Secreted Mutant Calreticulins As Rogue Cytokines Trigger Thrombopoietin Receptor Activation Specifically in CALR Mutated Cells: Perspectives for MPN Therapy

Abstract Background Mutant calreticulins carrying the sequence translated after a +1 frameshift at the C-terminus are major drivers of myeloproliferative neoplasms (MPNs). These mutant CALRs bind and activate TpoR/MPL in cells co-expressing TpoR and mutant CALRs, resulting in persistent JAK2-STAT5 signaling. Whether mutant CALR proteins are secreted, thus acting in trans on other cells, is not known. Aims Our objectives were to: 1) assess the direct TpoR-mutant CALR interaction both when expressed in the same or in different cells; 2) determine whether mutant CALRs are secreted; and 3) determine whether mutant CALR can act as extracellular cytokines. Methods Engineered CALR and TpoR mutants were analyzed by a combination of biochemical approaches (bioluminescence resonance energy transfer, recombinant protein production), functional assays (cell growth and transcriptional assays, flow cytometry, primary megakaryocytic clonogenic assay, analysis of CALR del52 knock-in mice) and cell imaging (confocal microscopy, flow cytometry and immuno-gold electron microscopy). Secreted CALRs were determined by ELISA using mutant specific antibodies. Results 1) Two systems provided evidence that mutant CALRs and TpoR directly interact. First, using Nano-BRET in cells co-expressing N-terminally fused TpoR or EpoR with Nano-luciferase and mutant or WT CALR C-terminally tagged with HaloTag that is bound to the 618-ligand fluorophore, we show that TpoR and mutant CALRs interact in a complex whether the two proteins are within 10 nm. The interaction does not occur between TpoR and WT CALR, or between EpoR and mutant or WT CALRs. Second, expressing mutant CALR and TpoR extracellular domain in S2 Drosophila Schneider cells showed that stable complex formation requires immature high mannose structure on TpoR. Lastly, we could detect surface expression of the TpoR/CALRdel52 complex using proximity ligation assay with anti-TpoR and anti-mutant CALR antibodies in CRISPR/Cas9 engineered UT7/Tpo cells that express endogenous mutant CALR and TpoR levels. 2) We used flow cytometry, confocal immunofluorescence and immunogold electron microscopy and could show that mutant CALRs are trafficking via cis-, medial- and trans-Golgi to the cell-surface and are secreted, independently from TpoR expression. Importantly, mutant CALRs are also secreted in CALR mutated MPN patients as determined by mutant CALR-specific ELISA assay in patient plasma (mean plasma level 24.6 ng/ml, range 0-156.5 ng/ml). In the 113 evaluated CALR mutated patients from different centers the plasma mutant CALR levels correlated with CALR mutant allele burden (P<0.001). Secreted mutant CALR can also be found in plasma from knock-in CALR del52 mice. 3) We show that recombinant mutant CALR can act as a cytokine and specifically stimulate JAK2-STAT5 pathway in cells that carry the TpoR at the surface. Using Nano-BRET, we could demonstrate that extracellular mutant Halo-tagged CALR can specifically bind in trans to the cell-surface TpoR fused with Nano-luciferase, but not to EpoR fused with Nano-luciferase. This binding and the subsequent JAK2 activation were obtained at levels of around 100-150 ng/ml only in cells exposing at the cell-surface TpoR with at least one immature N-linked sugar. This can be accomplished by co-expressing in the reporter cells non-tagged mutant CALR, which will promote cell-surface localization of partially immature TpoR. The effect of exogenous mutant CALR could involve both stabilization of the endogenous cell-surface mutant CALR-TpoR complexes and binding to unoccupied immature TpoRs. Conclusion We show that mutant CALRs directly interact with TpoR and also are secreted and can act as rogue cytokines, leading to activation of cells carrying TpoR. Activation of TpoR in trans is efficient at mutant CALR levels similar to those detected in patients when target cells co-express heterozygous mutant CALR and TpoR, where endogenous mutant CALR transports to the surface TpoR with immature glycosylation. Thus, secreted mutant CALRs is predicted to expand the MPN clone. Given that cell-surface mutant CALR in TpoR expressing cells is crucial for oncogenicity, and that mutant CALRs are also secreted correlating with allele burden, we discuss how antibodies and other immunotherapy approaches could specifically target the mutant CALR MPN clone. Disclosures Xu: MyeloPro Research and Diagnostics GmBH: Employment. Hug:MyeloPro Diagnostics and Research GmbH: Employment. Gisslinger:Janssen: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Shire: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership. Constantinescu:Personal Genetics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; AlsaTECH: Equity Ownership; Novartis: Honoraria; MyeloPro Research and Diagnostics GmbH: Equity Ownership.

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FLT3 Splice Variant (FLT3Va) As a Potential Immunotherapeutic Target in Patients with Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v128.22.1681.1681 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1681-1681

Author(s):

Sophia Adamia ◽

Jeffrey Nemeth ◽

Shruti Bhatt ◽

Sarah R Walker ◽

Natalie I Voeks ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Board Of Directors ◽

Research Funding ◽

Leukemic Cells ◽

Advisory Committees ◽

Gm Csf ◽

Nsg Mice ◽

Blocking Antibodies ◽

Pdx Models

Abstract Alternative pre-mRNA splicing (AS) is a normal epigenetic phenomenon, a key regulator of gene expression, yields multiple transcripts and thus a variety of proteins from a single gene. Mutations in the spliceosome components resulting in aberrant splicing isoforms are common in AML, and other myeloid neoplasms, and may generate leukemia-specific neoantigens targetable with an antibody-drug conjugates (ADCs) or blocking antibodies. Our previous studies revealed that the FLT3 cell surface receptor is one of the most commonly misspliced genes in AML (54-63% of ~400 AML patients). We conducted cloning and sequencing analyses in AML cells and identified multiple aberrant splice-variants of FLT3 that resulted from either skipping of one or more exons or activation of cryptic splicing sites. Transfection of cDNA with three of these variants in TF-1 (AML cell line) cells resulted in expression of Flt3 variant proteins on the cell surface. We successfully generated rabbit polyclonal antiserum against a unique peptide sequence present in the most commonly expressed abnormal splice variant, which we termed Flt3Va. Immunoblots performed with the polyclonal antibody identified a ~160 kDa protein expressed by TF-1 cells transfected with FLT3Va, and the antibody did not react with untransfected TF-1 cell lysate. Using standard techniques, we generated rabbit hybridomas and evaluated the clones by flow cytometry and western blotting experiments. Based on these data, we selected one antibody clone (15-7) for further experiments. The 15-7 anti-Flt3Va rabbit monoclonal antibody identified Flt3Va protein expressed on the cell surface and within the cytoplasm of transfected TF-1 cells by flow cytometry and western blotting. However, no Flt3Va protein was detected in untransfected TF-1 cells or normal CD34+ bone marrow cells. The 15-7 antibody bound to 26 of 52 primary AML samples and 5 of 10 primagraft samples (PDX models) of human AML. Immunoblotting analyses of PDX models and patient samples confirmed binding to a protein of the expected size (130-160 kDa). Additionally, multi-parameter flow cytometry in 10 PDX models and 52 primary demonstrated that putative AML stem cells (as defined by the CD45dim, CD34, CD38, CD33, c-Kit cell surface expression) co-expressed Flt3Va antigen in 50% samples evaluated. An analysis of Flt3Va protein localization by live cell imaging showed a punctate distribution of Flt3Va on the cell surface. Furthermore, we observed that overexpression of Flt3Va in TF-1 cells led to GM-CSF growth factor independence. Analysis of TF-1 cells in the absence of GM-CSF and Flt3 ligand demonstrated constitutive activation of STAT5, an important mediator of Flt3 signaling, in Flt3Va overexpressing cells. In addition, Erk1/2 phosphorylation was also increased in Flt3Va overexpressing cells, another downstream effector of Flt3. In an effort to determine if Flt3Va+ cells had tumor repopulating ability, we sorted 0.3X10^6 Flt3Va+ and Flt3Va- cells from a PDX sample and injected the sorted populations or unsorted bulk tumor cells into NSG mice. The human cell engraftment in the mice was detected by the expression of human CD45, CD33, CD34, CD38, and c-kit antigens in the peripheral blood. In two experiments, mice injected with Flt3Va+ cells had detectable circulating leukemic cells by ~18 days after injection, while those injected with Flt3Va- cells had detectable circulating leukemic cells after the 4th week. These results suggest both Flt3Va+ and Flt3Va- cell populations are able to reconstitute leukemia after transplantation in NSG mice. However, Flt3Va+ may be expressed by an aggressive AML clone that facilitate early tumor engraftment. Overall, these studies suggest that Flt3Va is a leukemia-specific neoantigen and is an attractive potential immunotherapeutic target in AML. Proteins such as Flt3Va generated by alternative splicing are common in AML and may be targets for of novel blocking antibodies or ADCs, minimizing effects on normal tissues. Disclosures Adamia: Janssen: Research Funding. Nemeth:Janssen: Employment. Attar:Janssen: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Steensma:Millenium/Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Ariad: Equity Ownership; Genoptix: Consultancy. Weinstock:Novartis: Consultancy, Research Funding. DeAngelo:Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Baxter: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Amgen: Consultancy. Stone:Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celator: Consultancy; Juno Therapeutics: Consultancy; Roche: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Merck: Consultancy; Xenetic Biosciences: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; Seattle Genetics: Consultancy. Griffin:Janssen: Research Funding; Novartis: Consultancy, Research Funding.

