Abstract: The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants severely limits available effective monoclonal antibody therapies. Effective drugs are also supply limited. COVID-19 convalescent plasma (CCP) qualified for high antibody levels effectively reduces immunocompetent outpatient hospitalization. The Food and Drug Administration currently allows outpatient CCP for the immunosuppressed. Viral-specific antibody levels in CCP can range 10- to 100-fold between donors, unlike the uniform viral-specific monoclonal antibody dosing. Limited data are available on the efficacy of polyclonal CCP to neutralize variants. We examined 108 pre-δ/pre-ο donor units obtained before March 2021, 20 post-δ COVID-19/postvaccination units, and 1 pre-δ/pre-ο hyperimmunoglobulin preparation for variant-specific virus (vaccine-related isolate [WA-1], δ, and ο) neutralization correlated to Euroimmun S1 immunoglobulin G antibody levels. We observed a two- to fourfold and 20- to 40-fold drop in virus neutralization from SARS-CoV-2 WA-1 to δ or ο, respectively. CCP antibody levels in the upper 10% of the 108 donations as well as 100% of the post-δ COVID-19/postvaccination units and the hyperimmunoglobulin effectively neutralized all 3 variants. High-titer CCP neutralizes SARS-CoV-2 variants despite no previous donor exposure to the variants.

Abstract: Progressive malignancy is a leading cause of death in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). To improve treatment of B-cell malignancies that persist despite alloHSCT, we conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Ten patients were treated on this trial. Four patients were recipients of human-leukocyte-antigen (HLA)-matched unrelated donor (URD) transplants and 6 patients were recipients of HLA-matched sibling transplants. T cells for genetic modification were obtained from each patient’s healthy alloHSCT donor. Patients received a single infusion of anti-CD19-CAR T cells. Cell doses ranged from 1x106 to 10x106 T cells/kg. A mean of 58% of the infused cells expressed the CAR. Patients did not receive chemotherapy or other anti-malignancy therapy with the CAR-T-cell infusions, so the responses observed in these patients are not confounded by the effects of chemotherapy. In contrast to other reports of successful treatment of B-cell malignancies with anti-CD19-CAR T cells, the patients on this study were not lymphocyte-depleted at the time of the CAR-T-cell infusions. Two patients with chronic lymphocytic leukemia (CLL) refractory to standard unmanipulated allogeneic donor lymphocyte infusions (DLIs) had regressions of large malignant lymph node masses after infusion of allogeneic anti-CD19-CAR T cells. One of these CLL patients obtained a complete remission that is ongoing 9 months after treatment with allogeneic anti-CD19-CAR T cells. This patient also had complete eradication of blood B cells within 9 days after her CAR-T-cell infusion. Another patient had tumor lysis syndrome requiring rasburicase treatment as his CLL dramatically regressed in lymph nodes, bone marrow, and blood within 2 weeks of his anti-CD19-CAR-T-cell infusion. A patient with mantle cell lymphoma obtained a partial remission that is ongoing 3 months after infusion of anti-CD19-CAR T cells. A fourth patient with diffuse large B-cell lymphoma has ongoing stable disease 11 months after infusion of anti-CD19-CAR T cells. The other 6 treated patients all had short periods of stable malignancy or progressive disease after their CAR-T-cell infusions. Specific eradication of blood B cells occurred after infusion of CAR T cells in 3 of 4 patients with measurable blood B cells pretreatment. None of the patients treated on this study developed GVHD after their anti-CD19-CAR-T-cell infusions, despite the fact that 6 of 10 treated patients had experienced GVHD at earlier time-points after their most recent alloHSCT. One patient, who had a history of cardiac dysfunction with prior acute illnesses, had temporary cardiac dysfunction after infusion of anti-CD19-CAR T cells. The most prominent toxicities experienced by patients were fever and hypotension; these peaked 5 to 12 days after CAR-T-cell infusions and resolved within 14 days after the T-cell infusions. Two patients had Grade 3 fever, and 2 patients had Grade 3 hypotension. No patients experienced Grade 4 toxicities that were attributable to the CAR-T-cell infusions. Elevated levels of serum interferon gamma were detected in 3 patients at the time that they were experiencing toxicities. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. The peak blood levels of CAR T cells varied from undetec to 2.8% of peripheral blood mononuclear cells. The persistence of the CAR T cells in the blood of patients was limited to one month or less. When we assessed T cells from the blood of patients ex vivo, we found elevated levels of the T-cell inhibitory molecule programmed cell death protein-1 (PD-1) on CAR+ T cells compared to CAR-negative T cells. These results show for the first time that small numbers of donor-derived allogeneic anti-CD19-CAR T cells can cause regression of highly treatment-resistant B-cell malignancies after alloHSCT without causing GVHD. Malignancies that were resistant to standard DLIs regressed after anti-CD19-CAR-T-cell infusions. Future goals for improving this approach include enhancing the persistence of anti-CD19-CAR T cells and reducing toxicities. Infusion of allogeneic T cells genetically modified to recognize malignancy-associated antigens is a promising approach for treating residual malignancy after alloHSCT. Disclosures: No relevant conflicts of interest to declare.

