽

Srikanth Talluri ◽

Megan Stekla ◽

Chaitanya Yenumula ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Cell Subpopulations

We have previously shown that immune cells, including regulatory T cells, Th17 cells, myeloid-derived suppressor cells and NK cells are abnormal in multiple myeloma (MM). It is well demonstrated that immune-modulatory drugs like pomalidomide (POM) have shown impact on various immune sub-populations in pre-clinical studies. Relapsed/refractory (RR) MM (N=540) patients were randomized to be bortezomib and dexamethasone (Vd) with or without pomalidomide POM (Vd) in a large Phase III CC4047-MM007 (OPTIMISMM, NCT01734928) study, allowing for critical investigation into the impact of POM on immune cell-subset. We have analyzed 197 RRMM patients utilizing 366 peripheral blood samples collected at screening, day 8 of cycle 1 and day 8 of cycle 3 using 98 immune biomarker-panel with multi-color flow to identify changes with POM exposure in various sub-populations of B, T, & NK cells. The primary objective was to investigate association of PFS with the modulation of predictive and prognostic immune bio-markers by POM. The analyzed patient-treatment cohorts had identical characteristics as the total patient population and was balanced for the 2-treatment arms, overall patient characteristics and response. Among B-Cell-subpopulations, we observed significant up-regulation of B1b cells (CD19+CD43+) and down-regulation of regulatory B cells (Bregs, CD19+CD5+CD43-) by the PVd arm as compared with the Vd arm. A significant increase in MZB cells were observed in both arms. Patients with a higher proportion of either CD19+ B cells or Bregs at screening showed significantly favorable PFS (logrank p value < 0.05) in the PVd arm as compared with the VD arm, which indicates that these markers are predictive of PFS for PVd treatment. Moreover, patients with a higher proportion of either [naïve B cells (CD19+CD27-IgD+)] or [CD95+ B cells that display germinal center differentiation features (CD19+ CD185+) at cycle 1 day 8] showed significantly favorable PFS in PVd arm (logrank p value < 0.05); however, they were not prognostic with the Vd arm alone. This indicates that the early impact of Pom on these cell types may provide a prognostic bio-marker outcome following PVd therapy in B cell-compartment at cycle 1. Among T-Cell subpopulations, we observed that patients with higher proportion of CD4+ T cells at screening showed significantly favorable PFS in PVd arm, indicating a predictive marker. We observed significantly favorable PFS in PVd arm in both patients with a higher number of CD8 T cells expressing OX-40 at cycle 3 day 8 and patients with lower number of CD4 T cells or Tfh cells (CD4+CD185+) expressing PD-1 or regulatory T cells expressing (low CD127+ and high CD25+) HLA-DR at cycle 3 day 8, indicating prognostic markers. The studied markers were neither predictive nor prognostic for the Vd arm alone. The total proportion of NK cells were significantly elevated following the Pom-containing regimen as early as day 8 of cycle 1 and persisted at cycle 3 as compared to the Vd arm. Among NK cell-subpopulations, we observed that the patients with either a higher proportion of NK cells without expressing KIR molecules or a lower proportion of NKT cells expressing CD158b KIR molecule at screening had significantly superior PFS with in the PVd, which indicates that each is a predictive marker. Finally, we showed that patients with either a higher proportion of NK cells expressing activation marker NKG2D, or a lower number of CD158b expressing NK cells at cycle 3 had significantly improved PFS with the PVd arm but not with the Vd arm, which indicates the prognostic nature of these markers. In summary, this large randomized study identified specific immunophenotypes at screening and after exposure to pomalidomide combinations, allowing for the identification of predictive and prognostic markers for favorable PFS, respectively. Disclosures Biyukov: Celgene: Employment, Equity Ownership. Oriol:Celgene, Amgen, Takeda, Jansse: Consultancy, Speakers Bureau. Pierceall:Celgene Corporation: Employment, Equity Ownership. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: 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, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Anderson:Janssen: Other: Advisory Board; Gilead Sciences: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder . Thakurta:Celgene: Employment, Equity Ownership. Munshi:Oncopep: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Takeda: Consultancy.