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CD200 Is a Stem Cell-Specific Immunosuppressive Target in AML

Blood ◽

10.1182/blood-2018-99-114818 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2768-2768

Author(s):

Shelley Herbrich ◽

Keith Baggerly ◽

Gheath Alatrash ◽

R. Eric Davis ◽

Michael Andreeff ◽

...

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

T Cells ◽

Stem Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Cd200 Receptor ◽

Blast Cells ◽

Equity Ownership

Abstract Acute myeloid leukemia (AML) stem cells (LSC) are an extremely rare fraction of the overall disease (likely <0.3%), largely quiescent, and capable of both long-term self-renewal and production of more differentiated leukemic blasts. Besides their role in disease initiation, they are also hypothesized as the likely source of deadly, relapsed leukemia. Due to the quiescent nature of the LSCs, they are capable of evading the majority of chemotherapeutic agents that rely on active cell-cycling for cytotoxicity. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML. We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression data to identify genes significantly over-expressed in LSCs when compared to their normal hematopoietic stem cell (HSC) counterparts (Herbrich et al Blood 2017 130:3962). These datasets contain gene expression arrays on human AML patient samples sorted by leukemia stem, progenitor, and blast cells (with normal hematopoietic cell subsets for comparison). We have since expanded our statistical model to identify targets that are both significantly overexpressed in AML LSCs when compared to HSC as well as LSCs compared to their corresponding, more differentiated blast cells. Instead of traditional methods for multiple testing corrections, we looked at the intersection of genes that met the above criteria in 3 independently generated datasets. This resulted in a list of 30 genes, 28 of which appear to be novel markers of AML LSCs. From this list, we first chose to focus on CD200, a type-1 transmembrane glycoprotein. CD200 is broadly expressed on myeloid, lymphoid, and epithelial cells, while the CD200 receptor (CD200R) expression is strictly confined to myeloid and a subset of T cells. CD200 has been shown to have an immunosuppressive effect on macrophages and NK cells and correlates with a high prevalence FOXP3+ regulatory T cells (Coles et al Leukemia 2012; 26:2146-2148). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget 2015; 6:30212-30221). To date, we have screened 20 primary AML patient samples by flow cytometry, 90% of which are positive for CD200. Expression is significantly enriched in the CD34+/CD123+ stem cell compartment. To examine the role of CD200 in AML, we established two in vitro model systems. First, we used CRISPR/Cas9 to knockout the endogenous CD200 protein in Kasumi-1. Further, we induced CD200 in the OCI-AML3 cell line that had no expression at baseline. Both cell lines did not express the CD200 receptor before or after manipulation, negating any autocrine signaling. In both systems, CD200 manipulation did not affect the proliferation rate or viability of the cells. To examine the immune function of CD200 in AML, we performed a series of mixed lymphocyte reactions. We cultured normal human peripheral blood mononuclear cells (PBMCs) with the CD200+ or CD200- cells from each line both. Cells were incubated in the culture media for 4-48 hours before being harvested and measured by flow cytometry for apoptosis or intracellular cytokine production. The presence of CD200 on the cell surface reduced the rate of immune-specific apoptosis among these leukemia cells. The difference in cell killing was most likely attributable to a CD200-specific suppression of CD107a, a surrogate marker or cytotoxic activity. In the OCI-AML3 model, PBMCs co-cultured with CD200+ cells produced approximately 40% less CD107a when compared to the CD200- co-culture. Additionally, we characterized our new cell lines using RNA sequencing. By comparing the CD200+ to the CD200- cells within each line, we observed that CD200+ cells significantly downregulate genes involved in defining an inflammatory response as well as genes regulated by NF-κB in response to TNFα. This indicates that CD200 may have an undiscovered intrinsic role in suppressing the immune microenvironment of AML LSCs. In conclusion, we have expanded our novel bioinformatics approach for robustly identifying AML LSC-specific targets. Additionally, we have shown that one of these markers, CD200, has a potential role as a stem cell-specific immunosuppressive target by reducing immune-mediated apoptosis and transcriptionally suppressing inflammatory cell processes. We are extending our study to explore CD200 in primary patient samples using a CD200-blocking antibody. Disclosures Andreeff: SentiBio: Equity Ownership; Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; Reata: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Jazz Pharma: Consultancy; Astra Zeneca: Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy. Konopleva:Stemline Therapeutics: Research Funding.

