Page 362 - Haematologica Vol. 109 - July 2024

CASE REPORT
Figure 2. Chimeric antigen receptor T-cell expansion and anti- SARS-CoV-2 T cells in two myeloma patients with COVID-19-re- lated acute respiratory distress syndrome. Panels (A-D) are the correlative studies of the first patient in the report. Panel (E) shows chimeric antigen receptor (CAR) T-cell expansion characteristics of the second patient. (A) Blood samples were collected under Institutional Review Board (IRB)-approved protocol 2043GCCC (IRB HP-00091736). Peripheral blood plasma was generated by centrifugation at 400 G and frozen immediately at -80°C. Peripheral blood mononuclear cells (PBMC) were isolated using den- sity gradient centrifugation and cryopreserved in liquid nitrogen. Proportions of CD4+ and CD8+ BCMA-targeted CAR T cells were determined in the peripheral blood of our patient at different time points using flow cytometry. Dot plots show percentage of CAR-expressing T cells versus all T cells. CAR T cells were identified by staining of the expression of the CAR on the cell surface using a recombinant BCMA protein as CAR detection reagent and co-staining with anti-CD3 and other T-cell markers (Online Supplementary Table S1). (B) CAR T-cell CD4+ and CD8+ subtypes were quantified at the different time points and CAR T-cell memory subtypes (naïve, central memory [CM], effector-memory [EM], effector-type [EFF]) were determined by co-staining for CD45RA and CD62L. T cells specific for the S (C) and the N (D) proteins of the SARS-CoV-2 virus were identified ex vivo after short-term stimulation of total PBMC using libraries of overlapping peptides covering the complete sequence of the respective proteins. Intracellular staining of cytokines followed by flow cytometry served as a read-out assay. SARS-CoV-2-specific CD4+ T cells (upper rows) were defined as TNFα/CD40L (CD154)-double positive CD3+CD4+ T cells and SARS-CoV-2-specific CD8+ T cells (lower rows) were defined as IFNγ/TNFα-double positive CD3+CD8+ T cells. (E) CAR T-cell expansion kinetics in the second patient. Dot plots show percentage of CAR-expressing T cells versus all T cells. CAR T cells were identified by staining for the expression of the CAR on the cell surface using a recombinant BCMA protein as CAR detection reagent and co-staining with anti-CD3.
(10 on mechanical ventilation), nine (75%) survived all of whom had a baseline pO2/FIO2 ratio (PFR) of >125 whereas three patients with baseline PFR <125 died.16 Although the primary mechanism of action in this setting appears to be endothelial stabilization, there is likely a multi-pronged suppression of non-specific inflammation and endothe- liitis, which is suggested by the inhibition of both donor neutrophil and T-cell trafficking resulting in reduced acute graft-versus-host disease severity in a murine mismatched allogeneic transplantation model, as well as other recently derived hypotheses for modulating activated endothelium and disrupting viral infection.17, 18 Our steroid- and remde- sivir-refractory patient showed rapid clinical improvement following initiation of defibrotide treatment (Figure 1B). Correlative studies demonstrated COVID-specific anti- body responses which were probably enhanced by IVIG administrations (Figure 1C-E). Importantly, we observed robust CAR T-cell expansion favoring a central memory phenotype at month 3 (Figure 2A, B). Similarly, despite the absence of full immune reconstitution at month 2 (CD4+ <200), there were clear CD4+ and CD8+ T-cell responses against the spike (S) and nucleocapsid (N) proteins at month 3 (Figure 2C, D).
Case 2
A 79-year-old man with an 8-year history of IgG-κ RRMM including upfront ASCT (Table 1) presented with cough and profound hypoxemia 45 days after ciltacel with SARS-CoV-2 infection. He was treated with paxlovid and was refractory to two separate inpatient courses of high-dose steroids and remdesivir administered 9 days apart. During 21-day ICU course, after persistent hypoxemia requiring 7-day high-flow oxygen support, he was started on defibrotide which led to a 30% drop in FIO2 immediately after the first dose. Unlike patient 1, he never received tocilizumab and IVIG was not given until 48 hours after administration of defibrotide, a time point when FIO2 was already reduced by 30%. Correlative studies also confirmed robust CAR T expansion (Figure 2E). He was
discharged on 2 lpm flow regulator. At 3 months, he did not require supplemental O2 and his myeloma was in remission with negative bone marrow biopsy and positron emission tomography (PET)/CT imaging.
Notably, both patients shared a historical triad of ASCT, CAR T treatment and SARS-CoV-2 infection all of which are well-es- tablished risk factors for endothelial activation and subsequent vascular damage. Although there are limited data on the im- pact of defibrotide on immune response in acute infectious inflammatory states, our clinical observations and correlative data suggest a prompt and clinically meaningful suppression of COVID-induced inflammatory responses with well-preserved SARS-CoV-2 specific antibody and T-cell immunity as well as sustained and effective anti-myeloma CAR T-cell kinetics. Notably, no hemorrhagic events were observed in either of these patients with defibrotide treatment. Patient 1 is 1 year out from CAR T-cell treatment, and remains with minimal exertional symptoms, without supplemental oxygen. He is PCR-negative for SARS-CoV-2 and his myeloma is in MRD-neg- ative (NGS) remission. Patient 2 is 5.5 months out from CAR T, his myeloma remains in remission and he does not require O2 support, as well as being PCR-negative for SARS-CoV-2. Given the complexity of CAR T-cell treatments, their unique adverse effect profiles such as CRS and potential infection profile including COVID-19, further investigation of defibrotide as a promising therapeutic option is clearly warranted.
Authors
Mehmet H. Kocoglu,1,2,3 Paul G. Richardson,4 Clifton C. Mo,4 Aaron P. Rapoport1,2,3 and Djordje Atanackovic1,2,3,5
1University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD; 2Department of Medicine, University of Maryland School of Medicine, Baltimore, MD; 3Transplant and Cellular Therapy Program, University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, MD; 4Dana Farber Cancer Institute, Boston, MA
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