First Evidence of Restoration of T and NK Cell Compartment after Venetoclax Treatment

Abstract Introduction Chronic lymphocytic leukemia (CLL) is characterized by a profound immune suppression. In addition, CLL cells evade immune destruction by interacting with cells of the adaptive immune system, resulting in dysfunctional T cells. CD4+ T cells are skewed towards a TH2-profile and the number of regulatory T (Treg) cells, that diminish cellular immune responses, is increased in CLL patients. CD8+ T cells resemble exhausted T cells and have reduced cytotoxic, yet increased cytokine production capacity. The cytotoxic function of NK cells is impaired in CLL patients, but in contrast to CD8+ T cells their cytokine production is also compromised, presumably induced by CLL cells. These data are chiefly obtained from studies on peripheral blood (PB). Although the lymph node (LN) compartment has a central role in the pathobiology of CLL, very little is known about the composition of non-malignant lymphocytes in LN tissue. The Bcl-2 inhibitor venetoclax (Ven) is highly effective in CLL and, especially in combination with anti-CD20 monoclonal antibodies such as obinutuzumab (O), results in high rates of minimal residual disease (MRD) undetectable responses. However, the prospective effects of venetoclax on non-malignant lymphocytes in patient samples remain largely unexplored. Methods PB and LN biopsy specimens were collected at baseline from patients enrolled in the 1st-line FCR-unfit HOVON 139 / GIVE trial. Study treatment consisted of O (cycle 1-2), Ven+O (cycle 3-8) and Ven (cycle 9-14). Immune composition was analyzed by 7-color flow cytometry. Baseline PB samples were compared to paired LN samples. Moreover, PB samples of the first patients that completed 6 cycles of Ven monotherapy (cycle 14) were compared to baseline. Cytokine production and degranulation of T and NK cells was studied after stimulation of PBMCs with PMA/Ionomycin. Results Comparison of LN (n=28) vs PB (n=48) revealed a larger proportion of T cells in LN (13.2% vs 5.1% of the lymphocytes), at the expense of CLL cells, with a skewed CD4:CD8 ratio (5.2 in LN vs 1.8 in PB). Within the CD4+ T cells, significantly higher levels of both follicular T helper cells (15. 7% vs 5.2%) and Tregs (11.5% vs 6.9%) were found in LN (see Table). CD4+ T cells mostly consisted of naïve and memory T cells in both PB and LN. There were fewer CD8+ T cells and especially fewer effector CD8+ T cells in the LN in comparison to PB. CD8+ T cells in LN mostly had a naïve and memory phenotype. An increased percentage of LN-residing CD8+ T cells expressed the exhaustion marker PD-1 as compared to PB CD8+ T cells (30.4% in LN vs 12.4% in PB). We then compared PB baseline samples to PB obtained after cycle 14 (n=11). Ten patients achieved MRD undetectable levels (<10-4, determined by flow cytometry) and 1 patient was MRD intermediate (10-4-10-2). As expected, the treatment regimen led to complete elimination of CD19+ B cells. In contrast, absolute numbers of CD4+ and CD8+ T cells did not change during treatment. Differentiation status of CD4+ and CD8+ T cells remained similar. Interestingly, the proportion and absolute number of Tregs decreased after treatment (6.1% vs 0.9% of CD4+ T cells). After stimulation with PMA/Ionomycin, the percentage of IL-2 producing CD4+ T cells increased after treatment, leading to a higher IL-2:IL-4 ratio, that suggests normalization towards a TH1-profile. Fewer CD8+ T cells expressed PD-1 after treatment. The fraction of CD8+ T cells that produced IFN-γ (69.8% vs 56.2%) and TNF-α (58.4% vs 40.3%) decreased. Degranulation of CD8+ T cells did not change upon treatment. After treatment, the capacity of NK cells to degranulate increased. In addition, a larger proportion of NK cells produced IFN-γ, suggesting recovery of NK cell function after treatment. Conclusion In conclusion, our data strengthen the view that CLL cells reside in an immune suppressive environment in the LN. Moreover, we provide the first evidence that the Ven+O regimen does not harm non-malignant lymphocyte populations other than B cells. Both the improved cytokine production of NK cells and diminished cytokine production of CD8+ T cells may point to normalization of immune function. Collectively, the phenotypical and functional changes observed may reflect the eradication of the immunosuppressive CLL clone by Ven+O and subsequent recovery of the immune microenvironment in CLL patients. Disclosures Eldering: Celgene: Research Funding. Mobasher:F. Hoffmann-La Roche Ltd: Other: Ownership interests non-PLC; Genentech Inc: Employment. Levin:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kater:Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

Blood ◽

10.1182/blood-2019-127261 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 621-621

Author(s):

Zachary Davis ◽

Martin Felices ◽

Todd R Lenvik ◽

Sujan Badal ◽

Peter Hinderlie ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Cytokine Production ◽