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High-Parameter Mass Cytometry (CyTOF) Evaluation of Relapsed/Refractory Multiple Myeloma (MM) Pts (Pts) Treated with Daratumumab Supports Immune Modulation As a Novel Mechanism of Action

Blood ◽

10.1182/blood.v128.22.4521.4521 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4521-4521 ◽

Cited By ~ 6

Author(s):

Homer Adams ◽

Frederik Stevenaert ◽

Jakub Krejcik ◽

Koen Van der Borght ◽

Tineke Casneuf ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Granzyme B ◽

Advisory Committees ◽

Activation Markers ◽

Equity Ownership

Abstract Introduction: Daratumumab (DARA) is a human CD38-targeting monoclonal antibody that induces deep clinical responses in MM pts through multifaceted mechanisms of action (MOA) including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis and induction of apoptosis. Flow cytometry analysis revealed a previously unknown immunomodulatory role of DARA, via T-cell induction expansion, T-cell activity enhancement, and reduction of immune suppressive cell populations including CD38+ myeloid-derived suppressor cells, CD38+ regulatory T cells (TRegs), and CD38+ regulatory B cells (BRegs). Next-generation mass cytometry (CyTOF), which allows high parameter evaluation of the immune system, was used to assess the effects of DARA alone or in combination on a more comprehensive profile of immune cell subpopulations. Methods: Relapsed/refractory MM pt samples from a subset of single agent studies; SIRIUS (32 pts; whole blood [WB] only; Lonial S et al. The Lancet, 2016) and GEN501 (5 pts; WB and bone marrow [BM], Lokhorst HM et al. NEJM, 2015) along with GEN503, a study of DARA plus lenalidomide and dexamethasone (9 pts; WB and BM; Plesner T et al. ASH 2015) were analyzed. Fluorochrome or metal-conjugated antibody panel stained samples were evaluated by flow cytometry or cytometry by time-of-flight (CyTOF®) platforms, respectively. FACS analyses were performed and analyzed by FACS Canto II flow cytometers and FACSDiva software. For CyTOF analysis, events were clustered by phenotype by a spanning tree progression of density normalized events (SPADE) algorithm, and each cluster was associated with an immune population via Cytobank® software. Differential analysis of population fractions and marker intensity, over time and between response groups, derived raw P values from t-tests and single cell level bootstrap adjusted P values corrected for multiple dependent hypothesis testing. Results were visualized using SPADE trees (Figure) and Radviz projections, a new method that allows for the comparison of populations and conditions while preserving the relation to original dimensions. Results: Flow cytometry and high-dimensional CyTOF analyses confirmed previous findings including higher CD38 expression on plasma cells compared with other immune populations of natural killer (NK), monocytes, B and T cells, and depletion of both plasma cells and NK cells upon DARA treatment. Interestingly, while NK cells were significantly reduced with DARA treatment, remaining active NK cells (CD16+CD56dim) demonstrated increased expression of activation markers CD69, CD25 and CD137 while also decreasing granzyme B and increasing naive marker CD27. Though functionality tests weren't performed, the ability to evaluate several markers simultaneously suggests these cells possess limited cytotoxicity. Additionally, these studies indicated depletion of CD38 positive immune suppressive subsets of Tregs and Bregs. CD38+ basophil reductions occurred independent of response and may provide insight to short-lived infusion related reactions. Several observations within the T-cell compartment were indicative of a DARA-mediated adaptive response in both WB and BM samples. T cells displayed increases in total numbers and shifted towards higher CD8:CD4 and effector:naïve ratios after 2 months of DARA treatment. Responders had higher expression levels of several activation markers including CD69 and HLA-DR along with increased production of cytolytic enzyme granzyme B in CD8+ T cells following DARA treatment. Interestingly, in the GEN503 sample set, pts who achieved a complete response presented with a distinct BM CD4 T-cell phenotype of high granzyme B positivity versus those that achieved a partial response or very good partial response. This observation suggests pts with an active immune phenotype may achieve deeper responses to DARA in combination with standard of care agents lenalidomide and dexamethasone. Conclusion: CyTOF analysis of pt samples from both single agent and combination DARA studies agree with flow cytometry and support the pharmacodynamics and immune modulatory MOA of DARA while providing additional insight into changes in T-cell subtypes and activation status. Future CyTOF analyses of clinical samples from phase 3 combination studies aim to confirm these observations and expand the understanding of the MOA of DARA. Disclosures Adams: Janssen Research & Development, LLC: Employment. Stevenaert:Janssen: Employment. Van der Borght:Janssen: Employment. Casneuf:Janssen R&D, Beerse, Belgium: Employment; Johnson & Johnson: Equity Ownership. Smets:Janssen: Employment. Bald:Janssen: Employment. Abraham:Janssen: Employment. Ceulemans:Janssen: Employment. Vanhoof:Janssen: Employment; Johnson & Johnson: Equity Ownership. Ahmadi:Janssen: Employment. Usmani:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Array: Research Funding; BioPharma: Research Funding; Pharmacyclics: Research Funding; Takeda: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Millenium: Membership on an entity's Board of Directors or advisory committees; Skyline: Membership on an entity's Board of Directors or advisory committees. Plesner:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lonial:Janssen: Consultancy; BMS: Consultancy; Merck: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Onyx: Consultancy; Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Lokhorst:Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mutis:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Celgene: Research Funding. van de Donk:Janssen: Research Funding; BMS: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Sasser:Janssen Pharmaceuticals R&D: Employment; Johnson & Johnson: Equity Ownership.

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Leukemic Cell Expressed CTLA-4 Suppresses T Cells Via Down-Modulation of CD80 By Trans-Endocytosis

Blood ◽

10.1182/blood.v128.22.3221.3221 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3221-3221 ◽

Cited By ~ 1

Author(s):

Priscilla Do ◽

Kyle A. Beckwith ◽

Larry Beaver ◽

Brittany G. Griffin ◽

Xiaokui Mo ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

Cell Surface ◽

Board Of Directors ◽

Tumor Cells ◽

Research Funding ◽

Leukemic Cell ◽

Advisory Committees

Abstract The function of CTLA-4 on non-T cells is largely ignored and currently ill defined despite rapidly growing interest in targeting this immune checkpoint protein in several cancers. While anti-CTLA-4 therapy is proposed to work through inhibition of the immunosuppressive effect of CTLA-4 on T cells, multiple examples of non-T cell expressed CTLA-4 have been reported. These cells include tumor cells of hematological and non-hematological origin and normal B cells. In this study, we have defined a novel immune suppressive role for non-T cell, tumor expressed CTLA-4 in Chronic Lymphocytic Leukemia (CLL). We have detected by microarray that CTLA-4 is in the top 5 most differentially expressed genes between pooled samples of healthy donor normal B cells (N=6) and pooled CLL leukemic B cells (N=5). Upregulation of CTLA-4 by CLL B cells compared to normal B cells was validated by RT-qPCR and flow cytometry. CTLA-4 was predominantly intracellular (42/46 CTLA-4+) and not on the cell surface (2/48 CTLA-4+) in primary CLL samples. B cell activating factors (CD40L, PMA/Ionomycin, LPS, IL4, LPS+IL4, CD40L+IL4, CpG, and anti-IgM) could not induce surface expression of CTLA-4; however, co-culture with anti-CD3/anti-CD28 or ConA activated T cells (autologous or allogeneic) resulted in detectable CTLA-4 on the cell surface of leukemic B cells. This induction did not occur with resting T cells. This finding suggests a role for CTLA-4+ tumor cells in sites of T cell activation, such as the lymph node, a site of leukemic cell proliferation in CLL. To mechanistically study leukemic B cell expressed CTLA-4, we generated CLL-derived Mec1 and OSU-CLL that inducibly express CTLA-4 upon doxycycline (dox) treatment. Mec1 and OSU-CLL cells highly express the ligands for CTLA-4, CD80 and CD86. Dox-induction of CTLA-4 resulted in decreased expression of Mec1 and OSU-CLL expressed CD80, a critical T cell co-stimulatory protein (N=3). Blockade of CTLA-4 using the anti-CTLA-4 therapeutic antibody, Ipilimumab, could restore CD80 on Mec1 and OSU-CLL cells (N=3). Because T cell-expressed CTLA-4 has been previously shown by others to down-modulate CD80 via trans-endocytosis, we co-cultured CTLA-4+ Mec1 and CTLA-4+ primary CLL cells with stably transfected CD80-GFP or CD86-GFP Hek293 cell lines to assess uptake of CD80/CD86 into CTLA-4 expressing tumor cells as the mechanism of CD80 down-modulation. Transfer of CD80-GFP and CD86-GFP was detected by flow cytometry in primary CLL cells and the Mec1 cell line, consistent with the ability of T cell expressed CTLA-4 to trans-endocytose CD80 and CD86. Furthermore, uptake of CD80-GFP or CD86-GFP by primary tumor cells was CTLA-4 dependent, demonstrated by inhibition of GFP uptake in the presence of Ipilimumab. Following determination of decreased CD80, we found that co-culture of primary T cells with Mec1 CTLA-4+ cells resulted in decreased IL2 production measured by Cytokine Bead Array. The loss of IL2 signified decreased co-stimulation as a result of tumor expressed CTLA-4. Studies are ongoing regarding dependence on CD80 or CD86. A minor subset of T cells, Tregs, are known to exert profound immunosuppressive effects through their expression of CTLA-4. Due to our results, tumor expressed CTLA-4 has an overlapping function with Treg CTLA-4, and it is imperative that we define the immunosuppressive effects as, in patients, the leukemic cells may comprise a much larger proportion of white blood cells than T cells. Efforts are now underway to address the effect of tumor expressed CTLA-4 in suppressing anti-tumor immunity in vivo utilizing a novel mouse model. Suppression of T cells by tumor expressed CTLA-4 is a novel finding that is broadly applicable to fields within and outside of cancer research as the pathway and mechanism described here are potentially applicable to CTLA-4 in diverse disease contexts and to the general biology of CTLA-4. [Funding: This work was supported by P01 CA95426. PD received the Pelotonia Graduate Fellowship. Any opinions, findings, and conclusions expressed in this material are those of the author(s) and do not necessarily reflect those of the Pelotonia Fellowship Program] Disclosures Jones: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Reductions in JAK2 V617F Allele Burden with Ruxolitinib Treatment in Comfort-II, a Phase 3 Study Comparing the Safety and Efficacy of Ruxolitinib with Best Available Therapy (BAT)