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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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Persistent Impairments in Humoral and Cellular Immunity in Patients with Immune Thrombocytopenia Treated with Rituximab: A Sub-Study of a Randomized Controlled Trial

Blood ◽

10.1182/blood.v120.21.492.492 ◽

2012 ◽

Vol 120 (21) ◽

pp. 492-492

Author(s):

Ishac Nazi ◽

John G. Kelton ◽

Mark Larche ◽

Denis P. Snider ◽

Jane C. Moore ◽

...

Keyword(s):

T Cells ◽

Placebo Group ◽

B Cells ◽

Antibody Response ◽

Board Of Directors ◽

Bactericidal Activity ◽

Research Funding ◽

Fold Increase ◽

Advisory Committees ◽

Adequate Antibody Response

Abstract Abstract 492 Background: The anti-CD20 antibody, rituximab, is increasingly being used as a treatment for immune thrombocytopenia (ITP). The concern about rare occurrences of progressive multifocal leukoencephalopathy in this population and the reliance on vaccine responses in patients who ultimately require splenectomy calls for additional studies into the integrity of the immune system after rituximab treatment. The objective of this study was to evaluate both the antibody and cellular responses to the Streptococcus pneumoniae polysaccharide vaccine and to the Haemophilus influenza type b (Hib) conjugate vaccine in rituximab-treated patients with ITP. Methods: Adults with primary ITP and a platelet count below 30 x109/L who had participated in a randomized trial of rituximab or placebo were eligible for this prospective sub-study. Six months after the study intervention, patients were given the S. pneumoniae polysaccharide vaccine (Pneumovax-23®, Merck) and the Hib conjugate vaccine (ActHIB®, Aventis). Antibodies against the pneumococcal capsular polysaccharide (anti-PCP) and Hib polyribosyl-ribitol phosphate (anti-PRP) were measured by EIA. A bactericidal assay was used to measure the ability to eradicate Hib in culture. An adequate antibody response was defined as a 4-fold increase in antibody concentration from baseline in the first month post vaccination. IgG anti-PRP ≥ 1 μg/mL was considered protective. For the bactericidal assay, a 4-fold increase from baseline was considered positive. CD3+ T-cells and total (CD19+), naïve (CD19+, CD27−), memory (CD19+, CD27+, CD38−) and pre-plasma (CD19low, CD27hi, CD38hi) B-cells were determined by flow cytometry before and after vaccinations. Interferon-γ (IFN-γ) secreting T-cells were measured by elispot. Results: Patients who had been randomized to receive rituximab (n=14) or placebo (n=6) participated in this prospective study. Compared with placebo, fewer patients in the rituximab group achieved an adequate antibody response to S. pneumonia vaccine (21.4% versus 66.7%) or the Hib vaccine (28.6% versus 83.3%). These results correlated with a reduced bactericidal effect of anti-PRP antibodies observed in patients treated with rituximab compared with placebo (14.2% versus 83.3%). Two patients in the rituximab group demonstrated an adequate rise in antibody titer but with no functional bactericidal activity. In addition, 3 patients who did not mount an adequate antibody response, had IgG anti-PRP titers below protective levels and did not demonstrate bactericidal activity in vitro were considered Hib vaccine failures. There were no Hib vaccine failures in the placebo group. In the rituximab group, total number of B-cells declined rapidly after treatment (1.895 × 104/mL) relative to the placebo group (5.65 × 104/mL) and never fully recovered even 1 year post-treatment. Resting memory B-cells were significantly reduced after rituximab and remained at low levels even up to 1 year. Conversely, naïve B-cells recovered to near normal levels within 6 months of treatment. Activated pre-plasma B-cells increased following vaccination in the placebo group; yet, this response was variable in patients who had received rituximab. CD3+ T-cell numbers were unaffected by rituximab and vaccination; however, the number of IFN-γ- secreting T-cells were lower in the rituximab group. Conclusions: Rituximab treatment was associated with an impaired antibody response to vaccination, which in some patients was only demonstrated by impaired bactericidal activity. The expected increase in B-cell subsets was not observed following vaccination in the rituximab group, and T-cells, while normal in number, demonstrated functional impairments. Our findings characterize subtle defects in immunity which may persist after rituximab treatment. Disclosures: Off Label Use: Rituximab is not licensed for use with ITP patient. Kelton:Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffman-LaRoche: Research Funding. Arnold:Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffman-LaRoche: Research Funding.