Nk Cell ◽

Fold Increase ◽

Advisory Committees ◽

Fund Research

Checkpoint blockade has become a promising immunotherapy for the treatment of a variety of malignancies. In particular, the receptor programmed death-1 (PD-1) has become a focus of intense study due to its expression on and negative regulation of T-cell function. The ligand for PD-1, PD-L1, is upregulated on many tumors and, as a result, can suppress antigen-specific T-cells thereby limiting their anti-tumor response. Pharmacological PD-1/PD-L1 axis disruption can occur with either Pembrolizumab and Nivolumab (PD-1 antagonists) and Avelumab and Atezolizumab (PD-L1 antagonists). These antibodies (mAbs) are being used to treat melanoma, non-small cell lung cancer, kidney, bladder and head and neck cancer with varying degrees of success. Like T-cells, natural killer cells (NK) also have potent antitumor cytolytic properties. The expression and functional effects of PD-1 on NK cells remain unclear due to difficulties in receptor detection and efficacy of receptor blockade by available commercial reagents. While some studies have been unable to detect PD-1 on resting NK cells, others have identified PD-1 expression only on specific NK populations under certain conditions (e.g. Cytokine stimulation or virus infection). Here, we identify PD-1 expression on peripheral blood NK cells. Using commercial reagents (Figure 1A) and a FITC-labeled clinical mAb (Pembrolizumab, Pembro), we detect low yet consistent PD-1 expression on all circulating, resting NK cells. Since FITC-Pembro mean fluorescent intensity was low and a high proportion of FITC labeled NK cells overlapped with the isotype control (Figure 1B), we designed a short-chain variable fragment (scFv) of the mAb to determine whether the smaller scFv molecule has better binding and functional activity than the intact mAb. The Pembro scFv bound to resting NK cells with a distinct fluorescent peak compared to the native Prembro from which the scFv was derived (Figure 1B). Compared to intact Prembro, use of the Pembro scFv as a PD-1 antagonist resulted in a 2-fold increase of NK cell cytolytic activity and a 3-4 fold increase in cytokine production against the PD-L1 expressing CML target, K562 (Figure 1C-D) and the AML target, THP-1 (Figure 1E-F). While PD-1 blockade enhanced NK cell degranulation and target cell killing, a greater functional enhancement was seen for interferon-γ production. PD-1 signaling inhibits PI3K induced pAkt and NK function. PD-1/PD-1 ligand blockade by the Pembro scFv resulted in increased NK cell pAKT in the presence of PD-L1 and NK activating NKG2D-ligand-expressing THP-1 cells. In addition to natural cytotoxicity, NK-mediated ADCC was also enhanced with PD-1 blockade. CD33 mAb immunoconjugates have been used to treat AML. Combined anti-CD33 mAb and PD-1 blockade against THP-1 cells resulted in a small but significant increase in NK cell degranulation and a 4-fold increase in cytokine production compared to anti-CD33 mAb without PD-1 blockade (Figure 1G-H). Since stimulation with IL-15, a cytokine that effectively lowers the NK activation threshold, abrogated the benefits of Pembro scFv in diminishing PD-1 inhibitory effects on NK cells, PD-1 control of NK function appears limited to be mostly relevant to resting NK cells. To understand the physiologic expression of PD-1 in vivo, we studied samples taken from AML patients receiving matched sibling donor transplantation at the University of Minnesota. Increased PD-1 on reconstituting NK cells in BMT recipients up to day 100 post-transplant was shown by both flow-cytometric (Figure 2A) and mass-cytometric (CyTOF) analyses (Figure 2B). Blockade of PD-1 on these cells significantly enhanced both NK degranulation (Figure 2C) and cytokine production (Figure 2D) against K562 targets. A similar increase in NK function was observed with PD-1 blockade in AML patients receiving umbilical cord transplants (not shown). These data indicate that PD-1 is present on human NK cells and PD-1 ligation negatively regulates NK function against PD-L1 expressing tumor targets. The observation that functional PD-1 is expressed on NK cells under resting conditions strongly suggests that the use of a PD-1 antagonist, in combination with NK cell therapy, should be clinically effective for treatment of cancer. Disclosures Felices: GT Biopharma.: Other: consulting funds, Research Funding. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie Inc: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Vallera:GT Biopharma, Inc.: Consultancy, Research Funding. Miller:Fate Therapeutics, Inc: Consultancy, Research Funding; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Keytruda. PD-1 blockade on NK cells for tumor immunotherapy

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CD4+ T Cell Fate in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117674 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4494-4494

Author(s):

Rachel Elizabeth Cooke ◽

Jessica Chung ◽

Sarah Gabriel ◽

Hang Quach ◽

Simon J. Harrison ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Flow Cytometric Analysis ◽

Advisory Committees ◽

Mitochondrial Mass ◽

Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, 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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Gene Set Enrichment Analysis Suggests That Increased Rituximab-Mediated NK Cell Cytotoxicity after Vitamin D Substitution Is Driven By Upregulation of Interferon Alpha (IFN-a) Isoforms

Blood ◽

10.1182/blood-2018-99-111817 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3696-3696

Author(s):

Konstantinos Christofyllakis ◽

Frank Neumann ◽

Stephan Stilgenbauer ◽

Dominic Kaddu-Mulindwa ◽

Evi Regitz ◽

...