Blood ◽

10.1182/blood.v120.21.802.802 ◽

2012 ◽

Vol 120 (21) ◽

pp. 802-802 ◽

Cited By ~ 7

Author(s):

Alessandro M. Vannucchi ◽

Francesco Passamonti ◽

Haifa Kathrin Al-Ali ◽

Giovanni Barosi ◽

Claire N Harrison ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Jak2 V617f ◽

Advisory Board ◽

Spleen Volume ◽

Employment Equity ◽

Phase 3 ◽

Advisory Committees ◽

Allele Burden ◽

Equity Ownership

Abstract Abstract 802 Background: Ruxolitinib is a potent oral JAK1 & JAK2 inhibitor that has demonstrated superiority over traditional therapies for the treatment of MF. In the two phase 3 studies, ruxolitinib demonstrated rapid and durable reductions in splenomegaly and improved disease-related symptoms and quality of life compared with placebo (COMFORT-I) and best available therapy (BAT; COMFORT-II) for pts with or without the JAK2 V617F mutation. Change in JAK2 V617F allele burden (%V617F) as a metric of molecular response to treatment in JAK2 V617 F–positive pts was investigated as an exploratory endpoint. Previously, we reported allele burden reductions in pts receiving ruxolitinib in the COMFORT-II study and demonstrated a positive correlation with reduction in spleen volume after 24 and 48 wk of treatment (Vannucchi, et al. Haematologica.2012); here, we evaluate the correlation between changes in %V617F and spleen size reduction after 72 wk of ruxolitinib therapy. Methods: COMFORT-II is a randomized (2:1), open-label, phase 3 study evaluating the safety and efficacy of ruxolitinib (n = 146) compared with BAT (n = 73) in pts with primary MF (PMF), post–polycythemia vera MF (PPV-MF), and post–essential thrombocythemia MF (PET-MF). Allele burden was measured from blood samples using allele-specific quantitative real-time polymerase chain reaction (qPCR) using the method outlined in Levine et al, 2006, using an Applied Biosystems ABI 7900 real-time PCR analyzer. Pts were stratified by absolute reduction in %V617F (< 10%, 10% to < 20%, ≥ 20%) and results were correlated with achievement and duration of a ≥ 35% reduction from baseline in spleen volume, as measured by magnetic resonance imaging (MRI) or computed tomography (CT) scans. Results: Overall, 110 (76%) pts in the ruxolitinib group and 49 (71%) pts in the BAT group were JAK2V617 F–positive at baseline. More pts in the ruxolitinib arm had ≥ 10% V617F reductions compared with BAT at wk 48 (Table; 41% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(2868\) \end{document} vs 5% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(122\) \end{document}) and at wk 72 (40% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(2153\) \end{document} vs 0). The majority of reductions ≥ 20% were gradual and progressive over the course of the study (Figure); 2 pts had rapid initial reductions in allele burden that were sustained over 72 wk, from absolute %V617F of 48% and 45% at baseline to < 10% at wk 72. The majority of patients who achieved a ≥ 20% reduction at wk 48 maintained their reduction at wk 72. Compared with wk 48, 4 additional pts were in the ≥ 20% group at wk 72: 1 pt achieved a > 20% reduction at wk 48 but the data were not available at the time of the 48-wk analysis, 2 pts did not have data at wk 48, and 1 pt achieved a 15% reduction at wk 48 that improved to a 21% reduction by wk 72. Among pts who achieved a ≥ 20% reduction in allele burden, 39% had PMF, 39% had PPV-MF, and 22% had PET-MF; this distribution was similar to that of the overall study population. In the ruxolitinib arm, significantly more pts with a ≥ 20% V617F reduction achieved a ' 35% reduction from baseline in spleen volume compared with pts with a < 10% reduction at both wk 48 (79% vs 30%) and wk 72 (69% vs 31%). Pts with a ≥ 20% reduction in allele burden maintained their spleen volume reductions from baseline out to 72 wk. In the 10% to < 20% group, a greater proportion of pts showed increases in spleen volume from nadir but spleen volumes still remained much reduced from baseline. Conclusions: Patients who received ruxolitinib had larger reductions in JAK2 V617F allele burden compared with BAT. %V617F reductions were gradual over the course of the study and continued between wk 48 and 72 in some pts. In JAK2 V617 F–positive pts, reductions in %V617F were associated with spleen responses; in pts with a ≥ 20% reduction, spleen volume reductions were sustained out to 72 wk. These results, along with findings from COMFORT-I and the phase 1/2 study, suggest that ruxolitinib has the potential to alter the course of disease through a reduction in the burden of JAK2V671 F–mutated cells. Disclosures: Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees. Passamonti:Sanofi: 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; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Al-Ali:Sanofi Aventi: Consultancy, Honoraria; celgene: Honoraria, Research Funding; Novartis: Consultancy, Honoraria. Harrison:Shire: Honoraria, Research Funding; Sanofi: Honoraria; YM Bioscience: Consultancy, Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau. Sirulnik:Novartis: Employment. Stalbovskaya:Novartis: Employment, Equity Ownership. Squires:Novartis : Employment. Burn:Incyte: Employment, Equity Ownership. Knoops:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cervantes:Bristol-Myers Squibb: Speakers Bureau; Teva Pharmaceuticals: Advisory Board, Advisory Board Other; Pfizer: Advisory Board, Advisory Board Other; Celgene: Advisory Board, Advisory Board Other; Sanofi-Aventis: Advisory Board, Advisory Board Other; Novartis: AdvisoryBoard Other, Speakers Bureau. Barbui:Novartis: Honoraria. Gisslinger:Celgene: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau; AOP Orphan Pharma AG: Consultancy, Speakers Bureau. Kiladjian:Incyte: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