Abstract: We have treated 20 patients and administered 23 total T-cell infusions on a clinical trial of autologous T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. This is the largest reported clinical trial of anti-CD19-CAR T cells. The first 9 CAR-T-cell treatments have been reported (Kochenderfer et al. Blood 2010 and Blood 2012). This abstract communicates unreported results from 14 patients who received anti-CD19-CAR T cells produced with a new 10-day culture process. These patients did not receive exogenous interleukin-2. Of these 14 patients, 5 obtained complete remissions (CR), and 6 obtained partial remissions (PR), (see table).TablePatientAge/GenderMalignancyNumberof priortherapiesTotal cyclo-phosphamidedose(mg/kg)Number ofCAR+ T cellsinfused(X106/kg)Response(timeafter cellinfusion inmonths)156/MSMZL41205PR (20+)243/FPMBCL4605CR (19+)361/MCLL2604CR (16+)430/FPMBCL31202.5NE563/MCLL41202.5CR (10+)648/MCLL1602.5CR (7+)742/MDLBCL5602.5CR (4+)844/FPMBCL10602.5PR (6+)938/MPMBCL31202.5SD (1)1057/FLow-grade NHL4601PR (4+)1158/FDLBCL from CLL13601PR (2)1260/FDLBCL3601SD (1+)1368/MCLL4601PR (2+)1443/MDLBCL2601PR (1+) The CAR used in this work is encoded by a gammaretrovirus and incorporates the variable regions of an anti-CD19 antibody, part of CD28, and part of CD3-zeta. A mean of 70.5% of the infused T cells expressed the CAR, and the infused cells produced cytokines and degranulated in a CD19-specific manner. Because prior chemotherapy has been shown to enhance the activity of adoptively-transferred T cells, patients received cyclophosphamide (total doses shown in table) plus fludarabine (25 mg/m2 daily for 5 days) before a single infusion of anti-CD19-CAR-transduced T cells. This is the first report of successful treatment of chemotherapy-refractory primary mediastinal B-cell lymphoma (PMBCL) and diffuse large B-cell lymphoma not otherwise specified (DLBCL) with anti-CD19-CAR T cells. All of the 8 treated patients with either PMBCL or DLBCL were chemotherapy-refractory, and 5 of these 8 patients obtained either a CR or PR on this trial. We defined chemotherapy-refractory as progression or no response 1 month after the end of the most recent chemotherapy. For example, Patient 2 had PMBCL that was refractory to 3 different chemotherapy regimens and that relapsed after radiation therapy. Patient 2 obtained a CR after infusion of anti-CD19 CAR T cells and remains in CR 19 months post-infusion. Blood B-cell depletion lasting more than 3 months occurred in 3 of 3 evaluable patients. Most patients were not evaluable for B-cell depletion due to B-cell depletion by prior treatments. One patient died suddenly of unknown etiology 16 days after infusion of CAR T cells. Acute toxicities including fever, hypotension, and delirium occurred after infusion of anti-CD19-CAR T cells. The toxicities resolved in less than 3 weeks after the cell infusion and were temporally associated with elevated serum interleukin-6 and interferon gamma levels in most patients. Peak blood levels of cells containing the CAR gene ranged from 2.3% to 66.5% of blood mononuclear cells. These results demonstrate the feasibility of treating patients with chemotherapy-refractory B-cell malignancies by using autologous anti-CD19 CAR T cells. The numerous remissions obtained should encourage further development of this approach. SMZL, splenic marginal zone lymphoma; PMBCL, primary mediastinal B-cell lymphoma; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma not otherwise specified. CR, complete remission; NE, not evaluable; PR, partial remission; SD, stable disease. (+) indicates an ongoing response. Disclosures: Rosenberg: Kite Pharma: Research Funding.

