Immune Microenvironment Analysis of Bone Marrow By Mass Cytometry and RNA Sequencing in Multiple Myeloma Patients Treated with Daratumumab and Durvalumab

Abstract Introduction Relapsed and refractory multiple myeloma (RRMM) remains a challenging disease to treat due to its heterogeneity and complexity. There is an urgent need for novel combination strategies, including immunotherapy. The study of the tumour and immune microenvironment before and after treatment with combination therapy is a crucial part of understanding the underpinning of disease response. Methods Longitudinal samples of bone marrow aspirates and whole blood were collected from a phase II clinical trial, MEDI4736-MM-003 (NCT02807454) where daratumumab and durvalumab naïve patients were exposed simultaneously to both these drugs. A combination of mass cytometry (CyTOF), RNAseq and flow cytometry were performed on a subset of samples from these subjects. Specifically, paired bone marrow mononuclear cells (BMMC) samples from nine patients taken at screening and six weeks post-treatment were analysed by mass cytometry (CyTOF) using a 37-marker pan-immune panel that included both lineage and functional intracellular/extracellular markers. In addition, whole blood sample specimens were collected at screening and on treatment (8, 15, 30, and 45 days after treatment) and analysed by flow cytometry. Flow cytometry panels were designed to allow interrogation of the abundance and activation status of immune cell subsets. Finally, RNA from bone marrow aspirates at screening and C2D15 were analysed by RNA sequencing. Expression profiles from the aspirates were used to estimate cell proportions by computational deconvolution. Individual cell types in these microenvironments were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results In a heavily pre-treated population with RRMM, treatment with durvalumab and daratumumab leads to shifts in a number of key immunological populations when compared to pre-treatment. In the bone marrow, CD8 and CD4 populations rise (by CyTOF and RNAseq), while NK, DC and B cell populations fall (by CyTOF). In the bone marrow within CD8+ T lymphocyte populations, we observed a post-treatment rise in markers of degranulation (granzyme p=0.0195, perforin p=0.0078, Wilcoxon signed-rank test). This is also accompanied by a fall in PD1 expression (p=0.0078) and rise in the co-stimulatory receptor DNAM1 (p=0.0273). These changes are most marked on cells with an effector memory CD45RA+ CD8+ T cell phenotype. In the blood, similar to the bone marrow, CD8+ T cells proliferate over the course of treatment (flow cytometry). A fall in both naïve and active NK cell populations is seen following treatment in bone marrow. NK cells express high levels of CD38 and are therefore depleted by daratumumab. Those NK cells which remain have an active phenotype with increased expression of TNFa (p=0.0039) and IFNg (p=0.0195) following treatment. Across the time points sampled in peripheral blood, NK cells were also decreased and those that remained were proliferating. Dendritic cells with a tolerogenic phenotype can be identified prior to treatment and are seen to fall in abundance following treatment with durvalumab and daratumumab. Conclusions The combination of durvalumab and daratumumab leads to several immune microenvironment changes that biologically portend clinical effect. We see increases in the abundance of cell populations with functional anti-tumour activity, including granzyme B+ CD8 T cells and a reduction in PD1high T cells. Despite the treatment expectedly reducing NK cell numbers, many functionally competent NK cells remain, as evidenced by the presence of anti-tumour cytokines. This combination strategy also reduces immunosuppressive tolerogenic DCs, which suppress CD4 and CD8 T cell activity. Taken together, this suggests that this chemotherapy free, doublet treatment has the potential to up-regulate anti-tumour immunological responses, which may restore immunosurveillance mechanisms critically needed in these highly refractory patients. Disclosures Seymour: Celgene: Research Funding. Young:Celgene Corporation: Employment, Equity Ownership. Tometsko:Celgene Corporation: Employment, Equity Ownership. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Thompson:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Danziger:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Gribben:Acerta Pharma: Honoraria, Research Funding; Cancer Research UK: Research Funding; TG Therapeutics: Honoraria; Roche: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Unum: Equity Ownership.

Download Full-text

Modeling Targeted Lymphodepletion with Radiolabeled CD45 Antibody As a Promising Preparative Regimen Prior to Adoptive Cell Therapy or CAR-T

Blood ◽

10.1182/blood-2018-99-119740 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5682-5682

Author(s):

Dale L Ludwig ◽

Mark S. Berger ◽

Dadachova Ekaterina ◽

Kevin Allen ◽

Wojciech Dawicki

Keyword(s):