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Rare Germline Mutations in Complement Regulatory Genes Make the Antiphospholipid Syndrome Catastrophic

Blood ◽

10.1182/blood-2019-129115 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4-4 ◽

Cited By ~ 1

Author(s):

Shruti Chaturvedi ◽

Evan M Braunstein ◽

Xuan Yuan ◽

Hang Chen ◽

Ravi Kumar Alluri ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Board Of Directors ◽

Complement Activation ◽

Research Funding ◽

Germline Mutations ◽

Functional Assay ◽

Advisory Committees ◽

Surface Deposition

Introduction: The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity along with persistent antiphospholipid antibodies (aPL). Despite adequate anticoagulation, 10-30% of patients have recurrent thrombosis. Catastrophic APS (CAPS) is associated with approximately 40% mortality despite treatment. The pathogenesis of APS complications is incompletely understood. Recent animal studies indicate that complement is required for aPL-associated thrombosis, and complement has emerged as an attractive therapeutic target for refractory thrombotic APS and CAPS. Methods: We first evaluated complement activation in sera of patients with thrombotic APS by ISTH criteria (N=53), catastrophic APS (CAPS; N=8, sera available for 6), and systemic lupus erythematosus (SLE; N=74) who presented to our institution from June 2015 to June 2019 (and four patients with CAPS from other institutions). We used the modified Ham (mHam) test, a functional assay for complement activation as described previously (Gavriilaki et al. Blood 2015). The mHam assay is based on the principle that a PNH cell line (PIGA-null TF-1 cells) lacking the cell surface complement regulators CD55 and CD59 undergoes lysis in serum containing activated complement. Cell death (measured by a cell viability assay) is a measure of complement activation. Cell surface deposition of complement products (C3c, C5b-9) is also detected by flow cytometry. We then evaluated whether adding purified patient-derived aPL (anti-β2 glycoprotein IgG) to normal serum induced complement activation. Finally, we performed targeted sequencing of 15 complement genes in the study subjects, as well as 22 patients with aHUS and 36 healthy individuals as positive and negative controls, respectively. Results: (A) Complement activation is associated with thrombotic APS. A positive mHam assay (>20% cell killing) was detected in 32.1% (17 of 53) patients with thrombotic APS and 100% (6 of 6 with available sera) of CAPS compared with 6.8% (5 of 74) with SLE, (P <0.001) (Fig. 1A). A history of thrombosis was present in 79.3% patients with a positive mHam and 38.4% with a negative mHam test. Among APS patients, mHam positivity was associated with triple positivity (lupus anticoagulant, anti-β2-glycoprotein-1 Ab and anti-cardiolipin Ab), which is associated with higher thrombotic risk (60%), than double (23%) or single positivity (10%) (P = 0.002) (Fig. 1B). APS patients were more likely to have a positive mHam closer to a thrombotic event (Fig. 1C). (B) aPL from patients activate complement in vitro. Patient-derived anti- β2 glycoprotein IgG from all four patients induced complement activation in the mHam assay (Fig 2A). Flow cytometry confirmed cell surface deposition of complement activation products (C4d, C5b-9), which was inhibited by adding anti-C5 monoclonal Ab or a factor D inhibitor (representative sample in fig. 1B). (C) Catastrophic APS is associated with complement mutations. Rare (minor allele frequency <0.01) germline mutations in complement genes were present in 62.5% (5 of 8) patients with CAPS, 22.6% (12 of 53) patients with thrombotic APS, and 23.8% (5 of 21) of SLE compared with 50% (11 of 22) of aHUS, and 19.4% (7 of 36) of normal individuals. The mutation rate in CAPS was significantly higher than in APS (P=0.019), SLE (P=0.051), and normal controls and similar to that seen in aHUS (P=0.36). Rare variants in CAPS included: (i) homozygous CFHR1-CFHR3 deletion, (ii) THBD P501L, (iii) CR1 S1982G and homozygous CFHR1-CFHR3 deletion, (iv) CFHR4 R287H, and (v) CR1 V2125L. Conclusions: APS serum activates complement in vitro shown by a functional assay (mHam) and increased C5b-9 deposition on the cell surface. A positive mHam test strongly associates with both recent thrombosis and triple positive APS. Purified human anti-B2GPI antibody from APS patients activates complement when added to normal human serum, suggesting that complement activation plays a pathophysiologic role in APS associated thrombosis. Finally, CAPS patients have a high rate of mutations in complement genes, which likely serves as a 'second-hit' (in addition to aPL) leading to uncontrolled complement activation and a more severe phenotype (Figure 3). Taken together, our results provide a rationale for complement inhibition as a therapeutic strategy in patients with CAPS and refractory thrombotic APS. Disclosures Chaturvedi: Shire/Takeda: Research Funding; Sanofi: Consultancy; Alexion: Consultancy. Streiff:Pfizer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Portola: Consultancy, Honoraria; Roche: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria. Petri:Astellas: Consultancy; Novartis: Consultancy; Exagen: Consultancy, Research Funding; Glenmark Pharmaceuticals: Consultancy; EMD Serono: Consultancy; Bristol-Myers Squibb: Consultancy; IQVIA: Consultancy; Janssen Pharmaceuticals: Consultancy; Aleon Pharmaceuticals: Consultancy; Momenta Pharmaceuticals: Consultancy; Blackrock Pharmaceuticals: Consultancy; Astrazeneca: Consultancy, Research Funding; UCB Pharmaceuticals: Consultancy; GSK: Consultancy; Qiagen: Consultancy; Abbive: Consultancy; Amgen: Consultancy; Decision Resources: Consultancy; Principia Biopharma: Consultancy; Eli Lilly: Consultancy; Kezaar Life Sciences: Consultancy. McCrae:Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Brodsky:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding; Achillion: Research Funding.

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Immunotherapy with T-Cells Engineered with a Chimeric Antigen Receptor Bearing a Human CD19-Binding Single Chain Variable Fragment for Relapsed or Refractory Acute Lymphoblastic Leukemia and B-Cell Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood-2018-99-111528 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1415-1415 ◽

Cited By ~ 3

Author(s):

Jordan Gauthier ◽

Alexandre V. Hirayama ◽

Kevin A. Hay ◽

Alyssa Sheih ◽

Barbara S. Pender ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Car T Cell ◽

