Antitransgene Rejection Responses Contribute to Attenuated Persistence of Adoptively Transferred CD20/CD19-Specific Chimeric Antigen Receptor Redirected T Cells in Humans

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 CD8, CD4, and TCR 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 4-hour 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 (cpm) of Jurkat T cells versus 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 versus the absence of rHuIL-2 for 14 days (UPN035 and UPN037). N.D., not done.

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 before administration of CD34⁺ autologous stem cells. BCNU, bis-chloronitrosourea; cytoxan, cyclophosphamide; VP-16, etoposide. For UPN035 and UPN037, administration of fludarabine (i.v. at 25 mg/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.

Transferred T cells do not persist long term in vivo. Using real time quantitative PCR, the percent of cells in the PBMC that were positive for the CD20R (A) or CD19R (B) genes 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.

Transgene rejection response detected when T cells administered after HSCT. In the trial targeting CD20⁺ diffuse large-cell lymphoma, PBMCs collected before 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.

Rejection response detected when T cells were administered following fludarabine administration. (A) In the trial targeting CD19⁺ FL, 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 nonreactive serum (black line) were used as negative controls. (B) TCR Vβ profiles of the infused T cell product, day 0 PBMCs (collected just before first T cell infusion; Pre-Trtmt), and day 14 PBMC (collected before the second T cell infusion, Post-Trtmt) were determined by spectratyping analysis. Alterations in Vβ usage that were observed pretreatment versus posttreatment are highlighted by red boxes. (C) The TCR Vβ23⁺ population of pretreatment and day 14 PBMC from UPN035 was further analyzed by flow cytometry for surface CD107 expression as a marker of degranulation on coculture with the infused T cell product (infused T), or nonmodified autologous T cells (neg T). (D) Pretreatment 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.

Product manufacturing strategy. (A) Plasmid vectors used to genetically redirect the patient's T cells to recognize CD20 (left) and CD19 (right). CD20- and CD19-specific CAR sequences (CD20R and CD19R) are indicated, as well as promoter (CMVp, EF-1p, SV40p), poly adenylation (BGHpolyA, SV40polyA), drug resistance (AmpR, NeoR, Hy), and origin of replication (ColE1ori, F1 ori) sequences. Note that the HyTK sequence is a fusion of the hygromycin resistance gene, and the HSV-1 thymidine kinase suicide gene. (B) Schematic of the CD20- and CD19-specific CAR molecules. Each has a murine single-chain variable fragment (scFv) which makes it specific for either CD20 or CD19, a human IgG hinge-Fc (hugFc) domain, a human CD4 transmembrane (huCD4tm) domain, and a human CD3z cytoplasmic (huCD3zcyt) domain. (C) Product manufacturing schema of genetically modified T cells that target CD20 (left) and CD19 (right). Patient PSMCs were activated with the CD3 agonist OKT3 and rHuIL-2 for 3 days and then electroporated with linearized CD20R_pcDNA3.1(-) (left) or CD19R_HyTK-pMG (right). Cells were placed in drug selection conditions 2-3 days later. Cloning (for CD20R expressing products only) and expansion were carried out in the presence of OKT3, rHuIL-2, irradiated LCL, and irradiated PBMC feeders. T-25, T-75, and T-150 refer to flask sizes that permit 25, 50, and 100 mL of culture media respectively. At certain steps, the target number of selected CD20-specific T cell clones that were to be generated are indicated in the schema on the left. Performance of tests for viability, sterility, the presence of mycoplasma or endotoxin, transgene copy number (via Southern blot and PCR), total and surface CAR expression (via Western blot and flow cytometry), IL-2 dependence, and cytolytic activity against CD20- (left) or CD19- (right) expressing targets (micro-CRA, CRA) are indicated.

Protocol treatment schema. (A) Treatment schema for recurrent/refractory CD20⁺ lymphoma using CD20R⁺ autologous T cells. Autologous HSCT was to be performed after a myeloablative preparative regimen of high dose chemotherapy with or without fractionated TBI; administration of escalating T cell dose infusions at 2-week intervals would then begin as early as 28 days later. (B) Treatment schema for CD19⁺ FL using CD19R⁺ HyTK⁺ autologous T cells. Lymphodepleting fludarabine was to be administered i.v. at 25 mg/m² for 5 days; T cell supportive rHuIL-2 was to be administered s.c. at 5 × 10⁵ IU/ m² twice daily for up to 5 days after infusions 3 and 4.