Nano-motion Dynamics are Determined by Surface-Tethered Selectin Mechanokinetics and Bond Formation

The interaction of proteins at cellular interfaces is critical for many biological processes, from intercellular signaling to cell adhesion. For example, the selectin family of adhesion receptors plays a critical role in trafficking during inflammation and immunosurveillance. Quantitative measurements of binding rates between surface-constrained proteins elicit insight into how molecular structural details and post-translational modifications contribute to function. However, nano-scale transport effects can obfuscate measurements in experimental assays. We constructed a biophysical simulation of the motion of a rigid microsphere coated with biomolecular adhesion receptors in shearing flow undergoing thermal motion. The simulation enabled in silico investigation of the effects of kinetic force dependence, molecular deformation, grouping adhesion receptors into clusters, surface-constrained bond formation, and nano-scale vertical transport on outputs that directly map to observable motions. Simulations recreated the jerky, discrete stop-and-go motions observed in P-selectin/PSGL-1 microbead assays with physiologic ligand densities. Motion statistics tied detailed simulated motion data to experimentally reported quantities. New deductions about biomolecular function for P-selectin/PSGL-1 interactions were made. Distributing adhesive forces among P-selectin/PSGL-1 molecules closely grouped in clusters was necessary to achieve bond lifetimes observed in microbead assays. Initial, capturing bond formation effectively occurred across the entire molecular contour length. However, subsequent rebinding events were enhanced by the reduced separation distance following the initial capture. The result demonstrates that vertical transport can contribute to an enhancement in the apparent bond formation rate. A detailed analysis of in silico motions prompted the proposition of wobble autocorrelation as an indicator of two-dimensional function. Insight into two-dimensional bond formation gained from flow cell assays might therefore be important to understand processes involving extended cellular interactions, such as immunological synapse formation. A biologically informative in silico system was created with minimal, high-confidence inputs. Incorporating random effects in surface separation through thermal motion enabled new deductions of the effects of surface-constrained biomolecular function. Important molecular information is embedded in the patterns and statistics of motion

Clinical Activity of DNA Methyltransferase Inhibitors In Therapy-Related Myelodysplastic Syndromes: A Retrospective Study