Bone Marrow ◽

T Cells ◽

Cell Therapy ◽

Research Funding ◽

Adoptive Cell Transfer ◽

Cell Transfer ◽

Immune Microenvironment ◽

Employment Equity ◽

Car T ◽

Equity Ownership

Abstract Prior to a patient receiving a dose of an adoptive cell transfer such as engineered autologous or allogeneic CAR-T cells, it is common to perform a lymphodepletion step often using high dose chemotherapy. This process is considered important to create sufficient space in the immune microenvironment, e.g. bone marrow, to allow the transferred cells to engraft. Further, it appears to elicit a favorable cytokine profile for establishment and proliferation of the donor lymphocytes. Anti-CD45 radioimmunotherapy (RIT) is being investigated in a Phase III clinical trial as a myeloablative targeted conditioning regimen prior to allogeneic hematopoietic cell transplantation in AML patients. In this study, a low 131I-CD45 RIT dosimetric dose is administered to each patient and imaging performed to define a personalized therapeutic dose. Results from studies performed post-dosimetric dose demonstrate that lower doses of 131I-anti-CD45 RIT may be suitable for use as a preparative conditioning or lymphodepleting regimen prior to cell therapy such as CAR-T. Significantly, targeted conditioning with pan-CD45 antibody, which selectively targets all nucleated immune cells, is anticipated to deplete not only lymphocytes, but also macrophages, as well as immune suppressive regulatory T cells (T-regs) and myeloid-derived suppressor cells in the immune microenvironment. It may also exert a direct anti-tumor effect on CD45+ hematopoietic cancers. We hypothesized that targeted lymphodepletion may result in a more suitable immune homeostatic environment for the reception of adoptive cell therapies, and possibly reduce the incidence of cytokine release syndrome. We have performed preclinical studies using a 131I- labeled surrogate anti-mouse pan-CD45 antibody (30F11) to investigate in a mouse model the response of targeted RIT lymphodepletion on particular immune cell types and resulting changes in immune cytokine expression. Following single dose administration of non-myeloablative doses of CD45-RIT, peripheral blood, bone marrow and spleen samples were collected from 8-12 week C57Bl/6 mice at 48 and 96 hours post-treatment for immunophenotyping to evaluate lymphoid and myeloid subsets for lymphodepletion, and serum for cytokine profiling. CD45-RIT was shown to effect a considerable reduction in both lymphocyte and myeloid cell counts, inclusive of immune suppressive T regs and MDSCs. Further, the cytoreduction by CD45-RIT was shown to induce the expression of immune homeostatic cytokines including IL-15. Studies are in progress to evaluate CD45-RIT as a targeted lymphodepletion regimen in E.G7 lymphoma tumor bearing mice prior to adoptive cell transfer with OVA-specific CD8+ T cells. Disclosures Ludwig: Actinium Pharmaceuticals: Employment, Equity Ownership. Berger:Actinium Pharmaceuticals: Employment, Equity Ownership. Ekaterina:Actinium Pharmaceuticals: Consultancy, Research Funding; Radimmune Therapeutics: Consultancy, Research Funding.

Download Full-text

Pharmacokinetic and Pharmacodynamic Study of NKTR-255, a Polymer-Conjugated Human IL-15, in Cynomolgus Monkey

Blood ◽

10.1182/blood-2018-99-115638 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2952-2952

Author(s):

Takahiro Miyazaki ◽

Peiwen Kuo ◽

Mekhala Maiti ◽

Palakshi Obalapur ◽

Murali Addepalli ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cynomolgus Monkey ◽

Memory T Cells ◽

Granzyme B ◽

Employment Equity ◽

Equity Ownership ◽

Dose Dependent

Abstract Introduction IL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey. Methods In vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry. Results NKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis. Conclusions Nektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

Download Full-text

Weekly Inotuzumab Ozogamicin in Adult Patients with Relapsed or Refractory CD22-Positive Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v120.21.2612.2612 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2612-2612 ◽

Cited By ~ 2

Author(s):

Daniel DeAngelo ◽

Wendy Stock ◽

Stephen Petersdorf ◽

Shaw-Ling Wang ◽

Angela Volkert ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Research Funding ◽