Abstract: Donor-derived anti-CD19-CAR T cells cause regressions of refractory malignancies after allogeneic transplantation.

Abstract: Neo-vascularization has been implicated in a number of inflammatory diseases as well as tumor growth. Both angiogenesis, the sprouting of resident tissue endothelial cells (ECs), and vasculogenesis, the recruitment of bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs), are thought to participate in neo-vascularization. EPCs have been implicated in tumor growth, however, the biologic significance of EPCs during inflammation is unclear. We studied neo-vascularization and the role of EPCs during inflammation in well-characterized murine models of graft-versus-host disease (GVHD). We found a significantly increased number of donor BM-derived EPCs in peripheral blood and BM in allogeneic bone marrow transplantation (allo-BMT) recipients with GVHD at different time points after BMT. We next quantified neo-vascularization during inflammation in GVHD target organs by immunofluorescence microscopy and by flow cytometry. We found significantly increased numbers of donor-derived ECs in the liver as well as a significantly higher vessel density in the liver, illeum and colon. We adoptively transferred selected GFP+ EPCs and observed incorporation into the neo-vasculature of the inflamed intestines (Fig. 1A) and liver during GVHD. Taken together, these data suggest that neovascularization during GVHD is due to vasculogenesis from donor EPCs. Next we used an antibody (E4G10) against the vascular endothelial adhesion molecule VE-cadherin, which recognizes a terminal epitope that is exposed on circulating EPCs, but is masked in the established vasculature, and found a significant reduction of EPCs in the peripheral blood and BM. We observed that depletion of EPCs was associated with a significant inhibition of donor BM-derived neo-vascularization in the liver, illeum and colon during GVHD. E4G10 treated recipients had significantly better survival and lower clinical GVHD scores in all tested models (B6BALB/c [1×106 T], B6B6D2F1 [1×106 T] , B6B6D2F1 [2×106 T], B6B6D2F1 [3×106 T] ). We found significantly reduced numbers of allo-reactive donor T cells in secondary lymphoid organs during GVHD, but no changes in the expression of activation markers and homing molecules, as a consequence of E4G10 administration. In blinded histopathological analyses we found significantly less GVHD and reduced numbers of tissue-infiltrating CD3+ T cells in the liver, illeum and colon in E4G10-treated allo-BMT recipients. To better emulate the clinical setting, we first assessed the role of EPCs in tumor growth in allo-BMT recipients. We transferred sorted GFP+ EPCs as well as renal carcinoma (RENCA) cells to BALB/c recipients and found that GFP+ EPCs were recruited to the neo-vasculature of lung metastases. We detected a significant inhibitory effect of E4G10 administration on tumor growth, as determined by in vivo bioluminescence imaging, in both tumors tested (RENCA, A20 lymphoma) as well as a significant survival prolongation in tumor-bearing mice that were treated with E4G10 in the RENCA and C1498 (AML) model. Finally, we performed experiments in which tumor-bearing allo-BMT recipients received allogeneic T cells, which mediate the favorable graft-versus-tumor (GVT) effect but also cause inflammation in GVHD target organs. We found that administration of E4G10 led to a significantly higher rate of tumor-free survival in all models (B6'BALB/c [1×105 B6 T and 2×105 RENCA], B10BR'B6 [1×105 B10BR T and 2×105 C1498] , B6'BALB/c [2×105 B6 T and 5×105 A20]), which was due to both attenuation of GVHD as well as inhibition of tumor growth (Fig. 1B). We conclude that depletion of EPCs is a strategy to simultaneously ameliorate inflammatory disease and tumors, providing a new approach to improve therapeutic outcome of allogeneic hematopoietic stem cell transplantation. This study demonstrates the biological significance of EPCs for neo-vascularization during inflammation and identifies the specific targeting of EPCs to disrupt neo-vascularization as a novel therapeutic concept to decrease inflammation. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group.