Abstract Abstract 1891 Therapy-related myelodysplastic syndrome (tMDS) is a poor-risk subtype of MDS with no standard treatment options, and yet patients (pts) with tMDS are often excluded from trials where they would have the opportunity to benefit from novel treatment approaches. DNA methyltransferase inhibitors are a treatment option for tMDS, and are being evaluated as a bridge to stem cell transplant in these often heavily pre-treated patients to avoid the organ toxicity of intensive chemotherapy. However, the response rate of tMDS to DNA methyltransferase inhibitor (DNMTI) therapy is unknown. In this retrospective study, adult patients tMDS were culled from a fully annotated, IRB-approved database of all MDS patients who received either decitabine (DAC) on both the 3-day and 5-day schedules or 5-azacytidine (5-aza) at our institution from 4/8/2002 to 6/18/2010. Patients who received interrupted therapy were only analyzed for response to their initial course of therapy. Patients who received sequential DNMTI therapy (i.e., DAC followed by 5-aza or 5-aza followed by DAC) were included, but response to only their initial therapy was assessed. Responses were determined using the modified International Working Group criteria (Cheson BD, et al, 2006). Of the 35 patients initially identified with tMDS who received DNMTI therapy, 4 were deemed inevaluable for response due to marrow involvement with the primary malignancy (n= 1), missing records (n=2), or delivery of < 1 full cycle of therapy (n=1). The 31 evaluable pts included 14 males and 17 females with a median age of 65 years (range 25–85). Therapy for the primary malignancy included chemotherapy alone (n=13), chemotherapy plus radiotherapy (n=14), radioactive iodine and radiotherapy (n=2), radioactive iodine and chemotherapy (n=2), and autologous stem cell transplant (n=3). Prior to DNMTI therapy, the MDS FAB subtypes were as follows: RA, n= 6; RARS, n= 3; RAEB, n= 19; RAEBt, n=2; CMMoL, n=1. Pre-DNMTI therapy included lenalidomide (n=4) and alloSCT (n=1). Of the 31 evaluable patients, 20 received DAC, including 7 pts who received tretinoin with DAC in a clinical trial, and 11 received 5-aza. DAC recipients received a median of 2 cycles of therapy (range, 1–12) and 5-aza recipients received a median of 5 cycles (range, 1–9). Best responses were as follows: CR, n=1; Marrow CR plus HI, n=6 (3 trilineage HI, 1 HI-P+ HI-N, 2 HI-P); Stable Disease, n=6; Progressive Disease, n=6; Failures (death during 1st cycle or before response evaluation), n=3. Rate of CR + mCR was 22% (n=7). Additional patients had inevaluable (aparticulate) marrows, or refused follow-up marrow studies, but showed signs of stable (n= 3), improved, (n=2; HI-P, HI-P+HI-N), or progressive cytopenias (n= 3). Median time to best response was 1.5 cycles (range 1–6). Fifty-eight percent (n=18) of 31 pts achieved stable disease or better responses (including 4 pts with stable cytopenias or HI with inevaluable marrow response). Four patients proceeded directly to transplant after DNMTI therapy. Two subsequently died from relapsed disease after transplant, while 1 pt is lost to follow-up and 1 pt is without evidence of MDS 2.5 years after transplant. Nine pts had persistence of their primary malignancy during DNMTI therapy, and 5/9 pts required interruption or cessation of their DNMTI therapy because of progressive primary malignancy. 24/31 pts died from complications of MDS (n=5) or subsequent AML (11), complications of MDS/AML with likely contribution from their primary malignancy (n=4), infection during DNMTI nadir (n=2), GVHD post AlloSCT (n=1), or unknown reasons (n=1). Living pts (n=7; median follow-up from start of DNMTI therapy = 12.5 months, range 4.1 – 35.1 months) include 5 who are not transplant candidates. In conclusion, DNMTI therapy produced modest clinical benefit in our tMDS cohort. In some patients, persistence of the primary malignancy interfered with our ability to deliver optimal DNMTI therapy and to assess response. Although DNMTIs represent a potential therapeutic option for tMDS, treatment of a larger cohort is required to clarify the response rate of these agents in tMDS. Disclosures: Klimek: Celgene: Consultancy

Efficacy of hypomethylating agents in therapy-related myelodysplastic syndromes

We retrospectively assessed morphologic and cytogenetic responses to 5-azacytidine and decitabine in a cohort of 42 adult therapy-related myelodysplastic syndromes (tMDS) patients treated at Memorial Sloan-Kettering Cancer Center and in 2 industry-sponsored decitabine trials (D0007 and DACO-020). The overall response rate (complete remission+marrow CR+hematologic improvement) was 38%, including 6 patients with complete remission (14%), 6 with marrow CR with or without hematologic improvement (14%), and 4 with hematologic improvement alone (10%). We conclude that DNA methyltransferase inhibitors showed activity in tMDS that is roughly comparable to that seen in de novo MDS

First-in-human data of ALLO-501A, an allogeneic chimeric antigen receptor (CAR) T-cell therapy and ALLO-647 in relapsed/refractory large B-cell lymphoma (R/R LBCL): ALPHA2 study