Response Rate ◽

Lymphoblastic Leukemia ◽

Hematologic Toxicity ◽

Employment Equity ◽

Inotuzumab Ozogamicin ◽

Equity Ownership ◽

Grade 3

Abstract Abstract 2612 Background: Inotuzumab ozogamicin (INO) is a humanized anti-CD22 antibody conjugated to calicheamicin, a potent cytotoxic antitumor agent. CD22 is expressed on a majority of B-cell acute lymphoblastic leukemia (ALL). An initial study suggested INO efficacy and tolerability in ALL (Lancet Oncol 2012;13:403-11). Objectives: The current phase 1, multicenter, dose-escalation study was performed to optimize the INO dose and schedule (weekly dosing) based on safety, efficacy, and pharmacokinetic data in CD22+ relapsed or refractory ALL. The safety and efficacy of INO at the recommended dose and schedule will subsequently be further evaluated in a 12-patient (pt) expanded cohort. Methods: Eligible pts were aged ≥18 y with CD22+ ALL (defined as ≥20% blasts CD22+ by flow cytometry) refractory to initial induction or in relapse (≥first relapse), with no evidence of central nervous system disease. INO was administered in 28-d cycles (see Table), with a maximum of 6 cycles. The final dose was to be determined based on both toxicity (ie, rate of dose-limiting toxicities [DLT] at each dose level) and evidence of efficacy using the EffTox V2.10 software (Biometrics 2004;60:684–693). Adverse event (AE) severity was assessed per CTCAE V3 with DLTs defined as any of the following INO-related events during Cycle 1: grade ≥4 non-hematologic toxicity; prolonged myelosuppression (absolute neutrophil count [ANC] <500/μL or platelets <25,000/μL in bone marrow) with no evidence of leukemia persisting >45 d from last dose; grade 3 non-hematologic toxicity persisting >7 d from the last dose; grade ≥3 elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST), or bilirubin persisting >7 d; or any toxicity resulting in permanent INO discontinuation. Weekly teleconferences with investigators were used to assess toxicity. Complete response (CR) was defined as <5% bone marrow blasts with absence of peripheral blasts, ANC ≥1,000/μL, platelets >100,000/μL, and no extramedullary disease; incomplete CR (CRi) was similar but permitted ANC <1,000/μL and/or platelets ≤100,000/μL. Results: We report preliminary data for 13 pts (see Table), with a median duration of follow-up of 147 d (range, 30–188 d). Median age was 56 y (range, 23–65 y), and 69% of pts were male. Five (39%) pts were in salvage 1, 2 (15%) were in salvage 2, and 4 (31%) were in salvage ≥3. Two pts had prior allogeneic stem cell transplant. Three (23%) pts were Ph+ and 7 (54%) pts had circulating blasts at baseline; median baseline WBC was 2.01×103/mm3 (range, 0.5–29.11×103/mm3). The single DLT observed to date was transient grade 4 elevated lipase occurring at INO dose level 3. The most frequent (≥10% of pts) treatment-related AEs were thrombocytopenia (31%, all grade 3/4), neutropenia (15%), and elevated ALT (15%). Treatment-related elevated AST and alkaline phosphatase were each reported for 8% of pts. Reported dose delays were due to thrombocytopenia (n = 3), neutropenia (n = 2), elevated LFT (n = 2), bacteremia, increased blood creatinine, periorbital cellulitis, and QTc prolongation (n = 1 each). Fourteen serious AEs were reported for 9 pts, including 2 cases each of febrile neutropenia and septic shock. Responses were observed across all INO doses explored to date (see Table). The preliminary response rate was 82% (9/11 evaluable pts), including 36% of pts with a CR and 45% with a CRi. Median time to response was 43 d (range, 28–56 d). Six of 9 (67%) pts who achieved CR/CRi also achieved minimal residual disease (<1 blast out of 104 mononuclear cells by flow cytometry). Seven pts discontinued treatment, including 1 each due to disease progression and an AE (acute renal failure, not treatment related), and 5 pts who proceeded to transplant. Four deaths were reported, including 1 due to disease progression and 3 due to sepsis occurring within 30 d after stem cell transplantation. Conclusions: INO had a safety profile consistent with prior reports, characterized by hematologic, gastrointestinal, and hepatic events and infection. The remarkable response rate of 82% for single-agent INO in this relapsed/refractory population warrants further exploration in CD22+ ALL. Updated results will be presented at the meeting. Disclosures: Stock: Tau for work done through the CALGB/ALLIANCE: Research Funding. Wang:Pfizer Inc: Employment, Equity Ownership. Volkert:Pfizer Inc: Employment, Equity Ownership. Vandendries:Pfizer Inc: Employment, Equity Ownership. Advani:Pfizer Inc: Consultancy, Honoraria, Research Funding.

Download Full-text

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.

Download Full-text

Flotetuzumab (FLZ), an Investigational CD123 x CD3 Bispecific Dart® Protein-Induced Clustering of CD3+ T Cells and CD123+ AML Cells in Bone Marrow Biopsies Is Associated with Response to Treatment in Primary Refractory AML Patients

Blood ◽

10.1182/blood-2019-128485 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1410-1410 ◽

Cited By ~ 1

Author(s):

John E. Godwin ◽

Carmen Ballesteros-Merino ◽

Nikhil Lonberg ◽

Shawn Jensen ◽

Tarsem Moudgil ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Refractory Aml

Introduction The infiltration of immune cells into tumors has been associated with therapeutic effects in preclinical models and patients with cancer. In AML, we have previously reported that immune infiltrated TME is predictive of failure to cytotoxic chemotherapy, but associated with response to immunotherapy, specifically FLZ (Uy ASH 2018, Rutella ASH 2018). Furthermore, FLZ also affects immune infiltration in the TME (Rutella ASH 2018). NK cells play an important role in AML control (Ruggieri Science 2012). FLZ (MGD006/S80880) is a humanized DART® molecule that bridges CD123 on AML with CD3 on T cells and mediates anticancer activity via T-cell activation and cytolytic activity against the bound cancer cell. While this is well described in vitro, little evidence of this interaction is available in vivo. Methods Patients (pts) were treated on the recommended phase 2 dose (RP2D) of FLZ (multi-step lead-in dose followed by 500ng/kg/day, in 28-day cycles). We studied the bone marrow (BM) tissue samples for 6 primary refractory pts at baseline and after treatment. Response assessment was performed at day 25±3 days of each cycle. Serial BM samples were evaluated using 2 different staining panels (PD-L1, FoxP3, CD8, CD3, CD103 / CD123, CD3, CD57, CD16) on consecutive slides. Slides were stained using a Leica BondRx autostainer and fluorescence imaged using a Polaris Vectra 3 and analyzed using inForm software. A density-based clustering algorithm developed and run in QuPath was used to quantify CD3+ T cell clusters. Results Six pts with primary refractory AML were included in this report. Pts were heavily pretreated (median prior lines of therapy was 3, range 2-9), and had adverse cytogenetic risk (ELN 2017). Three pts had a complete remission (CR) after 1 cycle of therapy (CR, CRh, CRi), two went on the receive allogeneic stem cell transplant (HSCT). In baseline BM samples, CD3 and CD8 cell infiltrates were higher in CR vs non-responders (CD3+ 18.3% ±6.9 vs 9.3% ±1.8; CD8+ 9.4% ±3.5 vs 4.8% ±1.2; mean±SEM). Two of the three CR patients, who underwent HSCT, developed clusters (Figure 1) in their on-treatment biopsies with 65 and 22 clusters of an average of 34 and 17 T cells per cluster, respectively. All clusters in CR pts were found on or adjacent to CD123+ cells. The BM biopsy of the CR pt with no detected clusters had no unequivocal evidence of residual/recurrent leukemic blasts. This pt had their dose interrupted early due to non-treatment related AE (infectious complication) and did not receive a full cycle of treatment; the response was transient and the pt relapsed shortly thereafter. NK cells (CD57+CD16+) were increased in post treatment biopsies of CR vs non-responders (0.93 ±0.31 vs 0.27 ±0.13; mean±SEM) with the largest fold increase in CR (28 vs 9). Lastly, post treatment biopsy PD-L1 expression was higher in non-responders than CR (23% vs 16%) with non-responders exhibiting the largest fold change in total PD-L1+ cells (10.9 vs 2.2). Summary Consistent with its proposed mechanism of action, these data highlight for the first time, the dynamic induction of an increase in T-cell infiltration, and clustering around CD123 AML cells in the bone marrow microenvironment of two AML patients that responded to FLZ. In pts with resistance to FLZ (non-responders) PD-L1 induction was significantly higher indicating that in some pts treatment with sequential check point inhibitor could obviate this mechanism of resistance A trial combining FLZ with sequential administration of a PD-1 inhibitor (MGA012) is currently recruiting pts. Figure 1. Baseline and on-treatment IHC of BM biopsies of a FLZ-treated CR pt showing cluster formation following treatment. Disclosures Bifulco: Ventana: Other: advisory board; PrimeVax: Equity Ownership, Other: ScientificBoard; BMS: Other: Advisory Board; Providnece: Patents & Royalties: Imaging processing; Halio Dx: Other: advisory board. Wigginton:macrogenics: Employment, Equity Ownership; western oncolytics: Consultancy, Other: consultancy. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership. Fox:Akoya: Research Funding; Bristol Myers Squibb: Research Funding; Definiens: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Research Funding; Ultivue: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Lenalidomide Exhibits Activity in Mantle Cell Lymphoma through Increased NK Cell Mediated Cytotoxicity