Car T ◽

Equity Ownership ◽

Grade 3

Abstract Background We previously reported high response rates and durable remissions in patients (pts) with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL; Turtle, JCI 2016) and non-Hodgkin lymphoma (NHL; Turtle, Sci Transl Med 2016) treated with CD19-specific chimeric antigen receptor T (CD19 CAR-T) cells. In a subset of pts, we identified CD8+ T cell responses to epitopes in the murine CD19-binding single chain variable fragment (scFv) of the CAR that could limit CAR-T cell persistence and responses to subsequent infusions. In an effort to reduce the potential for immune CAR-T cell rejection, the murine CD19-binding scFv of the CAR was replaced with a fully human scFv linked to 4-1BB and CD3z signaling domains (JCAR021; Sommermeyer, Leukemia 2017). Here we report the initial clinical results of immunotherapy with JCAR021. Methods We initiated a phase I trial investigating lymphodepletion with cyclophosphamide 300 mg/m2/d and fludarabine 30 mg/m2/d for 3 days (Cy/Flu) followed by infusion of JCAR021 in pts with R/R ALL and aggressive NHL (NCT03103971). Pts were enrolled into 1 of 3 cohorts: high marrow burden ALL (HMB; > 5% blasts in bone marrow [BM] before lymphodepletion); low marrow burden ALL (LMB; ≤ 5% blasts in BM before lymphodepletion); and NHL. The starting dose was 7x104 JCAR021 cells/kg for the HMB ALL cohort, and 7x105 JCAR021 cells/kg in both the LMB ALL and NHL cohorts. Dose escalation/de-escalation follows a modified toxicity probability interval algorithm (Guo, Contemp Clin Trials 2017). Responses in the NHL cohort and in the HMB/LMB ALL cohorts were determined by the Lugano criteria (Cheson, JCO 2014) and the 2018 NCCN guidelines, respectively. Cytokine release syndrome (CRS) was graded according to consensus criteria (Lee, Blood 2014) and neurotoxicity was graded according to CTCAE v4.03. Results Pt characteristics are detailed in Table 1. As of June 15, 2018, 9 pts were enrolled on the trial. Two pts did not receive JCAR021: one pt was excluded after aggressive NHL was reclassified as indolent after pathology review and one pt had no detectable disease upon pre-treatment restaging. The 7 pts who received JCAR021 had a median age of 63 years (range: 29 - 69). Both pts in the LMB ALL cohort had bulky extramedullary disease (> 5 cm diameter). One patient (LMB ALL cohort) had failed two allogeneic transplants and one patient (HMB ALL cohort) had failed an allogeneic transplant prior to treatment with JCAR021. Four of 4 pts in the NHL cohort and 2 of 2 pts in the LMB ALL cohort received 7x105 JCAR021 cells/kg. The pt treated in the HMB ALL cohort received 7x104 JCAR021 cells/kg. No pt in any cohort developed grade ≥ 3 CRS. All ALL pts developed grade 2 CRS. No pts with NHL developed CRS; one pt in the NHL cohort who had CNS disease prior to CAR-T cell immunotherapy developed grade 3 neurotoxicity in the absence of CRS. We did not observe other neurologic events. No other grade ≥ 3 non-hematopoietic organ toxicity was observed and all 7 treated pts have completed response evaluation. Four weeks after infusion of a low dose of JCAR021, both patients in the LMB ALL cohort had undetectable marrow disease by high resolution flow cytometry and regression of bulky extramedullary disease (1 complete response [CR] and 1 partial response [PR] by PET-CT). One pt treated with a low dose (7x104 cells/kg) of JCAR021 in the HMB ALL cohort did not achieve CR (decrease in BM blasts from 79.8% to 29.5%) but CNS disease was cleared by flow cytometry. In the NHL cohort, we observed objective responses in 2 of 4 patients (1 CR, 1 PR). JCAR021 was detected in blood by flow cytometry and/or quantitative PCR for up to 112 days after infusion. Conclusion JCAR021 appears to have a favorable toxicity profile in R/R ALL and NHL pts. JCAR021 cells expanded in vivo and have persisted in all pts. We observed responses at very low doses of CAR-T cells in ALL pts with bulky disease. This trial continues to enroll to define optimal dosing and determine the safety and efficacy of JCAR021. Disclosures Hirayama: DAVA Oncology: Honoraria. Hay:DAVA Oncology: Honoraria. Till:Mustang Bio: Patents & Royalties, Research Funding. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy. Shadman:TG Therapeutics: Research Funding; Mustang: Research Funding; Gilead: Research Funding; Pharmacyclics: Research Funding; AstraZeneca: Consultancy; Qilu Puget Sound Biotherapeutics: Consultancy; Acerta: Research Funding; Abbvie: Consultancy; Verastem: Consultancy; Genentech: Consultancy, Research Funding; Beigene: Research Funding; Celgene: Research Funding. Cassaday:Amgen: Consultancy, Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Adaptive Biotechnologies: Consultancy; Incyte: Research Funding; Pfizer: Consultancy, Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Jazz Pharmaceuticals: Consultancy. Acharya:Teva: Honoraria; Juno Therapeutics: Research Funding. Riddell:NOHLA: Consultancy; Adaptive Biotechnologies: Consultancy; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Maloney:Juno Therapeutics: Research Funding; Seattle Genetics: Honoraria; Janssen Scientific Affairs: Honoraria; GlaxoSmithKline: Research Funding; Roche/Genentech: Honoraria. Turtle:Aptevo: Consultancy; Nektar Therapeutics: Consultancy, Research Funding; Caribou Biosciences: Consultancy; Gilead: Consultancy; Juno Therapeutics / Celgene: Consultancy, Patents & Royalties, Research Funding; Bluebird Bio: Consultancy; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Rapid and Robust Mobilization of CD34+ HSCs without G-CSF Following Administration of Mgta-145 Alone or in Combination with Plerixafor

Blood ◽

10.1182/blood-2019-127523 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1961-1961

Author(s):

John F. DiPersio ◽

Jonathan Hoggatt ◽

Steven Devine ◽

Lukasz Biernat ◽

Haley Howell ◽

...

Keyword(s):