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The Influence Of KIR Haplotype In ITP Incidence, Treatment Response and Bleeding Symptoms

Blood ◽

10.1182/blood.v122.21.2316.2316 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2316-2316

Author(s):

Bethan Psaila ◽

Nayla Boulad ◽

Emily Leven ◽

Naznin Haq ◽

Christina Soo Lee ◽

...

Keyword(s):

Platelet Count ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Kir Genes ◽

Number Of Patients

Abstract The pathogenesis of immune thrombocytopenia (ITP) is multifactorial, with both cellular and humoural immune dysfunction. The role of NK cells has not been well defined in ITP but in other diseases NK cells have a role in rejecting “foreign” eg transplanted organ or tumor, and also acting against self as occurs in autoimmunity. NK cell activity is orchestrated by the balance of activating vs. inhibitory signalling, in particular via the killer cell immunoglobulin-like receptor (KIR) family of receptors. Significant variation exists in KIR allelic subtype and copy number for the KIR between individuals, and associations have been made with certain haplotypes and a number of autoimmune disorders including rheumatoid arthritis, scleroderma and diabetes. Previous reports have demonstrated a reduction in natural killer (NK) cell number and function in ITP and expression of inhibitory KIR genes is increased in patients in remission vs. active ITP. Methods To explore whether a particular KIR haplotype might predispose to ITP, and also affect response to ITP treatment, we performed KIR genotyping using the Invitrogen SSP kit on 92 patients attending a haematology centre in New York and compared the results to data from 213 controls taken from the USA Eastern Database. Genomic DNA was typed for the inhibitory KIR genes KIR2DL1, KIR2DL2, KIR2DL5A (alleles 001 and 002), KIR2DL5B (alleles 002-004, 06, and 007), KIR3DL1, KIR3DL3; the activating KIR genes KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1; the framework genes KIR2DL3, KIR2DL4, KIR3DL2, KIR3DP1; and the pseudogene KIR2DP1. The patients with ITP had been or were receiving treatment with IVIG (n=64), corticosteroids (72) and rituximab (37). Bleeding symptoms were recorded. Response to treatment was defined as complete - platelet count increase to > 100 x 109/mL; partial - platelet count increase to > 50 x 109/mL; or no response. For the purpose of analysis, PRs and CRs were combined. A comprehensive database allowed a logistic regression, assessing both responses to treatments, platelet counts, neutrophil counts, CRP, lymphocyte subsets and bleeding symptoms. Results The expression of two inhibitory KIR genes, 2DL1 and 3DL1, was significantly lower in the patients with ITP as compared to controls (87% 2DL1 and 87% 3DL1 compared to 99% in controls - P < 0.02). Response to rituximab was strongly related to KIR haplotype expression. 2DL1 expression was higher among nonresponders to Rituximab (100% of non responders compared to 82% of responders), whereas 2DL3 expression was significantly lower (79% compared to 90%) (P < 0.05, Figure 1B). Separately, patients with the 2DS3 allele, an activatory KIR, were 5.5 times more likely to have experienced significant bleeding. Conclusions Although these findings are preliminary and require further investigation, these data suggest that increased cytotoxic autoimmunity due to reduced KIR inhibition may be associated with the development of ITP and possibly contribute importantly to the pathogenesis. Anti-CD20 targeting therapy directed at B cells was strongly influenced by 2 different KIRs (1 upregulated and one down-regulated) emphasizing the potential role of NK cells in elimination of tissue-based (nodal) B cells. Finally a more pronounced clinical phenotype with a markedly higher incidence of severe bleeding associated with an increased activatory KIR expression demonstrates the role of NK cells in bleeding presumably via their effects on either endothelial cells or platelet function. These exciting findings will be pursued for confirmation in a larger number of patients. Disclosures: Bussel: Amgen: Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; GlaxoSmithKline: Family owns stock, Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity’s Board of Directors or advisory committees.