Blood ◽

10.1182/blood.v126.23.821.821 ◽

2015 ◽

Vol 126 (23) ◽

pp. 821-821 ◽

Cited By ~ 1

Author(s):

Patrick Hagner ◽

Hsiling Chiu ◽

Maria Ortiz-Estevez ◽

Tsvetan Biyukov ◽

Carrie Brachman ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Employment Equity ◽

Mantle Cell ◽

Apoptosis Assay ◽

Equity Ownership

Abstract Introduction: Lenalidomide (Len) is indicated for the treatment of relapsed/refractory (R/R) Mantle Cell Lymphoma (MCL) in the United States and Switzerland. Len binds to the cullin 4 ring E3 ubiquitin ligase complex resulting in ubiquitination and subsequent proteasomal degradation of lymphoid transcription factors Aiolos and Ikaros leading to stimulation of immune cells, such as T-cells. Clinical trial CC-5013-MCL-002 (NCT00875667) is a randomized open-label phase II study in R/R MCL patients in which Len was given orally at 25 mg/day on days 1-21 of each 28-day cycle until progression (N=170). The control arm consisted of investigator choice of single-agent rituximab, gemcitabine, fludarabine, chlorambucil, or cytarabine (N=84). We explored the immune effects of Len treatment in MCL patients enrolled in CC-5013-MCL-002 and further investigated our findings in in vitro MCL co-culture models. Methods: Peripheral blood samples for exploratory analysis were collected at Cycle 1 Day 1 (C1D1, pre-treatment), Cycle 1 Day 4 (C1D4), Cycle 2 Day 15 (C2D15) and at treatment discontinuation. Flow cytometric profiling of T, B and natural killer (NK) cell subsets was performed and differences were analyzed for correlation with clinical outcomes (response rate and progression free survival [PFS]). Cell dependent cytotoxicity was measured in 1) anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) treated with vehicle or 1-10000 nM Len for 3 days and incubated with target tumor cells for an additional 4 hours followed by an apoptosis assay as measured by Annexin V/ToPro-3 flow cytometry and 2) negatively selected CD56+ NK cells stimulated with IL-2 and treated with Len (1 nM to 10 μM) for 18 hrs and incubated with target tumor cells for an additional 4 hours followed by apoptosis assay. Results: At baseline, no significant differences were observed in the absolute levels of immune subsets when comparing non-responders (NR) and responders (R) in either Len (NR=11, R=23) or control (NR=4, R=5) arms. However, in the Len arm, significantly elevated (adj. p < 0.05) proportions of CD3-CD56+CD16+ NK cells (difference of means = 8.73; 95%CI [4.48, 12.98]) were observed at C1D4 compared to baseline in the R (N=19) outcome sub-group compared to NR (N=11). A similar trend in levels of NK subsets was observed at C2D15, however the difference was not significant. In addition, elevated proportions of CD3-CD56+CD16+ NK cells (p≤0.016) at C1D4 relative to total lymphocytes correlated significantly to longer PFS in the Len arm. Immune subset analysis in the control arm did not show any correlation to response or PFS at any visit. The mechanism whereby NK cell modulation contributes to clinical benefit demonstrated by Len in patients was further explored in in vitro co-culture systems with MCL cell lines. Len treated PBMC co-cultured with Jeko-1, Granta-519, and Mino MCL cell lines resulted in 38-47.5% more apoptosis compared to DMSO (p≤0.001). We examined the effect of Len on Aiolos and Ikaros protein expression in CD56+ NK and CD3+ T cells within anti-CD3 antibody stimulated PBMCs treated with DMSO or various concentrations of Len (1 nM to 10 μM) for 72 hours. Degradation of both Aiolos (40%) and Ikaros (95%) was observed after drug treatment in CD56+ NK cells. Aiolos and Ikaros levels were also monitored in CD3+ T cells and showed decreased levels after Len treatment, consistent with previous reports (Gandhi, 2014; Kronke, 2014). Furthermore, purified CD56+ NK cell mediated cytotoxicity produced a similar pro-apoptotic effect as the PBMC assay in all MCL cell lines versus DMSO (p≤0.01). Supernatants from co-cultures of NK cells with MCL cell lines showed significantly elevated granzyme B levels as compared to DMSO controls (p≤0.0001), suggesting that the apoptotic effects observed are induced by granzyme B. Conclusions: Lenalidomide is an immune modulating agent and NK cell modulation in particular may play a role in its clinical activity in MCL. A significant increase in proportions of NK cell subsets (vs total lymphocytes) at C1D4 versus baseline was observed and is a potential response indicator of favorable clinical outcome in R/R MCL patients treated with Len. In vitro, Len enhances cell mediated cytotoxicity of MCL cell lines in two co-culture model systems. Understanding NK cell mediated mechanism(s) has potential to enhance guiding patient selection strategies and rational combination therapies of lenalidomide in MCL. Disclosures Hagner: Celgene: Employment, Equity Ownership. Chiu:Celgene: Employment, Equity Ownership. Ortiz-Estevez:Celgene: Employment, Equity Ownership. Biyukov:Celgene: Employment, Equity Ownership. Brachman:Celgene: Employment, Equity Ownership. Trneny:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, expenses, Research Funding. Morschhauser:Genentech Inc./Roche: Other: Advisory boards. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding. Milpied:Celgene: Honoraria, Research Funding. Musto:Sandoz: Consultancy; Celgene: Honoraria; Roche: Honoraria; Sanofi: Consultancy; Genzyme: Consultancy; Novartis: Honoraria; Janssen: Honoraria; Mundipharma: Honoraria. Martinelli:AMGEN: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Carmichael:Celgene: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment. Gandhi:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