2529 Background: Allogeneic CAR T cell therapy addresses logistical/manufacturing challenges inherent in autologous (auto) CAR T therapy. ALLO-501A, which uses Cellectis technologies, is an allogeneic anti-CD19 CAR T cell product whose a) disrupted TCRα gene may reduce GvHD risk, and b) edited CD52 gene may permit use of ALLO-647 (a humanized anti-CD52 mAb) to selectively deplete host T cells. Methods: The ongoing ALPHA2 study is a single-arm, open-label, 2 phase study of ALLO-501A in non-HLA matched patients (pts) with R/R LBCL and ≥2 prior lines of therapy. Prior auto CD19 CAR T therapy is allowed if tumors remain CD19 + . Following lymphodepletion (LD) with ALLO-647 (60 mg or 90 mg), fludarabine 30 mg/m 2 /d x 3d (Flu), and cyclophosphamide 300 mg/m 2 /d x 3d (Cy), escalating doses of ALLO-501A (40 [DL1] or 120 [DL2] x 10 6 viable CAR T cells) were administered. Retreatment was allowed for PD or SD with suboptimal CAR T expansion. Pts who had ≥SD at D28 could receive a second dose in a consolidation cohort. Phase 1 assessed safety/tolerability and cell kinetics of escalating doses of ALLO-501A following LD. Results: By 1/15/21, 11/11 enrolled pts received ALLO-647 (60 mg: n=6; 90 mg: n=5). Mean duration from enrollment to start of therapy was 6 days. After LD, 1 and 9 pt(s) were treated with ALLO-501A at DL1 and DL2, respectively; 1 pt developed CNS lymphoma and was not treated. Of 10 pts treated, 1 pt received retreatment and 4 pts were enrolled in the consolidation cohort. Pts had a median age of 60 years; 8 were ≥ stage III at diagnosis, 5 had IPI scores ≥3, and 3 had baseline LDH > 2x ULN. Median number of prior therapies was 3 (range 2 – 7); 3 pts had received auto CD19 CAR T cell therapy. 4/8 evaluable pts had rapidly PD at study entry. Median FU was 1.7 months. No dose modifications were required and no pt experienced DLTs. The most common AEs were anemia, leukopenia, neutropenia and thrombocytopenia (73%); and lymphopenia (64%). No GvHD or ICANS were reported. CRS was seen in 2 (18%) pts, both Grade < 3. Infusion-related reactions, all grade <3, were observed in 4 (36%) pts. D28 response data are available for 8 pts: 1 died of PD before D28; 4 additional pts had PD, including 2 who progressed 2 and 3 mos. after auto CAR T; 1 had SD; and 2 (both DL2) had CR. Of those in CR, 1 had peak ALLO-501A expansion at D14, persistence until D42, and ongoing CR at 4 mo; 1, with a 4-mo response to prior auto CAR T, had peak expansion at D28 and remains in ongoing CR at D56 after ALLO-501A with pending persistence. Conclusions: This dose escalation cohort contained heavily pretreated, actively progressing pts, some of whom had failed auto CAR T. Preliminary data suggest an acceptable safety profile following ALLO-501A and ALLO-647 and early signs of efficacy in LBCL. Enrollment into the consolidation cohort is ongoing; updated clinical/biomarker data of resistance and clinical activity will be presented. Clinical trial information: NCT04416984

Remission of severe myasthenia gravis after autologous stem cell transplantation

Abstract Objective Myasthenia gravis (MG) is an autoantibody‐mediated neuromuscular junction disorder involving the acetylcholine receptors on the motor endplate. The safety and response to high‐dose chemotherapy (HDIT) and autologous hematopoietic cell transplantation (HCT) were assessed in a patient with severe refractory MG. Methods As part of a pilot study of HDIT/HCT for patients with treatment‐resistant autoimmune neurological disorders, a patient with severe refractory MG underwent treatment. After mobilization of hematopoietic stem cells with rituximab, prednisone, and G‐CSF, the patient had HDIT consisting of carmustine, etoposide, cytarabine, melphalan, and rabbit antithymocyte globulin, followed by autologous HCT. The effect of treatment on the autoantibody to the acetylcholine receptor (AChR) was assessed. Results The patient had been diagnosed with AChR antibody‐positive MG 14 years before HDIT/HCT and had failed thymectomy, therapeutic plasma exchange, and multiple immunomodulatory agents. The Myasthenia Gravis Foundation of America (MGFA) clinical classification was IVb before HDIT/HCT. She tolerated HDIT/HCT well and started to improve clinically within days of treatment. At both 1 and 2 years after HDIT/HCT, patients remained symptom‐free. After HDIT/HCT, AChR‐binding autoantibodies persisted, and the relative frequency of immune cell subtypes shifted. Interpretation HDIT/HCT induced a complete response of disease activity in a patient with severe refractory MG. This response may suggest that a cell‐mediated etiology may be a significant contributing factor in refractory MG cases. A phase 2 clinical trial is warranted to establish if HDIT/HCT can be an effective therapy for severe refractory MG and to gain a further understanding of disease pathogenesis