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Expression of S100A9 in Bone Marrow Cells Differentiates Refractory Cytopenia with Multilineage Dysplasia (RCMD) from Refractory Anemia with Excess Blasts (RAEB) and Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v128.22.4303.4303 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4303-4303 ◽

Cited By ~ 1

Author(s):

Paul Brent Ferrell ◽

Caroline R. Maier ◽

Mikael Roussel ◽

Michael R. Savona ◽

Jonathan Michael Irish

Keyword(s):

Bone Marrow ◽

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Bone Marrow Cells ◽

Myeloid Cells ◽

Mass Cytometry ◽

Immune Microenvironment ◽

Advisory Committees ◽

Marrow Cells

Abstract Introduction: Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow disorders with a yearly incidence of approximately 13,000 in the United States. It has been observed that both genetic mutations within stem and progenitor cells and a disordered immune microenvironment are present early in MDS. Abnormal levels of inflammatory cytokines as well increased numbers of suppressive cell types, such as regulatory T cells and myeloid derived suppressor cells (MDSC) have been noted in MDS bone marrow. MDSC are recently discovered subset of myeloid cells with specific immune regulatory functions, such as T cells suppression, seen in pathological conditions, such as cancer. Recent data suggest MDSC may play a critical role in MDS pathogenesis, and that S100A9, a danger-associated molecular pattern (DAMP) produced by some myeloid cells, including neutrophils, monocytes and MDSC, is a key signal for bone marrow immune dysregulation. Here, we report a systems immunology approach to cell type discovery within MDS bone marrow using high dimensional mass cytometry. Methods: Bone marrow aspirate samples with informed consent from MDS (n=19) and AML (n=4) patients were collected and cryopreserved following red blood cell lysis for storage by the Vanderbilt Hematology Tissue Repository, a tissue repository approved by the local Institutional Review Board (IRB). Samples were acquired for the study and stained with a 35-marker panel of metal tagged mass cytometry antibodies and analyzed with a mass cytometer (CyTOF). Cellular populations were then characterized using biaxial gating as well as viSNE, SPADE and hierarchical clustering as has been previously reported (Diggins et al. Methods 2015, Ferrell et al. PLoS One, 2016). Results: Unsupervised viSNE analysis of 35-markers per cell revealed distinct cellular subsets within each sample. Interestingly, one of the strongest marker signals was expression of S100A9, which was seen in multiple cells types including phenotypic MDSC. Further analysis revealed that as a percentage of bone marrow cells, S100A9 expression was significantly more common in RCMD vs. RAEB and AML (30.0% (n=10) vs. 10.9% (n=9) and 2.4% (n=4), respectively, p<0.05 for each comparison) (Figure 1A). Additionally, three paired RCMD/AML samples were available for analysis. Within these patients, the percentage of S100A9+ cells dropped from a mean of 41.7% in RCMD to a mean of 1.84% in AML bone marrow (Figure 1B&C). Conclusion: S100A9 is both a distinguishing feature of RCMD and of disease progression within MDS. Because of its important role inflammation and cellular recruitment, S100A9 may correlate with bone marrow cellular inflammation and could represent a viable target in treatment of the disordered immune microenvironment present in MDS, especially RCMD. Disclosures Savona: Celgene: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Takeda: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Irish:Incyte: Research Funding; Janssen: Research Funding; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Optimizing Ex-Vivo Expanded NK Cell- Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Combined with NKTR-255 in Chronic Lymphocytic Leukemia (CLL), Follicular Lymphoma (FL), and Burkitt Lymphoma (BL)