Download Full-text

Deep Sequencing Approach for Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.1388.1388 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1388-1388

Author(s):

Malek Faham ◽

Jianbiao Zheng ◽

Martin Moorhead ◽

Victoria Carlton ◽

Patricia Lee Stow ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Research Funding ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Clonal Evolution ◽

Employment Equity ◽

Equity Ownership ◽

Minimal Residual

Abstract Abstract 1388 Background: The clinical management of patients with acute lymphoblastic leukemia (ALL) relies on accurate prediction of relapse hazard to determine the intensity of therapy and avoid over- or under-treatment.1 The measurement of minimal residual disease (MRD) during therapy has now emerged as the most important predictor of outcome in ALL.2 We developed the LymphoSIGHT platform, a high-throughput sequencing method, which universally amplifies antigen-receptor gene segments and can identify all leukemia-specific sequences at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In this study, we determined the sensitivity and specificity of this method, delineated the extent of clonal evolution present at diagnosis, and compared its capacity to measure MRD to that of flow cytometry and allele-specific oligonucleotide PCR (ASO-PCR) in follow-up samples from >100 patients with ALL. Methods: Using the sequencing assay, we analyzed diagnostic bone marrow samples from 100 ALL patients for clonal rearrangements of immunoglobulin (IgH@) and T cell receptor (TRB@, TRD@, TRG@) genes, as well as the extent of clonal evolution present at diagnosis. We assessed the capacity of the sequencing assay to detect MRD using diagnostic samples from 12 ALL patients carrying 13 leukemic IgH clonal rearrangements. Serial dilutions were prepared in normal peripheral blood mononucleated cells, at a range between <1 in 1 million to >1 in 1,000 cells. We also assessed MRD in follow-up samples from 106 ALL patients and analyzed concordance between MRD results obtained by the sequencing assay, flow cytometry and ASO-PCR. Results: In diagnostic bone marrow samples, we detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 100 ALL samples; 94 samples had at least 2 calibrating receptors at diagnosis, with 51 having 3 or more. We also detected a variable degree of clonal evolution: the number of evolved clones in each sample ranged from 0 to 6933, with 39 (37%) samples having 1–50 evolved clones and 17 (16%) >50 (Figure 1). In experiments with mixtures of normal and leukemic cells, the sequencing assay unequivocally and accurately detected leukemic signatures in all dilutions up to a concentration of at least one leukemic cell in 1 million leukocytes. In direct comparisons with established MRD assays performed on follow-up samples from patients with B-ALL, sequencing detected MRD in all 28 samples positive by flow cytometry, and in 35 of the 36 positive by ASO-PCR; it also revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively (Figure 2). Conclusions: The sequencing assay is precise, quantitative, and can detect MRD at levels below 1 in 1 million leukocytes (0.0001%), i.e., represents sensitivity 1–2 orders of magnitude higher than standard flow cytometric and ASO-PCR methods. Our assay also allows monitoring of all leukemic rearrangements regardless of their prevalence at diagnosis, which abrogates the risk of false-negative MRD results due to clonal evolution. Finally, the sequencing assay utilizes a set of universal primers and does not require development of patient-specific reagents. These data, together with the results of our comparison with standard MRD assays in clinical samples, strongly support the use of the sequencing assay as a next-generation MRD test for ALL. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Research Funding. Zheng:Sequenta: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta: Employment, Equity Ownership, Research Funding. Carlton:Sequenta: Employment, Equity Ownership, Research Funding.