Single Dose ◽

Adverse Events ◽

Board Of Directors ◽

Preliminary Data ◽

Research Funding ◽

Fold Change ◽

Employment Equity ◽

Advisory Committees ◽

Cd34 Cells ◽

Equity Ownership

Background Granulocyte colony-stimulating factor (G-CSF) is the standard of care for mobilization of hematopoietic stem cells (HSCs). G-CSF requires 4-7 days of injections and often multiple aphereses to acquire sufficient CD34+ cells for transplant. The number of CD34+ HSCs mobilized can be variable and patients who fail to mobilize enough CD34+ cells are treated with the combination of G-CSF plus plerixafor. G-CSF use is associated with bone pain, nausea, headaches, fatigue, rare episodes of splenic rupture, and is contraindicated for patients with autoimmune and sickle cell disease. MGTA-145 (GroβT) is a CXCR2 agonist. MGTA-145, in combination with plerixafor, a CXCR4 inhibitor, has the potential to rapidly and reliably mobilize robust numbers of HSCs with a single dose and same-day apheresis for transplant that is free from G-CSF. MGTA-145 plus plerixafor work synergistically to rapidly mobilize HSCs in both mice and non-human primates (Hoggatt, Cell 2018; Goncalves, Blood 2018). Based on these data, Magenta initiated a Phase 1 dose-escalating study to evaluate the safety, PK and PD of MGTA-145 as a single agent and in combination with plerixafor. Methods This study consists of four parts. In Part A, healthy volunteers were dosed with MGTA-145 (0.0075 - 0.3 mg/kg) or placebo. In Part B, MGTA-145 dose levels from Part A were selected for use in combination with a clinically approved dose of plerixafor. In Part C, a single dose MGTA-145 plus plerixafor will be administered on day 1 and day 2. In Part D, MGTA-145 plus plerixafor will be administered followed by apheresis. Results MGTA-145 monotherapy was well tolerated in all subjects dosed (Table 1) with no significant adverse events. Some subjects experienced mild (Grade 1) transient lower back pain that dissipated within minutes. In the ongoing study, the combination of MGTA-145 with plerixafor was well tolerated, with some donors experiencing Grade 1 and 2 gastrointestinal adverse events commonly observed with plerixafor alone. Pharmacokinetic (PK) exposure and maximum plasma concentrations increased dose proportionally and were not affected by plerixafor (Fig 1A). Monotherapy of MGTA-145 resulted in an immediate increase in neutrophils (Fig 1B) and release of plasma MMP-9 (Fig 1C). Neutrophil mobilization plateaued within 1-hour post MGTA-145 at doses greater than 0.03 mg/kg. This plateau was followed by a rebound of neutrophil mobilization which correlated with re-expression of CXCR2 and presence of MGTA-145 at pharmacologically active levels. Markers of neutrophil activation were relatively unchanged (<2-fold vs baseline). A rapid and statistically significant increase in CD34+ cells occurred @ 0.03 and 0.075 mg/kg of MGTA-145 (p < 0.01) relative to placebo with peak mobilization (Fig 1D) 30 minutes post MGTA-145 (7-fold above baseline @ 0.03 mg/kg). To date, the combination of MGTA-145 plus plerixafor mobilized >20/µl CD34s in 92% (11/12) subjects compared to 50% (2/4) subjects receiving plerixafor alone. Preliminary data show that there was a significant increase in fold change relative to baseline in CD34+ cells (27x vs 13x) and phenotypic CD34+CD90+CD45RA- HSCs (38x vs 22x) mobilized by MGTA-145 with plerixafor. Mobilized CD34+ cells were detectable at 15 minutes with peak mobilization shifted 2 - 4 hours earlier for the combination vs plerixafor alone (4 - 6h vs 8 - 12h). Detailed results of single dose administration of MGTA-145 and plerixafor given on one day as well as also on two sequential days will be presented along with fully characterized graft analysis post apheresis from subjects given MGTA-145 and plerixafor. Conclusions MGTA-145 is safe and well tolerated, as a monotherapy and in combination with plerixafor and induced rapid and robust mobilization of significant numbers of HSCs with a single dose in all subjects to date. Kinetics of CD34+ cell mobilization for the combination was immediate (4x increase vs no change for plerixafor alone @ 15 min) suggesting the mechanism of action of MGTA-145 plus plerixafor is different from plerixafor alone. Preliminary data demonstrate that MGTA-145 when combined with plerixafor results in a significant increase in CD34+ fold change relative to plerixafor alone. Magenta Therapeutics intends to develop MGTA-145 as a first line mobilization product for blood cancers, autoimmune and genetic diseases and plans a Phase 2 study in multiple myeloma and non-Hodgkin lymphoma in 2020. Disclosures DiPersio: Magenta Therapeutics: Equity Ownership; NeoImmune Tech: Research Funding; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Consultancy; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Macrogenics: Research Funding, Speakers Bureau; Bioline Rx: Research Funding, Speakers Bureau; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Devine:Kiadis Pharma: Other: Protocol development (via institution); Bristol Myers: Other: Grant for monitoring support & travel support; Magenta Therapeutics: Other: Travel support for advisory board; My employer (National Marrow Donor Program) has equity interest in Magenta. Biernat:Medpace, Inc.: Employment. Howell:Magenta Therapeutics: Employment, Equity Ownership. Schmelmer:Magenta Therapeutics: Employment, Equity Ownership. Neale:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Goncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Raffel:Magenta Therapeutics: Employment, Equity Ownership. Falahee:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Davis:Magenta Therapeutics: Employment, Equity Ownership.

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Treatment Patterns and Outcomes in Elderly Patients with Newly Diagnosed Multiple Myeloma: Results from the Connect® MM Registry

Blood ◽

10.1182/blood-2019-126205 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3129-3129

Author(s):

Hans C. Lee ◽

Sikander Ailawadhi ◽

Cristina Gasparetto ◽

Sundar Jagannath ◽

Robert M. Rifkin ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Causes Of Death ◽

Age Groups ◽

Treatment Patterns ◽

Age Group ◽

Newly Diagnosed ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Background: Multiple myeloma (MM) is common among the elderly, with 35% of patients (pts) diagnosed being aged ≥75 years (y). With increasing overall life expectancy, the incidence and prevalence of newly diagnosed and previously treated MM patients ≥80 y is expected to increase over time. Because elderly pts are often excluded from clinical trials, data focused on their treatment patterns and clinical outcomes are lacking. The Connect® MM Registry (NCT01081028) is a large, US, multicenter, prospective observational cohort study of pts with newly diagnosed MM (NDMM) designed to examine real-world diagnostic patterns, treatment patterns, clinical outcomes, and health-related quality of life patient-reported outcomes. This analysis reviews treatment patterns and outcomes in elderly pts from the Connect MM Registry. Methods: Pts enrolled in the Connect MM registry at 250 community, academic, and government sites were included in this analysis. Eligible pts were adults aged ≥18 y with symptomatic MM diagnosed ≤2 months before enrollment, as defined by International Myeloma Working Group criteria; no exclusion criteria were applied. For this analysis, pts were categorized into 4 age groups: <65, 65 to 74, 75 to 84, and ≥85 y. Pts were followed from time of enrollment to the earliest of disease progression (or death), loss to follow-up, or data cutoff date of February 7, 2019. Descriptive statistics were used for baseline characteristics and treatment regimens. Survival outcomes were analyzed using Cox regression. Time to progression (TTP) analysis excluded causes of death not related to MM. Results: Of 3011 pts enrolled (median age 67 y), 132 (4%) were aged ≥85 y, and 615 (20%) were aged 75-84 y at baseline. More pts aged ≥85 y had poor prognostic factors such as ISS stage III disease and reduced hemoglobin (<10 g/dL or >2 g/dL <LLN) compared with other age groups, although no notable differences between creatinine and calcium levels were observed across age groups (Table). A lower proportion of elderly pts (75-84 and ≥85 y) received triplet regimens as frontline therapy. More elderly pts received a single novel agent, whereas use of 2 novel agents was more common in younger pts (Table). The most common frontline regimens among elderly pts were bortezomib (V) + dexamethasone (D), followed by lenalidomide (R) + D, whereas those among younger pts included RVD, followed by VD and CyBorD (Table). No pt aged ≥85 y, and 4% of pts aged 75-84 y received high-dose chemotherapy and autologous stem cell transplant (vs 61% in the <65 y and 37% in the 65-74 y age group). The most common maintenance therapy was RD in pts ≥85 y (although the use was low) and R alone in other age groups (Table). In the ≥85 y group, 27%, 10%, and 4% of pts entered 2L, 3L, and 4L treatments respectively, vs 43%, 23%, and 13% in the <65 y group. Progression-free survival was significantly shorter in the ≥85 y age group vs the 75-84 y age group (P=0.003), 65-74 y age group (P<0.001), and <65 y age group (P<0.001; Fig.1). TTP was significantly shorter in the ≥85 y group vs the <65 y group (P=0.020); however, TTP was similar among the 65-74 y, 75-84 y, and ≥85 y cohorts (Fig. 2). Overall survival was significantly shorter in the ≥85 y group vs the 75-84 y, 65-74 y, and <65 y groups (all P<0.001; Fig. 3). The mortality rate was lowest (46%) during first-line treatment (1L) in pts aged ≥85 y (mainly attributed to MM progression) and increased in 2L and 3L (47% and 54%, respectively); a similar trend was observed in the younger age groups. The main cause of death was MM progression (29% in the ≥85 y vs 16% in the <65 y group). Other notable causes of death in the ≥85 y group included cardiac failure (5% vs 2% in <65 y group) and pneumonia (5% vs 1% in <65 y group). Conclusions: In this analysis, elderly pts received similar types of frontline and maintenance regimens as younger pts, although proportions varied with decreased use of triplet regimens with age. Considering similarities in TTP across the 65-74 y, 75-84 y, and ≥85 y cohorts, these real-world data support active treatment and aggressive supportive care of elderly symptomatic pts, including with novel agents. Additionally, further clinical studies specific to elderly patients with MM should be explored. Disclosures Lee: Amgen: Consultancy, Research Funding; GlaxoSmithKline plc: Research Funding; Sanofi: Consultancy; Daiichi Sankyo: Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Ailawadhi:Janssen: Consultancy, Research Funding; Takeda: Consultancy; Pharmacyclics: Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy; Cellectar: Research Funding. Gasparetto:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Jagannath:AbbVie: Consultancy; Merck & Co.: Consultancy; Bristol-Myers Squibb: Consultancy; Karyopharm Therapeutics: Consultancy; Celgene Corporation: Consultancy; Janssen Pharmaceuticals: Consultancy. Rifkin:Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Durie:Amgen, Celgene, Johnson & Johnson, and Takeda: Consultancy. Narang:Celgene: Speakers Bureau. Terebelo:Celgene: Honoraria; Jannsen: Speakers Bureau; Newland Medical Asociates: Employment. Toomey:Celgene: Consultancy. Hardin:Celgene: Membership on an entity's Board of Directors or advisory committees. Wagner:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; American Cancer Society: Other: Section editor, Cancer journal. Omel:Celgene, Takeda, Janssen: Other: Patient Advisory Committees. Srinivasan:Celgene: Employment, Equity Ownership. Liu:TechData: Consultancy. Dhalla:Celgene: Employment. Agarwal:Celgene Corporation: Employment, Equity Ownership. Abonour:BMS: Consultancy; Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