Keyword(s):

Vitamin D ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Cytokine Production ◽

Nk Cell ◽

Cell Cytotoxicity ◽

Advisory Committees ◽

Vitamin D Levels ◽

Nk Cell Cytotoxicity

Abstract Introduction: We recently showed that vitamin D deficiency leads to decreased overall survival of DLBCL-patients treated with rituximab-chemotherapy (Bittenbring et al, JCO, 2014). We hypothesized that rituximab-mediated NK cell-cytotoxicity is more effective at higher vitamin D levels. This was confirmed by vitamin D substitution of healthy volunteers, which increased their rituximab-mediated cytotoxicity in vitro against the Daudi lymphoma cell line. To unveil the molecular mechanisms behind this finding, resting NK cells before and after vitamin D supplementation were isolated from those volunteers and a whole transcriptome analysis was performed. Methods: We collected PBMCs from eight healthy volunteers with vitamin D deficiency before and after vitamin D substitution to > 30 ng/ml 25-OH vitamin D3. NK cells were isolated from PBMCs by magnetic depletion of all non-NK cells. Purity of the CD16+ cells was confirmed by flow cytometry. After isolating total RNA, we performed a microarray analysis using an Affymetrix Gene-Chip 2.0 ™. The signals were normalized using the LMA algorithm. For pathway analysis, gene set enrichment analysis (GSEA) was used. A two-step approach was chosen. Firstly, we separated 7.705 genes due to their involvement in the NK cell-mediated immune response according to the Gene Ontology database, irrespective of their differential expression. This dataset was used separately for specific analysis of the NK cell-cytotoxicity pathway to increase sensitivity. Secondly, the complete data set of 48.145 genes was used in an exploratory analysis in an attempt to screen for other dysregulated pathways involved in the immune response and vitamin D homeostasis. We used gene sets provided from the Molecular Signature Database. A significance level of < 0.05 for p and False Discovery Rate (FDR) was chosen. Real-time quantitative PCR was performed to confirm the results. Results: The NK cell-associated cytotoxicity pathway was found to be significantly upregulated after restoration of normal vitamin D levels in the specific analysis. The most significantly overexpressed genes in the gene set were five IFN-α subtypes (IFN-α2, IFN-α4, IFN-α6, IFN-α7, and IFN-α10) as well as IFN-κ. The exploratory analysis showed an upregulation of the response to type I interferon pathway and regulation of type I interferon mediated signaling pathway. The most upregulated genes in those pathways were again the IFN-α subtypes mentioned above. Other pathways involved in the immune response were found to be downregulated after vitamin D substitution, like interferon gamma response; cytokine production and chemotaxis. The common denominator of these pathways was the downregulation of three toll-like receptor genes (TLR-8, TLR-7, TLR-2). Conclusion: The increased expression of specific IFN-α subtypes could explain the increased rituximab-mediated NK cell-cytotoxicity after vitamin D substitution in deficient individuals. To the best of our knowledge, this is the first study to suggest a role for vitamin D in IFN-α regulation. TLRs are known to stimulate cytokine production in NK cells including IFN-α. It can be assumed, that the observed upregulation of IFN-α genes after vitamin D substitution leads to a negative feedback on positive regulators of cytokine production like TLR, causing their downregulation once vitamin D levels are restored. This implies a comprehensive role of vitamin D in IFN-α biosynthesis in human NK cells. Disclosures Stilgenbauer: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: 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; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: 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; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Combination of Melphalan Flufenamide and Anti-PD-L1 or Anti-CD38 Antibodies Enhances Anti-Myeloma Immunity

Blood ◽

10.1182/blood-2021-153705 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4357-4357

Author(s):

Arghya Ray ◽

Ting DU ◽

Nina N. Nupponen ◽

Fredrik Lehmann ◽

Jakob Lindberg ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Tumor Cells ◽