Download Full-text

CAR T Cell Therapy Targeting G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), a Novel Target for the Immunotherapy of Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-110471 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 589-589 ◽

Cited By ~ 3

Author(s):

Eric L. Smith ◽

Kimberly Harrington ◽

Mette Staehr ◽

Reed Masakayan ◽

Jon Jones ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Employment Equity ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership ◽

G Protein Coupled

Abstract Early clinical results using BCMA targeted CAR T cell therapies for advanced multiple myeloma (MM) have shown promise. However, BCMA expression can be variable, and BCMA downregulation has been correlated with relapse (Brudno J. JCO. 2018; Cohen A. ASH. 2017). Targeting multiple antigens may enhance response durability. We report that the orphan seven transmembrane G protein coupled receptor, GPRC5D, is an attractive additional target for CAR T cell therapy of MM. GPRC5D mRNA expression was previously identified in bone marrow cells from patients with MM; however its protein expression could not be detected with available FACS reagents (Frigyesi I. Blood. 2014). We evaluated 83 primary marrow samples by quantitative immunofluorescence (Q-IF) for CD138, BCMA, and GPRC5D. In 98% of the samples, CD138+ cells expressed surface GPRC5D. In most samples, the majority of CD138+ cells expressed both BCMA and GPRC5D, however, in several cases the dominant CD138+ population expressed only BCMA or GPRC5D, with GPRC5D expression independent of BCMA across samples (R2=0.156; Figure 1). The potential for "on target/off tumor" binding by targeting GPRC5D was evaluated by screening 30 essential normal tissue types by IHC (n=3 donors/type) followed by validation of any positive findings by RNA in situ hybridization and quantitative PCR. Results in non-plasma cell normal tissue were consistent with prior reports of GPRC5D off target expression restricted to cells from the hair follicle, a potentially immune privileged site. We developed GPRC5D-targeted CARs considering immunogenicity, spacer length, and tonic signaling. To minimize potential anti-CAR immunity, a human B cell-derived phage display library was screened. Seven diverse and highly specific human single chain variable fragments (scFvs) were identified. 42 CARs were derived from the 7 scFvs by modifying scFv orientation (VH/VL; VL/VH) and incorporating a short, medium, or long IgG4 based spacer. To monitor CAR-mediated signaling, we transduced each CAR into a Jurkat reporter line with RFP inserted in-frame, downstream of endogenous NR4A1 (Nur77). Nur77 expression is an immediate-early, specific marker of CD3z signaling (Ashouri J. J Immunol. 2017). Using this reporter, we observed that (1) a long spacer provided enhanced antigen-dependent activation across all anti-GPRC5D CARs; and (2) the use of different scFvs resulted in vastly different levels of tonic signaling. We selected potential lead and backup constructs and evaluated CAR activity on primary human T cells. When co-cultured specifically with MM cell lines, anti-GPRC5D CAR T cells secreted a polyfunctional cytokine profile, proliferated, and effectively lysed target cells. CD138+/CD38hi primary MM bone marrow aspirate cells were also specifically lysed. In vivo efficacy of GPRC5D-targeted CAR T cells was evaluated in NSG mice engrafted with a human MM cell line (ffLuc+) bone marrow xenograft. Donor T cells were gene-modified to express anti-GPRC5D CARs with either a 4-1BB or a CD28 co-stimulatory domain and membrane-anchored Gaussia luciferase (GLuc). Compared to control CAR T cells specific for an irrelevant target, anti-GPRC5D CAR T cells with either co-stimulatory domain proliferated and homed to the site of MM (Gluc imaging), eradicated MM xenograft (ffLuc imaging), and increased survival (Figure 2). One scFv that was highly functional in our GPRC5D CAR screen was evaluated for off-target binding against either >200 G protein-coupled receptors (cell based), or >4000 human transmembrane proteins (scFv-Fc based), and demonstrated binding only to GPRC5D. Studies with murine and cynomolgus cross-reactive GPRC5D targeting CARs did not show signs of alopecia or other unexpected toxicity in either species. In a murine model of post-BCMA CAR T cell treated antigen escape (CRISPR BCMA KO of a subpopulation of MM cells), anti-GPRC5D CAR T cells rescue BCMA- relapse. These results indicate that GPRC5D will be an important target for the immunotherapy of MM. We are translating this 4-1BB-containing, human-derived, GPRC5D-targeted CAR construct to the clinic. Disclosures Smith: Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Harrington:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Masakayan:Agentus Inc: Employment. Jones:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Long:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Ghoddusi:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Do:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Pham:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Wang:Eureka Therapeutics: Employment, Equity Ownership. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Xu:Eureka Therapeutics: Employment, Equity Ownership. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Sather:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Download Full-text

Preliminary Clinical Data from a Phase 1 Trial with CPI-818, a Selective ITK Inhibitor That Preferentially Blocks the Growth of T Lymphoma Cells

Blood ◽

10.1182/blood-2019-127338 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1571-1571

Author(s):

