Antitransgene rejection responses contribute to attenuated persistence of adoptively transferred CD20/CD19-specific chimeric antigen receptor redirected T cells in humans

Abstract

Immunotherapeutic ablation of lymphoma is a conceptually attractive treatment strategy that is the subject of intense translational research. Cytotoxic T lymphocytes (CTLs) that are genetically modified to express CD19- or CD20-specific, single-chain antibody-derived chimeric antigen receptors (CARs) display HLA-independent antigen-specific recognition/killing of lymphoma targets. Here, we describe our initial experience in applying CAR-redirected autologous CTL adoptive therapy to patients with recurrent lymphoma. Using plasmid vector electrotransfer/drug selection systems, cloned and polyclonal CAR(+) CTLs were generated from autologous peripheral blood mononuclear cells and expanded in vitro to cell numbers sufficient for clinical use. In 2 FDA-authorized trials, patients with recurrent diffuse large cell lymphoma were treated with cloned CD8(+) CTLs expressing a CD20-specific CAR (along with NeoR) after autologous hematopoietic stem cell transplantation, and patients with refractory follicular lymphoma were treated with polyclonal T cell preparations expressing a CD19-specific CAR (along with HyTK, a fusion of hygromycin resistance and HSV-1 thymidine kinase suicide genes) and low-dose s.c. recombinant human interleukin-2. A total of 15 infusions were administered (5 at 10(8)cells/m(2), 7 at 10(9)cells/m(2), and 3 at 2 x 10(9)cells/m(2)) to 4 patients. Overt toxicities attributable to CTL administration were not observed; however, detection of transferred CTLs in the circulation, as measured by quantitative polymerase chain reaction, was short (24 hours to 7 days), and cellular antitransgene immune rejection responses were noted in 2 patients. These studies reveal the primary barrier to therapeutic efficacy is limited persistence, and provide the rationale to prospectively define T cell populations intrinsically programmed for survival after adoptive transfer and to modulate the immune status of recipients to prevent/delay antitransgene rejection responses.

Copyright (c) 2010 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. T cell products meet release requirements

Depicted from left to right: Southern blots of T cell genomic DNA using an HyTK specific probe showing existence of single bands as indicated by arrows; Western blots revealing both the 16-kDA endogenous CD3ζ and the 66-kDA CE7R chimeric ζ bands detected with anti-human CD3ζ cytoplasmic tail specific antibody; flow cytometry analysis for surface expression of the chimeric receptor using anti-Fc antibody, or for the T cell markers TCR and, CD8, CD4 and/or CD3, where isotype control staining is indicated with the open histogram; ability of CTL clones to lyse CD19+ CD20+ Daudi targets was determined in a 4hr 51Cr release assay; ganciclovir (GCV) sensitivity using a flow cytometry based assay for viable cell numbers after 14 days of culture with either rhuIL-2 or rhuIL-2 + GCV; Assays for IL-2 dependence were performed using 3H-thymidine incorporation measurements (c.p.m) of Jurkat T cells vs. the indicated T cell clones after 11 days of culture in the absence of rhuIL-2 (UPN006, UPN009), or using a flow cytometry based assay for viable cell numbers after the T cell products were cultured in the presence vs. the absence of rhuIL-2 for 14 days (UPN035, UPN037).

Figure 2. Treatment regimens for each patient

First i.v. infusions of T cells were administered on day 0 for each patient. For UPN006 and UPN009, fractionated total body irradiation (TBI) and/or myeloablative chemotherapies administered to UPN006 and UPN009 are indicated just prior to administration of CD34+ autologous stem cells. BCNU, bis-chloronitrosourea; Cytoxan, cyclophosphamide; VP-16, etoposide. For UPN035 and UPN037, administration of fludarabine (i.v. at 25mg/m2) occurred between days 4 and 8 after the first T cell infusion, and rhuIL-2 administration (5×105 IU/m2 BID) was initiated after the third T cell infusion.

Figure 3. Transferred T cells do not persist long term in vivo

Using real time quantitative PCR, copy numbers of the CD20R (A) or CD19R (B) genes per μg of genomic DNA were determined as an indicator of the relative amount of chimeric receptor expressing T cells in the PBMC samples collected at the indicated days during the treatment schedule. Escalating infusion doses are indicated by arrows. *, cells not harvested.

Figure 4. Transgene rejection response detected when T cells administered following hematopoietic stem cell transfer

In the trial targeting CD20+ diffuse large cell lymphoma, PBMC collected prior to treatment and at day 75 (UPN006) or day 77 (UPN009) after initiation of treatment were stimulated in vitro with irradiated LCL as a control (xLCL) or the corresponding irradiated CTL clone that had been administered (i.e., x1A11 or x6D10). Effectors were then used in a 4-hour 51Cr-release assay using either LCL or the corresponding CTL as targets (A) or, in the case of UPN009, using LCL that had been transfected with the pcDNA3.1(−) vector lacking the CD20R transgene as targets (B). (C), Clones derived from UPN009 day 98 PBMC were also stimulated in vitro with irradiated 6D10 CTL and then analyzed for cytolytic activity against 51Cr-labeled LCL, 6D10 or pcDNA3.1(−) vector transfected LCL to determine specificity of transgene-specific response. Percent 51Cr-release at an E:T of 25:1 in each case is depicted for four representative clones.

Figure 5. Rejection response detected when T cells administered following fludarabine administration

(A), In the trial targeting CD19+ follicular lymphoma, serum collected at the time of patient enrollment (Pre-Trtmt) and at day 50 (UPN035) or day 42 (UPN037) after initiation of treatment was examined for immunoreactivity against Jurkat cells expressing the CD19R (red line) in a flow cytometry based assay. Parental Jurkat cells (grey histogram), and a known non- reactive serum (black line) were used as negative controls. (B), TCR Vβ profiles of the infused T cell product, day 0 PBMC (collected just prior to first T cell infusion; Pre-Trtmt), and day 14 PBMC (collected prior to the second T cell infusion, Post-Trtmt) were determined by spectratyping analysis. Alterations in Vβ usage that were observed pre- vs. post-treatment are highlighted with red boxes. (C), The TCR Vβ23+ population of pre-treatment and day 14 PBMC from UPN035 was further analyzed by flow cytometry for surface CD107 expression as a marker of degranulation upon co-culture with the infused T cell product (infused T), or non-modified autologous T cells (neg T). (D), Pre-treatment and day 50 PBMC from UPN035 were stimulated in vitro with irradiated LCL as a control (xLCL) or with the irradiated T cell product (xTcells). Effectors were then used in a 4-hour 51Cr-release assay using either LCL or T cells as targets.