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Impact of Modified Thalidomide Dosing in Bortezomib/Thalidomide/Dexamethasone for Patients with Newly Diagnosed Multiple Myeloma Who Are Transplant-Eligible: A Matching-Adjusted Indirect Comparison

Blood ◽

10.1182/blood-2019-122775 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4739-4739

Author(s):

Pieter Sonneveld ◽

Maria-Victoria Mateos ◽

Adrián Alegre ◽

Thierry Facon ◽

Cyrille Hulin ◽

...

Keyword(s):

Peripheral Neuropathy ◽

Board Of Directors ◽

Research Funding ◽

Indirect Comparison ◽

Employment Equity ◽

Advisory Committees ◽

Post Transplant ◽

Post Induction ◽

Adjusted Indirect Comparison ◽

Equity Ownership

Introduction: For patients with newly diagnosed multiple myeloma (NDMM) who are transplant-eligible, bortezomib/thalidomide/dexamethasone (VTd) is a standard of care (SoC) for induction and consolidation therapy. Clinical practice has evolved to use a modified VTd dose (VTd-mod; 100 mg thalidomide daily), which is reflected in recent treatment guidelines. As VTd-mod has become a real-world SoC, a matching-adjusted indirect comparison (MAIC) of the VTd-mod dose from recent clinical trials versus the dose included in the label (VTd-label; ramp up to 200 mg thalidomide daily) was performed to understand the effect on efficacy of modified VTd dosing for patients with NDMM who are transplant-eligible. Methods: For each outcome (overall survival [OS], progression-free survival [PFS], overall response rates [ORR] post-induction and post-transplant, and rate of peripheral neuropathy), a naïve comparison and a MAIC were performed. Data for VTd-label were obtained from the phase 3 PETHEMA/GEM study (Rosiñol L, et al. Blood. 2012;120[8]:1589-1596). Data for VTd-mod were pooled from the phase 3 CASSIOPEIA study (Moreau P, et al. Lancet. 2019;394[10192]:29-38) and the phase 2 NCT00531453 study (Ludwig H, et al. J Clin Oncol. 2013;31[2]:247-255). Patient-level data for PETHEMA/GEM and CASSIOPEIA were used to generate outcomes of interest and were validated against their respective clinical study reports; aggregate data for NCT00531453 were extracted from the primary publication. Matched baseline characteristics were age, sex, ECOG performance status, myeloma type, International Staging System (ISS) stage, baseline creatinine clearance, hemoglobin level, and platelet count. Results: Patients received VTd-mod (n = 591) or VTd-label (n = 130). After matching, baseline characteristics were similar across groups. For OS, the naïve comparison and the MAIC showed that VTd-mod was non-inferior to VTd-label (MAIC HR, 0.640 [95% CI: 0.363-1.129], P = 0.121; Figure 1A). VTd-mod significantly improved PFS versus VTd-label in the naïve comparison and MAIC (MAIC HR, 0.672 [95% CI: 0.467-0.966], P = 0.031; Figure 1B). Post-induction ORR was non-inferior for VTd-mod versus VTd-label (MAIC odds ratio, 1.781 [95% CI: 1.004-3.16], P = 0.065). Post-transplant, VTd-mod demonstrated superior ORR in both the naïve comparison and MAIC (MAIC odds ratio, 2.661 [95% CI: 1.579-4.484], P = 0.001). For rates of grade 3 or 4 peripheral neuropathy, the naïve comparison and MAIC both demonstrated that VTd-mod was non-inferior to VTd-label (MAIC rate difference, 2.4 [⁻1.7-6.49], P = 0.409). Conclusions: As naïve, indirect comparisons are prone to bias due to patient heterogeneity between studies, a MAIC can provide useful insights for clinicians and reimbursement decision-makers regarding the relative efficacy and safety of different treatments. In this MAIC, non-inferiority of VTd-mod versus VTd-label was demonstrated for OS, post-induction ORR, and peripheral neuropathy. This analysis also showed that VTd-mod significantly improved PFS and ORR post-transplant compared with VTd-label for patients with NDMM who are transplant-eligible. A limitation of this analysis is that unreported or unobserved confounding factors could not be adjusted for. Disclosures Sonneveld: Takeda: Honoraria, Research Funding; SkylineDx: Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; Amgen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding. Mateos:Janssen, Celgene, Takeda, Amgen, Adaptive: Honoraria; AbbVie Inc, Amgen Inc, Celgene Corporation, Genentech, GlaxoSmithKline, Janssen Biotech Inc, Mundipharma EDO, PharmaMar, Roche Laboratories Inc, Takeda Oncology: Other: Advisory Committee; Janssen, Celgene, Takeda, Amgen, GSK, Abbvie, EDO, Pharmar: Membership on an entity's Board of Directors or advisory committees; Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Takeda Oncology.: Speakers Bureau; Amgen Inc, Janssen Biotech Inc: Other: Data and Monitoring Committee. Alegre:Celgene, Amgen, Janssen, Takeda: Membership on an entity's Board of Directors or advisory committees. Facon:Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hulin:celgene: Consultancy, Honoraria; Janssen, AbbVie, Celgene, Amgen: Honoraria. Hashim:Ingress-Health: Employment. Vincken:Janssen: Employment, Equity Ownership. Kampfenkel:Janssen: Employment, Equity Ownership. Cote:Janssen: Employment, Equity Ownership. Moreau:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

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