Patrick P. Ng ◽

Mehrdad Mobasher ◽

Kitman S. Yeung ◽

Andrew N. Hotson ◽

Craig M. Hill ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Phase 1 ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Introduction ITK is a tyrosine kinase critical to T cell receptor (TCR) signaling. Overexpression of this gene has been reported in cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL). Genomic analyses have demonstrated the contribution of aberrant TCR signaling in the pathogenesis of T-cell lymphomas (TCL). RLK, a closely related kinase, is co-expressed with ITK in T and NK cells, and is partially functionally redundant with ITK signaling. In NK cells, ITK has been shown to be involved in FcγRIII signaling and antibody-dependent cellular cytotoxicity (ADCC). However, the relative contribution of ITK vs RLK in ADCC is not well understood. Thus, selective inhibition of ITK, but not other signal transduction components such as RLK, may be an effective strategy to treat TCL while preserving normal T and NK cell functions. CPI-818 is an orally bioavailable, covalent inhibitor of ITK with >100-fold selectivity over RLK and BTK. It was well tolerated and exhibited anti-tumor activity in companion dogs with spontaneous TCL (2019 AACR Annual Meeting Abstract #1313). A phase 1/1b trial with CPI-818 in human TCL has been initiated (NCT03952078). Here we present preclinical evidence that CPI-818 inhibits the proliferation of human malignant T cells with relative sparing of normal lymphocytes and report early results from the clinical trial. Methods Eligible patients for the dose-escalation/expansion trial of CPI-818 have relapsed/refractory TCL (PTCL, CTCL and others). Starting dose of CPI-818 is 100 mg BID continuously. The objectives of the study are to evaluate the safety and tolerability of CPI-818 in ascending dose levels; evaluate pharmacokinetics/pharmacodynamics and potential biomarkers. In in vitro studies, T cells from the blood of Sézary syndrome patients were stimulated for 6 days with αCD3/CD28. Sézary cells were identified by antibodies to specific TCR Vβ. For assays of ADCC, αCD20-coated lymphoma B cells were cultured with NK cells from multiple healthy donors for 18 h with inhibitors. In animal studies, mice received control or CPI-818-formulated diet (300 mg/kg/day). C57BL/6 mice were vaccinated with keyhole limpet hemocyanin (KLH) or subcutaneously implanted with the TCL line EL4. MRL/lpr mice began treatment at 9 weeks old. Lymph nodes were calipered weekly. Spleens and lungs were harvested at 22 weeks. Results Mouse models were studied to assess the impact of CPI-818 on normal, autoreactive and malignant T cells in vivo. No changes in total blood cell counts or T, B, NK cell subsets in lymphoid organs were seen in normal mice receiving daily doses of CPI-818 sufficient to continuously inhibit ITK for 28 days. Immune responses to antigen re-challenge were not affected in these mice, as determined by levels of antibody or CD4 T cell response to vaccination with KLH. In mice with established EL4 lymphoma, administration of CPI-818 reduced the growth of tumors at the primary site and in the draining lymph nodes (P values <0.033). CPI-818 also reduced lymphadenopathy and expansion of autoreactive T cells in the spleens of MRL/lpr mice (P values <0.0001), without affecting CD4 or CD8 cells. Sézary cells from 3 of 3 patients tested in vitro were more sensitive to growth inhibition with CPI-818 than autologous normal CD4 or CD8 cells, or T cells from a healthy donor (Figure 1). CPI-818 showed minimal inhibition of NK-mediated ADCC (5%), whereas CP-2193, an ITK/RLK dual inhibitor with an IC50 for ITK comparable to CPI-818, reduced ADCC by 50%. CPI-818 has been administered to two patients at the first dose level cohort (100 mg BID) with no DLTs, and with no changes to B, T, and NK cell counts in blood during the first dosing cycle (21 days). Pharmacokinetic and occupancy studies have revealed 80% and 50% occupancy of ITK at peak and trough drug levels, respectively in peripheral blood T cells. Conclusions CPI-818 is a selective covalent ITK inhibitor that has greater antiproliferative effects on malignant and autoreactive T cells compared to normal T cells. The drug has a minimal impact on NK mediated ADCC compared with a less selective inhibitor that also blocks RLK. Preliminary data from a phase 1/1b study shows CPI-818 at 100 mg BID was tolerable with acceptable bioavailability and ITK occupancy. Further dose escalation is ongoing. Disclosures Ng: Corvus Pharmaceuticals, Inc.: Employment, Equity Ownership. Mobasher:Corvus Pharmaceuticals: Employment, Equity Ownership. Yeung:Corvus Pharmaceuticals: Employment, Equity Ownership. Hotson:Corvus Pharmaceuticals: Employment, Equity Ownership. Hill:Corvus Pharmaceuticals: Employment, Equity Ownership. Madriaga:Corvus Pharmaceuticals: Employment, Equity Ownership. Dao-Pick:Corvus Pharmaceuticals: Employment, Equity Ownership. Verner:Corvus Pharmaceuticals: Employment, Equity Ownership. Radeski:Corvus Pharmaceuticals: Research Funding. Khodadoust:Corvus Pharmaceuticals: Research Funding. Kim:Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Horizon: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Galderma: Research Funding; Elorac: Research Funding; Soligenix: Research Funding; Medivir: Honoraria, Membership on an entity's Board of Directors or advisory committees; miRagen: Research Funding; Forty Seven Inc: Research Funding; Neumedicine: Research Funding; Portola Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Corvus: Honoraria, Membership on an entity's Board of Directors or advisory committees; Trillium: Research Funding. Miller:Corvus Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Buggy:Corvus Pharmaceuticals: Employment, Equity Ownership. Janc:Corvus Pharmaceuticals: Employment, Equity Ownership.

