Sequential CD19- and CD22-CART Cell Therapies for Relapsed B-Cell Acute Lymphoblastic Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Liu Shuangyou , Deng Biping , Lin Yuehui , Yin Zhichao , Pan Jing , Wu Tong , Gao Zhiyong , Song Yanzhi , Zhao Yongqiang , Tong Chunrong 
Published: October 2019

Abstract
With traditional therapies, the prognosis of relapsed acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is extremely poor. Chimeric antigen receptor (CAR) T cell therapy targeting at CD19 has demonstrated a significant efficacy on refractory/relapsed (r/r) B-ALL, but single-target CART could not maintain a long-term remission. Recently, CD22-CART has also shown an exciting result in r/r B-ALL. Here we sequentially applied CD19- and CD22-specific CART cells to treat relapsed B-ALL post-HSCT and observed the therapeutic effect.
From June 30,2017 through May 31,2018, twenty-four B-ALL patients (pts) relapsing after allo-HSCT with both antigens CD19 and CD22 expression on blasts were enrolled, the median age was 24 (2.3-55) years. Seventeen pts had hematologic relapse, 6 with both bone marrow and extramedullary (EM) involvements and 1 with EM disease (EMD) only. Fourteen pts had failed to previous therapies including chemotherapy, donor lymphocyte infusion, interferon and even murinized CD19-CART in other hospitals.
Recipient-derived donor T cells were collected for producing CAR-T cells, which were transfected by a lentiviral vector encoding the CAR composed of CD3ζ and 4-1BB. Eighteen pts were initially infused with murinized CD19-CART, then humanized CD22-CART; while 6 pts (5 failed to prior murinized CD19-CART and 1 had bright CD22-expression) were initially infused with humanized CD22-CART, then humanized CD19-CART. The time interval between two infusions was 1.5-6 months based on patients' clinical conditions. The average dose of infused CAR T cells was 1.4×105/kg (0.4-9.2×105/kg) for CD19 and 1.9×105/kg (0.55-6.6×105/kg) for CD22. All patients received fludarabine with or without cyclophosphamide prior to each infusion, some pts accepted additional chemo drugs to reduce the disease burden. Treatment effects were evaluated on day 30 and then monthly after each CART, minimal residual disease (MRD) was detected by flow cytometry (FCM) and quantitative PCR for fusion genes, EMD was examined by PET-CT, CT or MRI.
Sixteen patients finished sequential CD19- and CD22-CART therapies. Three cases could not undergo the second round of CART infusion (1 died, 1 gave up and 1 developed extensive chronic graft-versus-host disease (GVHD)). The rest of 5 pts are waiting for the second CART. After first T-cell infusion, 20/24 (83.3%) pts achieved complete remission (CR) or CR with incomplete count recovery (CRi), MRD-negative was 100% in CR or CRi pts, 3 (12.5%) cases with multiple EMD obtained partial remission (PR), and 1 (4.2%) died of severe cytokine release syndrome (CRS) and severe acute hepatic GVHD.
Sixteen patients (15 CR and 1 PR) underwent the second CART therapy. Before second infusion, 3/15 pts in CR became MRD+ and others remained MRD-. On day 30 post-infusion, 1 of 3 MRD+ pts turned to MRD-, 1 maintained MRD+ ( BCR/ABL+) and 1 had no response then hematologic relapse later. The PR patient still had not obtained CR and then disease progressed. As of 31 May 2018, at a median follow-up of 6.5 (4-10) months, among 16 pts who received sequential CD-19 and CD-22 CART therapies, 1 had disease progression, 2 presented with hematological relapse and 2 with BCR/ABL+ only, the overall survival (OS) rate was 100% (16/16), disease-free survival (DFS) was 81.3% (13/16) and MRD-free survival was 68.8% (11/16).
CRS occurred in 91.7% (22/24) pts in the first round of T-cell infusion, most of them were mild-moderate (grade I-II), merely 2 pts experienced severe CRS (grade III-IV). The second CART only caused grade I or no CRS since the leukemia burden was very low. GVHD induced by CART therapy was a major adverse event in these post-HSCT patients. After the first CART, 7/24 (29.2%) pts experienced GVHD, of them, 4 presented with mild skin GVHD, 2 with severe hepatic GVHD (1 recovered and 1 died), and 1 developed extensive chronic GVHD. No severe GVHD occurred in the second infusion.
Our preliminary clinical study showed that for B-ALL patients who relapsed after allo-HSCT, single CD19- or CD22- CART infusion resulted in a high CR rate of 83.3%, sequentially combined CD19- and CD22-CART therapies significantly improved treatment outcome with the rate of OS, DFS and MRD-free survival being 100%, 81.3% and 68.8%, respectively, at a median follow-up of 6.5 months. The effect of CART on multiple EMD was not good and CART induced GVHD needs to be cautious.

Disclosures
No relevant conflicts of interest to declare.