Abstract: BACKGROUND: Mantle cell lymphoma (MCL) typically has a poor outcome with overall survival of only 3–4 years. Higher treatment response and event-free survival has been demonstrated with aggressive high dose chemotherapy followed by autologous hematopoietic stem cell support, though long term cure rates remain unclear(Dreger P. Hematol J. 2000;vol.2). Modest response rates have also been reported with the monoclonal antibody (MoAb) rituximab and ALEMTUZUMAB (Foran, JM. JCO 2000; vol. 2. Faderl S. Blood 2003; vol. 9). We therefore combined dose-dense therapy with MoAbs to explore response rate and event free survival (EFS) in mantle cell lymphoma. The strength of this trial design is ability to follow all patients from induction chemotherapy through high dose therapy and transplant in order to gauge clinical outcome on all enrolled patients, not just the subpopulation who is able to proceed to high dose therapy. PATIENTS AND METHODS: Induction therapy consisted of 1 cycle of high dose cytarabine (3gm/m2 IV over 1 hour Q12H for 8 doses), mitoxantrone (10mg/m2 daily for 3 days), and ALEMTUZUMAB 30mg IV 3 times a week for 6 weeks with growth factor support. All responding patients were mobilized with cyclophosphamide 4gm/m2 and G-CSF 10 mcg/kg/day and/or bone marrow harvest. The transplant preparative regimen was carmustine 15mg/kg over 2 hours day -6, etoposide 60mg/kg over 4 hours day -4, and cyclophosphamide 100mg/kg over 2 hours day -2 followed by autologous reinfusion on day zero. Consolidation was given with rituximab 375mg/m2 weekly for 4 doses at 6 weeks and 6 months post transplant. RESULT: 9 patients with advanced disease (7 stage IV, 1 stage III, 1 stage IIA) and median age of 60 (48 – 65 years) have been accrued and treated since February 2003. Four were newly diagnosed and 5 had relapsed/refractory disease. Seventy eight percent (7/9) had complete response and 22% (2/9) had partial response (PR) following induction therapy. One patient had severe infection after induction and was unable to proceed to transplant. Another had constitutional decline preventing further therapy and each died within 4 months of withdrawal from the protocol. Both had relapse/refractory disease at accrual. The remaining 7 patients proceeded to the transplant phase. With a median follow-up of 7 months (range 3–16 months), all 7 patients remain in CR for 1 –16 months. Significant induction therapy toxicity included neutropenia in all 9 patients with average duration of 10.7 days, non-disseminated CMV reactivation in 44% of patients, one overwhelming fungal infection, and one patient with delay in engraftment. Figure Figure CONCLUSION: Our preliminary data show a high induction and transplant phase completion rate, manageable toxicity, and excellent overall response rate in this group of elderly patients with advanced disease. Larger numbers of patients and longer follow-up is needed to confirm these promising results.