Download Full-text

Immune Profiling of Relapsed or Refractory Multiple Myeloma Patients Treated with Pomalidomide and Low-Dose Dexamethasone in Combination with Daratumumab

Blood ◽

10.1182/blood-2018-99-111554 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2012-2012 ◽

Cited By ~ 2

Author(s):

William E. Pierceall ◽

Nizar Bahlis ◽

David S Siegel ◽

Gary J. Schiller ◽

Christy J. Samaras ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Employment Equity ◽

Advisory Committees ◽

Ki 67 ◽

Equity Ownership

Abstract Background: Multiple myeloma clinical trial CC-4047-MM-014 (NCT01946477) is a Phase II study designed to test the safety and efficacy of pomalidomide and low-dose dexamethasone alone (arm A) or in combination with daratumumab, an anti-CD38 antibody, (arm B) subjects with relapsed or refractory multiple myeloma who have received a first or second line treatment of lenalidomide-based therapy. Immunomodulatory agents (IMiD® compounds) continue to be the backbone of multiple myeloma therapy especially when combined with monoclonal antibodies, more specifically pomalidomide had been shown previously to enhance T cell- and NK cell-mediated immunity. We sought to characterize on-treatment pharmacodynamic changes of immune biomarkers associated with POM + LoDEX + DARA administration (arm B) using multicolor flow cytometry panels designed to characterize T-cell subsets and CD38+ expressing cells. IMiD agents are the backbone of combination regimens in the treatment of patients with newly diagnosed or relapsed and/or refractory multiple myeloma. The anti-myeloma properties of these agents derive from a dual mechanism of pro-apoptotic effects on tumor cells as well as enhanced immune stimulation. An understanding of how IMiD agents interact with new monoclonal antibodies to modify patient immune profiles offers key insights into the role of such in innate and adaptive immunity in determining patient outcomes. Methods and Results: Peripheral blood samples were collected at screening, Cycle1 Days 1, 8, and 15, and Cycle 2 Days 1 and 15 to monitor pharmacodynamic changes in populations of T cells, NK cells, monocytes and MDSCs by flow cytometry. From 112 patients enrolled in Arm B, 98 patients had baseline and post-treatment specimens available for these analyses. As expected, combination treatment with POM + LoDEX + DARA led to decreased peripheral counts of CD56+CD16+ NK cells as well as CD4+CD38+ and CD8+CD38+ T cell subpopulations. Decreased counts were also noted in CD3-CD19+ B cells. In contrast, total counts of CD14+ monocytes and CD3+CD4+ or CD3+CD8+ T cells were stably maintained and pronounced increases were observed in proliferating CD4+Ki-67+ and CD8+Ki-67+ T cells. Further, when examined as a percent of total counts, increases were observed in CD14+ monocytes, CD3+CD4+ and CD3+CD8+ T-cells, with decreases in CD3-CD19+ B-cells and CD3-CD56+CD16+ NK cells. Correlation of these pharmacodynamic changes with clinical outcomes will be presented. In addition, baseline immune profiling of specific cell population subsets and associations with best overall response and progression-free survival is currently being analyzed. Conclusions: The triplet regimen POM + LoDEX + DARA has shown notable clinical activity with deep and durable responses in relapsed multiple myeloma patients progressed and are or refractory to lenalidomide. Immune characterization here is consistent with a model for clinical activity in which the loss of CD56+CD16+ NK cells along with a concomitant immune suppression by loss of CD38+CD4+ and CD38+CD8+ T- cells is offset by an increase in proliferating cytotoxic CD4+Ki-67+ and CD8+Ki-67+ T-cell populations. Our results demonstrate that patients treated with the POM + LoDEX + DARA combination do not demonstrate impairment in the innate and adaptive immune compartments and, in contrast, show significant proliferative activity in the subsets of CD4, CD8 and NK cells following treatment. Pomalidomide had been shown previously to enhance T cell- and NK cell-mediated immunity; these data are consistent with a mechanism of action in which pomalidomide administration facilitates the ability to overcome immunosuppressive effects of Dara and LoDex. Potential associations of immune biomarkers with patient outcomes is ongoing and will be updated. Disclosures Pierceall: Celgene Corporation: Employment, Equity Ownership. Bahlis:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Siegel:Merck: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau. Schiller:Astellas Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; bluebird bio: Research Funding. Sebag:Amgen Canada: Membership on an entity's Board of Directors or advisory committees; Takeda Canada: Membership on an entity's Board of Directors or advisory committees; Janssen Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene Canada: Membership on an entity's Board of Directors or advisory committees. Berdeja:Takeda: Research Funding; Genentech: Research Funding; Sanofi: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Glenmark: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Bluebird: Research Funding; Teva: Research Funding. Ganguly:Amgen: Consultancy; Daiichi Sankyo: Research Funding; Janssen: Consultancy; Seattle Genetics: Speakers Bureau. Matous:Celgene: Consultancy, Honoraria, Speakers Bureau. Srinivas:VAHCSNJ: Employment. Bar:Celgene: Consultancy. Quick:CTI BioPharma: Research Funding. Fonseca:Celgene: Speakers Bureau. Reece:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Merck: Honoraria, Research Funding; Otsuka: Research Funding. Serbina:Celgene: Employment. Zafar:Celgene: Employment. Agarwal:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

Download Full-text