Research Funding ◽

Mononuclear Cells ◽

Nk Cell ◽

Cytolytic Activity ◽

Publicly Traded ◽

Advisory Committees

Abstract Introduction Melphalan flufenamide (Melflufen; Oncopeptides AB) is a novel enzyme-activated analogue of melphalan that enables a more rapid and higher intracellular accumulation of melphalan in tumor cells than is achievable by direct exposure to equimolar doses of melphalan. Our preclinical study showed that melflufen is a more potent anti-myeloma (MM) agent than melphalan, overcomes drug-resistance, and induces synergistic anti-MM activity in combination with bortezomib, lenalidomide, or dexamethasone (Chauhan et al, Clinical Cancer Res 2013;19:3019). However, the effect of melflufen on the immunosuppressive and tumor-promoting MM-host bone marrow (BM) accessory cells such as immunologically dysfunctional plasmacytoid dendritic cells (pDCs; CD123/IL-3Rα) remains unclear. Here, we utilized our coculture models of pDCs, T-, and NK cells with autologous patient MM cells to examine whether a combination of melflufen and immune checkpoint inhibitor anti-PD-L1 Ab, or daratumumab (anti-CD38 Ab), restores anti-MM immunity. Methods MM patient BM and PB samples (N=10; obtained after informed consent), and cell lines were used for the study. Minimally cytotoxic concentration of melflufen (0.1 µM) was used to assess immune functions. CTL/NK activity assays MM CD8 + T- or NK-cells were cultured with autologous pDCs (1:10 pDC:T/NK ratio) with melflufen (0.1 μM) alone, and with anti-PD-L1 (5 μg/ml) or anti-CD38 (0.5 μg/ml) Abs for 3-5 days; cells were washed to remove the drugs, and then cultured for another 24h with pre-stained target MM cells (10:1 E/T ratio; T/NK:MM), followed by quantification of viable MM cells by flow. Results 1) Both MM tumor cells and pDCs showed higher PD-L1 and CD38 levels vs normal plasma cells; 2) Treatment of MM patient total BM mononuclear cells or purified MM cells with melflufen (0.1 µM) increased PD-L1 expression on MM cells (1.84-fold, treated vs untreated; p<0.05). Importantly, treatment of MM cells with melflufen and anti-PD-L1 Abs enhanced anti-MM cytotoxicity; 3) Combination of melflufen and anti-PD-L1 Ab triggers activation of CD3 + T cells, evidenced by an increase in CD69 expression on CD3 + T cells (1.15-fold, treated vs untreated, p<0.05); 4) Combination of melflufen and anti-PD-L1 Ab induced a more robust autologous MM-specific CD8 + cytotoxic T lymphocyte (CTL) activity than melflufen alone (% MM lysis: melflufen: 20%; melflufen plus anti-PD-L1 Ab: 60%; n=5; p=0.013); 5) Meflufen and anti-PD-L1 also triggered pDC-induced NK cell-mediated MM-specific cytolytic activity (p<0.05); and finally, 6) Low doses of melflufen and anti-CD38 Abs enhanced pDC-induced NK cell-mediated MM-specific cytolytic activity (%Viability: melflufen: 75%; melflufen + anti-CD38 Ab: 12.5%; n=4; p=0.001). Conclusions The combination of melflufen and anti-PD-L1 increases pDC-induced T- and NK cell-mediated cytolytic activities against MM. Moreover, combined melflufen and anti-CD38 Abs modestly enhance pDC-induced NK cell-mediated MM-specific cytolytic activity. Our preclinical data suggest targeting PD-L1 in combination with melflufen as well as support an ongoing clinical trial of melflufen with anti-CD38 Abs to enhance anti-MM immunity. Disclosures Nupponen: Oncopeptides AB: Consultancy. Lehmann: Oncopeptides AB: Current Employment. Lindberg: Oncopeptides: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Other: Travel, Accommodations, Expenses; Camurus: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Affibody: Membership on an entity's Board of Directors or advisory committees. Gullbo: Oncopeptides AB: Consultancy. Richardson: Takeda: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Janssen: Consultancy; Sanofi: Consultancy; Protocol Intelligence: Consultancy; Karyopharm: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Regeneron: Consultancy; AstraZeneca: Consultancy; Secura Bio: Consultancy; AbbVie: Consultancy; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Oncopeptides: Consultancy; Stemline Therapeutics: Consultancy. Anderson: Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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Immunological Monitoring of CML Patients during First-Line Bosutinib and Imatinib Treatment

Blood ◽

10.1182/blood.v128.22.3069.3069 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3069-3069

Author(s):