Blood ◽

10.1182/blood-2020-139822 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 23-24

Author(s):

Yaya Chu ◽

Susiyan Jiang ◽

Jian Jiang ◽

Meijuan Tian ◽

Dean Anthony Lee ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Type I ◽

Type Ii ◽

Advisory Committees ◽

Anti Cd20

Background: The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Rituximab, a monoclonal chimeric anti-CD20 antibody, has been widely used as a chemoimmunotherapeutic regimen in the frontline therapy for patients with CD20+ BL and diffuse large B-cell lymphoma. The addition of rituximab to the CHOP backbone or to standard FAB/LMB therapy has greatly improved outcomes without significantly increasing toxicity in patients with B-NHL (Goldman/Cairo, Leukemia, 2013, Coiffier et al, NEJM, 2002). However, patients who relapse have a poor clinical response to rituximab retreatment. Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully expanded functional and active peripheral blood NK cells PBNKwith irradiated feeder cells to target B-NHL (Chu/Cairo, et al, Can Imm Res 2015). We previously demonstrated that obinutuzumab has significantly enhanced expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and expand natural killer (NK) cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, and enhances long-term immunological memory which may lead to sustained anti-tumor immune response. Objective: To investigate the effects of NKTR-255 on the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Dean Lee, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFNg, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, ASH, 2018). MEC-1 (CLL), PGA-1 (CLL), DOHH2 (FL) and Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Results: NKTR-255 significantly enhanced the in vitro cytotoxicity of expanded NK cells when combined with rituximab against MEC-1 (E:T=3:1, p<0.001), PGA-1 (E:T=3:1, p<0.001), and DOHH2 (E:T=3:1, p<0.001) as compared to the control groups (Fig.1A). NKTR-255 also significantly enhanced granzyme and perforin release from expanded NK cells when combined with rituximab against MEC-1 (granzyme: p<0.05; perforin: p<0.001), PGA-1(granzyme: p<0.05; perforin: p<0.05), DOHH2 (granzyme: p<0.05; perforin: p<0.001) as compared to controls. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab agains rituximab-resistant BL cells like Raji-2R (E:T=3:1, p <0.01), and Raji-4RH (E:T=3:1, p<0.01) as compared to the control groups (Fig.1B). NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.001 and perforin: p<0.001) and Raji-4RH (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.01 and perforin: p<0.01) as compared to controls. Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells in vitro with enhanced IFN-g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells using humanized NSG models are under investigation. Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Madakamutil:Nektar Therapeutics: Current Employment. Marcondes:Nektar Therapeutics: Current Employment. Klein:Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Cairo:Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding.

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CD20-TCB, a Novel T-Cell-Engaging Bispecific Antibody, Induces T-Cell-Mediated Killing in Relapsed or Refractory Non-Hodgkin Lymphoma: Biomarker Results From a Phase I Dose-Escalation Trial

Blood ◽

10.1182/blood-2019-123979 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5319-5319 ◽

Cited By ~ 2

Author(s):