Abstract: BACKGROUND: With the poor prognosis and outcome described in mantle cell lymphoma (MCL), there is a continuing need to explore treatment options that might overcome suspected underlying drug resistance and improve the current median survival of about 24 months. One strategy to overcome drug resistance has been the use of high dose therapy followed by autologous transplant. Newer agents such as the monoclonal antibodies (MoAb) rituximab and alemtuzumab which target CD20 and CD52 respectively have recently become available. We have therefore incorporated both a dose dense approach in combination with these antibodies to treat newly diagnosed and relapsed MCL patients. PATIENTS AND METHODS: A total of 16 patients have been enrolled since February 2003. Induction therapy consisted of 1 cycle of cytarabine 3gm/m2 IV Q12H for 8 doses, mitoxantrone 10mg/m2 daily for 3 days, and Alemtuzumab 30mg IV 3 times a week for 6 weeks with growth factor support. All responding patients were mobilized with cyclophosphamide 4gm/m2 and G-CSF 10 mcg/kg/day and/or bone marrow harvest. The transplant preparative regimen was carmustine 15mg/kg on day -6, etoposide 60mg/kg on day -4, and cyclophosphamide 100mg/kg on day -2 followed by autologous re-infusion. Consolidation was given with rituximab 375mg/m2 weekly for 4 doses at 6 weeks and 6 months post transplant. RESULT: Of the 16 patients, 12 had stage IV, 1 stage III, 3 stage IIA, and 1 stage I disease. The median age was 60 (48 66 years). Eight were newly diagnosed and 8 had relapsed disease with at least 2 prior chemotherapy treatments. In the induction phase, overall response rate was 94% (15/16 patients) with 73% complete response (CR) and 27% partial response (PR). Response rate were 100% and 88% in the newly diagnosed and relapsed patients respectively but CR was similar (75%) in both groups. Nine patients have been transplanted and one patient is awaiting transplant. Six patients were not transplanted due to death in 2 patients with relapsed disease at study entry, and one each due to progression of disease, prolonged cytopenias of & gt; 60days, mental status changes and inability to collect peripheral or bone marrow stem cell respectively. Among the transplanted patient, 78% (7/9) remain in CR with 2-year lymphoma progression free survival of 67% after a median follow-up of 487 days (range 175–787 days). Induction therapy toxicities included average neutropenia duration of 11.6 days and CMV reactivation of 50% that was equally distributed in the transplanted and non-transplanted patients. Peripheral stem cell collection was inadequate in 5 of the transplanted patients requiring bone marrow harvest. CONCLUSION: Our preliminary data continue to show a high induction response rate with majority of patients who are able to proceed to the transplant phase remaining free of lymphoma at 24 months. Toxicity was manageable although bone marrow harvest was needed to obtain adequate stem cells in majority of transplanted patients. While CMV reactivation was observed in half of the patients, its effect on the ability of subjects to complete the study and on survival appear to be minimal. Although a small study, multimodal dose dense strategy with maintenance MoAb for patients with mantle cell lymphoma is a promising strategy that needs to be confirmed in larger number of patients with prolonged follow-up.

Abstract: Deletion of both Rap1a and Rap1b impairs platelet production and abolishes platelet adhesion at sites of mechanical trauma. Platelet RAP1 signaling is dispensable for vascular integrity during development and at sites of inflammation.

Abstract: Following reports of childhood acute myeloid leukemia (AML) showing that patients with t(9; 11)(p22; q23) have a better prognosis than those with translocations between 11q23 and other chromosomes, we compared response to therapy and survival of 24 adult de novo AML patients with t(9; 11) with those of 23 patients with other 11q23 translocations [t(11q23)]. Apart from a higher proportion of French-American-British (FAB) M5 subtype in the t(9; 11) group (83% v 43%, P = .006), the patients with t(9; 11) did not differ significantly from patients with t(11q23) in terms of their presenting clinical or hematologic features. Patients with t(9; 11) more frequently had an extra chromosome(s) 8 or 8q as secondary abnormalities (46% v 9%, P = .008). All patients received standard cytarabine and daunorubicin induction therapy, and most of them also received cytarabine-based intensification treatment. Two patients, both with t(9; 11), underwent bone marrow transplantation (BMT) in first complete remission (CR). Nineteen patients (79%) with t(9; 11) and 13 (57%) with t(11q23) achieved a CR (P = .13). The clinical outcome of patients with t(9; 11) was significantly better: the median CR duration was 10.7 versus 8.9 months (P = .02), median event-free survival was 6.2 versus 2.2 months (P = .009), and median survival was 13.2 versus 7.7 months (P = .009). All patients with t(11q23) have died, whereas seven (29%) patients with t(9; 11) remain alive in first CR. Seven of eight patients with t(9; 11) who received postremission regimens with cytarabine at a dose of 100 (four patients) or 400 mg/m2 (2 patients) or who did not receive postremission therapy (2 patients) have relapsed. In contrast, 7 (64%) of 11 patients who received intensive postremission chemotherapy with high-dose cytarabine (at a dose 3 g/m2) (5 patients), or underwent BMT (2 patients) remain in continuous CR. We conclude that the outcome of adults with de novo AML and t(9; 11) is more favorable than that of adults with other 11q23 translocations; this is especially true for t(9; 11) patients who receive intensive postremission therapy.

Abstract: Deletion of Arp2/3 leads to marked microthrombocytopenia due to abnormal platelet release and increased platelet clearance. Arp2/3 is critical for platelet lamellipodia formation and spreading, but plays a minor role for platelet adhesion and hemostasis.