Anna Kreutzman ◽

Perttu Koskenvesa ◽

Kasanen Tiina ◽

Ulla Olsson-Strömberg ◽

Jesper Stentoft ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Nk Cell ◽

T Cell Subsets ◽

Imatinib Treatment ◽

First Line

Abstract Background: Tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) are not entirely selective for the BCR-ABL1 kinase but also inhibit a variety of other kinases, sometimes triggering unpredicted biological effects. As an example, the TKIs dasatinib and bosutinib both inhibit Src-kinases, which are important mediators of T-cell function. Earlier in vitro data has shown that dasatinib can suppress activation and proliferation of T and NK cells, but it can also elicit signs of immunostimulation in patients, including rapid mobilization of lymphocytes and LGL lymphocytosis. No extensive analyses of the immunological in vivoeffects of bosutinib have been performed thus far. Therefore, we aimed at characterizing T and NK cell phenotypes and functional features in CML patients in a clinical setting in the context of first-line bosutinib and imatinib treatment. Methods:Peripheral blood samples were obtained from newly diagnosed CML CP patients enrolled in the BFORE clinical trial (NCT02130557), receiving bosutinib (n=13) or imatinib (n=20) as frontline TKI treatment. Samples were drawn at diagnosis and following 3 and 12 months of therapy. Detailed immunophenotyping of NK and T cells was performed with multicolor flow cytometry. In addition, mononuclear cells were used to study the function of NK and T cells (CD107ab degranulation upon stimulation with K562 cells and detection of IFN-γ/TNF-α secretion after stimulation with anti-CD3/anti-CD28 antibodies, respectively). Moreover, blood differential counts were taken before and 2 hours after drug intake at 3 and 12 months to examine the direct effects on lymphocyte counts (mobilization). Results: No significant changes were observed in absolute white blood cell or lymphocyte counts directly (2 hours) after bosutinib or imatinib intake, in contrast to what has been observed in dasatinib treated patients. Analysis of T cell subsets during bosutinib treatment revealed that the proportion of CD4+ cells increased after the start of treatment (median dg. 60.0% vs. 3 months 62.0% p=0.06; vs. 12 months 72.8% p=0.03), but no significant changes were observed in the phenotype. Correspondingly, the proportion of CD8+ T-cells decreased moderately (dg. 31.6% vs. 3 months 25.5% p=0.01) after the therapy start. Interestingly, the proportion of PD1+ (dg. 19.6% vs. 3 months 11.9%, p=0.06; vs. 12 months 14.3%, p=0.11) and DNAM+ CD8+ T-cells decreased (dg. 73.1% vs. 3 months 66.2% p=0.004; vs. 12 months 64.6% p=0.02). No changes in the cytokine production of any of the studied subgroups of T-cells was observed. Moreover, the proportion, phenotype and function of NK-cells were not affected by bosutinib treatment. In contrast, during imatinib treatment the proportion of CD56+CD16+ NK-cells significantly increased (dg 4.3% vs. 3 months 9.9% p=0.0005; vs 12 months 14.4% p=0.002; 8.1% in bosutinib treated patients). Moreover, in imatinib patients NK-cells downregulated CD27 (dg 9.0% vs. 3 months 5.2% p=0.004; vs. 12 months 4.9%; p=0.002). Further, NK-cells from imatinib-treated patients expressed more CD107ab upon stimulation with K562 at 3 and 12 months, when compared to samples from diagnosis (dg 13.0% vs. 3 months 16.1%, p=0.01; vs. 12 months 23.2%, p=0.008). The proportion of CD4+ T-cells increased 3 months after the start of imatinib treatment (dg 60.1% vs. 3 months 63.5% p=0.01), whereas the percentage of CD8+ T-cells decreased (dg. 38.6% vs. 3 months 31.5% p=0.02). Decreased expression of DNAM (dg 73.5% vs. 3 months 67.9% p=0.0008; vs. 12 months 62.4% p=0.002) was observed in the CD4+ T-cells. Similarly as in bosutinib treated patients, the proportion of PD1+ CD8+ cells decreased during imatinib treatment (dg 18.2% vs. 3 months 14.7%, p=0.02; vs. 12 months 14.8%, p=0.03). Both CD4+ and CD8+ T-cell subsets from imatinib-treated patients secreted less cytokines after the start of treatment when compared to the pre-treatment samples. Conclusions: Despite of the Src-kinase inhibitory profile of bosutinib, no major changes were observed in T- or NK-cell phenotype or function during first-line bosutinib treatment. In contrast, in imatinib treated patients the proportion of NK-cells increased and their degranulation responses were significantly higher than in untreated CML patients. Comparison of these data with the clinical variables and treatment outcome is warranted. Disclosures Stentoft: Novartis: Research Funding; Bristol-Myers-Squibb: Research Funding; Pfizer: Research Funding; Ariad: Research Funding. Gjertsen:BerGenBio AS: Consultancy, Research Funding. Janssen:Pfizer: Honoraria; Novartis: Research Funding; Ariad: Honoraria; BMS: Honoraria. Brümmendorf:Pfizer: Research Funding; Novartis: Research Funding. Richter:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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A High Dose of Naïve CD8+ T-Cells Increases the Incidence of Graft-Versus-Host Disease (GvHD) after Donor Lymphocyte Infusions (DLIs) from HLA Identical Sibling Donors

Blood ◽

10.1182/blood-2019-124358 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3292-3292

Author(s):

Guillermo Ortí ◽

Carlos Palacio ◽

Irene García-Cadenas ◽

Isabel Sánchez-Ortega ◽

María-José Jimenez ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Mixed Chimerism ◽