Ann-Marie E Bröske ◽

Ian James ◽

Anton Belousov ◽

Enrique Gomez ◽

Marta Canamero ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Board Of Directors ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Introduction: CD20-TCB (RG6026) is a novel T-cell-engaging bispecific (TCB) antibody with a '2:1' molecular format that comprises two fragment antigen binding regions that bind CD20 (on the surface of B cells) and one that binds CD3 (on the surface of T cells). CD20-TCB offers the potential for increased tumor antigen avidity, rapid T-cell activation, and enhanced tumor cell killing versus other bispecific formats. The safety, tolerability, pharmacokinetics, biomarkers, and antitumor activity of CD20-TCB are currently being investigated in a multicenter Phase I dose-escalation trial (NP30179; NCT03075696). We recently presented preliminary clinical data demonstrating promising clinical activity in relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) patients with indolent or aggressive disease (Dickinson et al. ICML 2019). Here, we present preliminary blood and tissue biomarker analyses to explore modes of action, support optimal biological dose selection, and identify potential outcome predictors. Methods: For biomarker analyses, we performed immune profiling of peripheral blood by flow cytometry, analyzed plasma cytokine levels by ELISA, and characterized baseline and on-treatment tumor biopsies by immunohistochemistry/immunofluorescence assays and RNA sequencing. Biomarker data were obtained from 122 patients dosed with 0.005-25mg CD20-TCB. Results: CD20-TCB infusion led to a rapid and transient reduction in T cells in the peripheral circulation (T-cell margination) in all patients. T-cell margination reached nadir 6 hours after the first CD20-TCB infusion, and showed a strong association with CD20-TCB dose and receptor occupancy (RO%; as determined by Djebli et al. ASH 2019). Interestingly, rebound of T cells 160 hours after the first CD20-TCB infusion was associated with response to treatment. Responding patients showed long-term T-cell activation after the first infusion of CD20-TCB at doses from 0.6mg and above. T-cell activation was demonstrated by 2-4-fold elevation of T-cell activation markers such as Ki67, HLA-DR, PD-1, ICOS, OX40, and 4-1BB, which was sustained up to Cycle 5 (105 days). Analysis of paired pre- and on-treatment tumor biopsies (n=6) obtained before and 2-3 weeks after the first dose of CD20-TCB showed evidence of T-cell-mediated tumor cell killing. Analysis of archival and pre-treatment tumor biopsies (n=80) revealed that clinical responses were achieved irrespective of the amount of tumor T-cell infiltration at baseline. In contrast, preliminary baseline bulk tumor RNA sequencing data (n=46) showed upregulation of gene signatures associated with cell proliferation/Myc and T-cell subsets (effector vs exhausted-like) in non-responding patients. Conclusions: In this study, we demonstrated the mode of action of CD20-TCB, a novel bispecific antibody with promising clinical activity in R/R NHL. We also demonstrated that biomarker data on T-cell activation can support dose finding in conjunction with pharmacokinetics. Additional analysis is ongoing to evaluate response predictors and better characterize the population that will benefit most from T-cell mediated therapies. Disclosures Bröske: Roche: Employment, Equity Ownership. James:A4P Consulting Ltd: Consultancy. Belousov:Roche: Employment. Gomez:F. Hoffmann-La Roche Ltd: Employment. Canamero:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Ooi:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Grabole:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Wilson:F. Hoffmann-La Roche Ltd: Employment. Korfi:F. Hoffmann-La Roche Ltd: Consultancy. Kratochwil:F. Hoffmann-La Roche Ltd: Employment. Morcos:Roche: Employment, Equity Ownership. Ferlini:Roche: Employment, Equity Ownership. Thomas:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Dimier:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Moore:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Bacac:Roche: Employment, Equity Ownership, Patents & Royalties: Patents, including the one on CD20-TCB. Weisser:Pharma Research and Early Development Roche Innovation Center Munich: Employment, Equity Ownership, Patents & Royalties. Dickinson:Merck Sharpe and Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: CD20-TCB (also known as RG6026, RO7082859) is a full-length, fully humanized, immunoglobulin G1 (IgG1), T-cell-engaging bispecific antibody with two fragment antigen binding (Fab) regions that bind to CD20 (on the surface of B cells) and one that binds to CD3 (on the surface of T cells) (2:1 format). The 2:1 molecular format of CD20-TCB, which incorporates bivalent binding to CD20 on B cells and monovalent binding to CD3 on T cells, redirects endogenous non-specific T cells to engage and eliminate malignant B cells. CD20-TCB is an investigational agent.

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