High Dose ◽

Cellular Composition ◽

Advisory Committees

Introduction DLIs represent a major therapeutic approach for relapse and mixed chimerism (MC) after allogeneic hematopoietic cell transplant (AlloHCT). DLI studies have identified several variables with impact on response and GvHD. Despite some studies having explored the role of T-cells and other cell subsets, such as mononuclear cells (MNCs), comprehensive data regarding the cellular composition of DLI and its role in GvHD remains incomplete, as the development of GvHD post DLI is often unpredictable. Herein we analyzed the cellular composition of DLI from fully human leukocyte antigen (HLA) identical sibling (HLA Id Sib) donors and its impact on the development of GvHD in patients who underwent AlloHCT for hematological malignancy, and its impact on the development of GvHD. Methods Inclusion criteria were as follows: 1) Patients ≥ 18 years-old, 2) AlloHCT, 3) HLA Id Sib donor; 4) treatment with DLI; and 5) signed informed consent of patient and donor. Exclusion criteria were: 1) unrelated or mismatched related donors, 2) HCT2 prior to DLI, or 3) GvHD at DLI. For the purpose of avoiding bias, only the cell composition of the first DLI (DLI1) was analyzed. The following cell subsets of the DLI were studied: CD3+, CD4+, CD8+, CD16+CD56+CD3+ (NKT-cell), CD3+CD45RA+CCR7+ (TN), CD3+CD45RA+CCR7+CD31+ (TRTE), CD3+CD45RA+CD95+CD27+ (TSCM), CD3+CD45RA-CCR7+ (TCM), CD3+CD45RA-CCR7- (TEM), CD3+CD45RA+CCR7- (TTE), CD3+CD4+CD25brightCD127dim (TREG), CD3+CD4+CD25brightCD45RA+CD127dim (naïve TREG). The TN, TCM, TEM and TEM compartment was analyzed for both CD4+ and CD8+. We also analyzed the MNCs, CD19+ (B-cell), CD27+CD19+ (mature B-cell), CD16+CD56+CD27- (natural killer (NK+) cell) and CD16+CD56+CD27+ (CD27+NK+cell). Results Fifty-six DLIs were infused in 36 patients; the median number of DLI was 1 per patient (range, 1-3). Diagnoses were as follows: 13 AML/MDS, 6 HL, 5 MPN, 4 NHL, 4 CLL, 3 MM and 1 B-ALL. For the study, a landmark analysis was performed from the DLI date. The median follow up from DLI was 282 days (range, 9-5,560 days). Overall response rate in relapsed patients was 29% (9 of 31 patients; 6 CR and 3 PR, most responses being observed after DLI1. Further, five patients had DLI for MC and full donor chimerism was achieved in all patients. Thirteen patients (36%) developed GvHD post DLI. Two patients had GvHD before DLI, but there was no case of GvHD at DLI. The median time interval form DLI to GvHD was 76 days (range, 7-261). As per clinical presentation, 10 patients (27%) had acute GvHD, whereas eight patients (22%) had chronic GvHD. The 6-month and 1-year cumulative incidence (CI) of GvHD was 33% and 46%, respectively. When the risk of GvHD was analyzed according to DLI cell subsets, we observed that a DLI1 containing >3x106 CD8+TN correlated with an increased incidence of GvHD (Figure 1a). Also, a DLI1 with >0.8x108 MNCs/Kg (Figure 1b), >2.6x106 mature B-cell/Kg, or >0.35x106 CD27+NK+cells/Kg were linked to the development of GvHD (Table 1). Noteworthy, CD3+, TN (both CD4+ and CD8+ combined) or CD4+TN had no impact on the development of GvHD; and a high proportion of TREG was not protective for the development of GvHD (Table 2). Finally, there was no statistically significant association between any clinical variable and GvHD. Conclusion In conclusion, in this cohort of AlloHCT patients from HLA Id Sib donors, a DLI1 containing a high proportion of CD8+TN, but not CD4+TN, increased the probability of developing GvHD. Further, a DLI1 containing a high dose of MNCs, CD27+NK+cells and mature B-cell also associated with GvHD. These data provide novel insight for the understanding of GvHD post DLI. A DLI1 containing a lower dose of CD8+TN could reduce the risk of GvHD, but this asset warrants further validation in larger cohorts, and within a controlled randomized trial setting. Disclosures Bosch: F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Characterization of Peripheral Blood Mononuclear Cells Addback Following CD34 Enrichment, Engraftment and T and NK Cells Immune Reconstitution in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Blood ◽

10.1182/blood-2020-140425 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 20-21

Author(s):

Yaya Chu ◽

Julie-An Talano ◽

Lee Ann Baxter-Lowe ◽

Carolyn A. Keever-Taylor ◽

Erin Morris ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Immune Reconstitution ◽

Mononuclear Cells ◽

Nk Cell ◽

Advisory Committees ◽

Hla Dr

Background: CD3/CD19 cell depletion (Barfiled RC, et al, Cytotherapy, 2004), αβ T-cell/CD19 cell depletion (Locatelli F, et al, Blood, 2017), CD34+ positive selection (Aversa F, et al, NEJM, 1998) are designed to deplete T cells and reduce AGVHD following allogeneic stem cell transplantation (AlloSCT). These approaches achieved low rates of AGVHD, but the grafts had few T and B cells. To improve immune reconstitution we undertook an alternative approach to addback small numbers and percentages of immune cells in the final HSCT product. We previously reported a very low incidence of AGVHD in pediatric recipients receiving CD34 enriched HPC products with peripheral blood mononuclear cells (PBMNC) addback containing a fixed dose of 2 x 105 CD3/kg from MUD donors (Geyer/Cairo et al, BJH, 2012). Recently we demonstrated that despite a 5 log depletion of T cells, PBMNC addback (fixed at 2 x 105 CD3/kg) facilitated rapid hematopoietic engraftment, high levels of donor chimerism and immune reconstitution with a low probability of Grade II-IV AGVHD. Patients had a 1 yr OS of 90% following familial haploidentical (FHI) CD34 Enriched Stem Cell Transplantation in patients with SCD (Cairo, JAMA Pediatr, 2020). Objective: To determine the final immune cell concentration following CD34 enrichment and PBMNC (2 x 105 CD3/kg) addback and determine the effect on engraftment and T and NK cell immune reconstitution. Methods: Patients and/or their guardians signed written informed consents and/or assents (NCT NCT02675959). CD34+ enrichment was performed using a CD34+ reagent system (CliniMACS; Miltenyi Biotec). Mononuclear cells (2 × 105 CD3 cells/kg of recipient body weight) were removed from the leukapheresis collection prior to CD34+ enrichment and were cryopreserved as a source of MNC addback (T cells). The addback products were analyzed for CD3+CD56- T cells, CD3-CD56+ NK cells, CD3+CD56+ NKT cells, Lin-CD123+ HLA-DR+ DC cells and Lin-CD11c+ HLA-DR+ DC cells by multicolor flow cytometry analysis. Th1/Th2 cytokines were measured by multiplex assays. T cell activity was measured by viral T cells IFN-g and plasma cytokines. NK function was measured by NK receptor expression by flow cytometry analysis and in vitro cytotoxicity. Results: We identified in the PBMNC addback, mean+SEM white blood cell (WBC) percentage of: CD3+ CD56- T cells = 56.4±5%; CD3- CD56+ NK cells = 4.6±1%; CD3+ CD56+ NKT cells = 5.1±0.6%; CD19+ B cells = 29.9±3.5%. Lin- WBC consisted of: CD123+ HLA-DR+ DC cells = 18.4±8.2%; CD11c+ HLA-DR+ DC cells = 6.0±3.0%. There were 20.0+9.1e6 T cells, 1.1+0.3e6 NK cells, 1.6+0.7 e6 NKT cells, 8.6+2.5e6 B cells, 1.2+0.6e6 CD123+DC and 0.8+0.5e6 CD11c DC in the final infused products (Fig.1). We found that percentages of IFN-g+ in CD4 cells in response to CMV (pp65), ADV (hexon) and EBV (BZLF1), ranged from 0.2%+0.1% to 0.5%+0.1%, while percentages of IFN-g+ in CD8 cells in response to the antigens ranged from 0.7%+0.3% to 3.7%+1.8% when examined at days 180, 270 and 365. NK (CD3- CD56+) reconstitution was extremely rapid and occurred as early as day 30 (35.5±8.6%, 2710+1624.4 cells/ul total cells; p<0.01 vs pre-t). There were no significant differences pre-HSCT vs day 365 in plasma cytokines (Th1 and Th2) and growth factors released including IFN-g, TNF-a, IL-18, IL-4, IL-5, IL-6, IL-10, G-CSF, MCP-1 and MIP1a. There was also robust expression of NK receptor expression including NK cytotoxicity receptors, NK KIR receptors, and C-type lectin-like receptors at day 30 as compared to pre-HSCT. NK cytotoxicity, as measured using PBMC cells from recipients at different time points against K562 (E:T=10:1), was also significantly increased at day 30 (26.2±2.8%) and day 180 (28.3±3%) vs pre-HSCT (16.1±2.1%) (p<0.01). As a NK cell activation marker, CD107a expression and granzyme B levels in gated NK cells peaked at day 30. Conclusion: PBMNC addback to CD34 enriched HPC products, with a final dose of 2 × 105 CD3 cells/kg, led to stem cell products with a diverse mixture of T, NK, NKT, DC1, and DC2 cells. Immune reconstitution following PBMNC addback to CD34 enriched cells resulted in excellent CD4 and CD8 responses to CMV, ADV and EBV, and rapid functional NK cell reconstitution (Supported by FDA R01FD004090 (MSC)). Disclosures Baxter-Lowe: CHLA: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Patents related to HLA typing, Research Funding. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Cairo:Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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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.

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Enhanced Natural Killer and Cytotoxic T Lymphocyte Responses, with Decreased Monocytic Myeloid Derived Suppressor Cells May Promote Treatment Free Remission in Chronic Myeloid Leukaemia Patients Following Tyrosine Kinase Inhibitor Cessation

Blood ◽

10.1182/blood.v128.22.1122.1122 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1122-1122 ◽

Cited By ~ 1

Author(s):

Amy Hughes ◽

Jade Clarson ◽

Deborah L White ◽

David M. Ross ◽

Timothy P. Hughes ◽

...

Keyword(s):

T Cells ◽

Immune Responses ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Receptor Expression ◽

Advisory Committees ◽

Ctl Responses

Abstract Introduction:An attempt at tyrosine kinase inhibitor (TKI) withdrawal in deep molecular remission leads either to treatment free remission (TFR) or early molecular relapse (MolR) in chronic myeloid leukaemia (CML) patients. We hypothesise that immune responses promote sustained TFR and immunological markers may predict response following TFR attempt. Methodology: We studied 54 CML patients (from ALLG trials CML 8, median follow-up 66 mo, and CML 10, median follow-up 24 mo) at baseline on TKI (minimum 24 mo MR4.5) and 3 mo and 6 mo following TKI discontinuation. MolR was defined as any single sample on follow-up with BCR-ABL1 >0.1% or two consecutive BCR-ABL1 positive samples at any value. Effector immune responses of CD56dim natural killer (NK) cells and NK cell receptor repertoire were characterised by flow cytometry and cytotoxic T lymphocyte (CTL) responses to leukaemia-associated-antigens (LAAs) WT1, BMI-1, PR3 and PRAME by interferon-gamma ELISPOT. Immune suppressor regulatory T cells (Treg; CD4+CD25brightCD127-FoxP3+), Granulocytic and Monocytic Myeloid-Derived Suppressor Cells (MDSCs; HLA-DR-Lin-CD11b+CD33+CD66b+CD15+ and HLA-DR-Lin-CD11b+CD33+CD66b-CD14+, respectively), Programmed cell death-1 (PD-1) expression on T cells, NK cells, B cells and Monocytes, and major B cell subsets were characterized by flow cytometry. Results: TFR patients displayed increased CD3-CD56dimCD16bright cytolytic NK cells as a proportion of total lymphocytes at baseline (n=23, 27.1% ± 2.9) vs MolR (n=23, 19.1% ± 2.0, p=0.02). TFR patients displayed a more mature CD56dim CD57+ NK cell phenotype at baseline (74.5% ± 2.2 of total NK cells) vs MolR (66.3% ± 2.7, p=0.04). Extensive characterisation of NK cell receptor repertoire revealed NKG2D activating receptor expression was increased in TFR patients (baseline= 56.8% ± 3.8, 3 mo= 61.4% ± 5.0, 6 mo= 49.9% ± 5.8) vs MolR (baseline= 44.2% ± 3.7, 3 mo= 42.2% ± 5.5, 6 mo= 22.0% ± 8.3, all p=0.02). KIR2DL2/DL3/DS2-positive NK cells were increased in MolR patients at 3 and 6 mo vs TFR. (MolR; 3 mo= 44.8% ± 4.6, 6 mo= 48.8% ± 4.9. TFR; 3 mo= 31.5% ± 4.0 p=0.05, 6 mo= 31.1% ± 2.1, p=0.001). No significant differences were observed in CD56brightCD16-/dim immunoregulatory NK cells, C-type lectin receptor expression (CD94/NKG2A/NKG2C, CD161, CD69), Natural cytotoxicity receptors (NKp30, NKp44, NKp46), CD62L (on T cells and NK cells) and KIR2DL5 expression. No difference in NK Cell-mediated K562 degranulation as a surrogate marker of NK cell function was observed between TFR and MolR patients. Functional CTL immune responses were observed in TFR and MolR patients. BMI-1 CTL responses were increased at baseline in TFR (23%) vs MolR (9%). PR3 CTL responses were not detected in TFR at baseline, 3 mo or 6 mo (0%) vs MolR (baseline= 18%, 3 mo= 50%, 6 mo= 50%). No difference was observed in WT1 or PRAME CTLs. Quantification of immune suppressor cell types revealed decreased Monocytic MDSCs in TFR patients at baseline (10.0% ± 2.3) vs MolR (17.7% ± 3.1, p=0.02). There was no difference in granulocytic MDSCs or Treg between TFR and MolR. No difference in PD-1 expression was observed on NK cells, T cells, B cells and Monocytes. Extensive characterisation of B cell subsets revealed no difference in TFR vs MolR (Table 1). Conclusion: In keeping with STIM and EURO-SKI trials, a threshold level of particular NK cell subsets may be important in maintaining TFR. We found additionally that enhanced NK and CTL effector responses and decreased inhibitory NK KIR2DL2/DL3/DS2 expression, in combination with reduced monocytic MDSC may promote sustained TFR. Methods to enhance nett immune effector responses, such as mature CD56dimCD57+ NK cells and BMI-1 CTL responses or targeting inhibitory KIR may increase TFR success rates. Disclosures White: Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria. Yong:Celgene: Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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