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. 2016 Jun 3:6:27130.
doi: 10.1038/srep27130.

T cells expressing CD19-specific Engager Molecules for the Immunotherapy of CD19-positive Malignancies

Mireya Paulina Velasquez  1   2   3 , David Torres  1   2 , Kota Iwahori  1   2 , Sunitha Kakarla  1   2 , Caroline Arber  1   2 , Tania Rodriguez-Cruz  1   2 , Arpad Szoor  1   2 , Challice L Bonifant  1   2   3 , Claudia Gerken  1   2 , Laurence J N Cooper  4 , Xiao-Tong Song  1   2   5 , Stephen Gottschalk  1   2   3   5
Affiliations

T cells expressing CD19-specific Engager Molecules for the Immunotherapy of CD19-positive Malignancies

Mireya Paulina Velasquez et al. Sci Rep. .

Abstract

T cells expressing chimeric antigen receptors (CARs) or the infusion of bispecific T-cell engagers (BITEs) have shown antitumor activity in humans for CD19-positive malignancies. While BITEs redirect the large reservoir of resident T cells to tumors, CAR T cells rely on significant in vivo expansion to exert antitumor activity. We have shown that it is feasible to modify T cells to secrete solid tumor antigen-specific BITEs, enabling T cells to redirect resident T cells to tumor cells. To adapt this approach to CD19-positive malignancies we now generated T cells expressing secretable, CD19-specific BITEs (CD19-ENG T cells). CD19-ENG T cells recognized tumor cells in an antigen-dependent manner as judged by cytokine production and tumor killing, and redirected bystander T cells to tumor cells. Infusion of CD19-ENG T cells resulted in regression of leukemia or lymphoma in xenograft models and a survival advantage in comparison to control mice. Genetically modified T cells expressing engager molecules may present a promising addition to current CD19-targeted immunotherapies.

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Conflict of interest statement

Baylor College of Medicine has a research collaboration with Celgene and Bluebird Bio and MD Anderson Cancer Center has a research collaboration with Intrexon and ZIOPHARM Oncology to develop cell therapies for cancer. MPV, KI, SK, CA, LJNC, XTS, SG have patent applications in the field of cancer immunotherapy.

Figures

Figure 1
Figure 1. Generation of CD19-ENG T-cells.
(a) Retroviral construct used to generate CD19 ENG T cells. (b,c) Five to 7 days after transduction, mOrange expression was measured by flow cytometry, and 60.4 ± 16% of the T-cells were mOrange positive (n = 24; range 35–87%; 95% CI 53.7–67.2). (D) A CD19-specific scFv Id antibody was used to detect CD19-ENG molecules. mOrange-positive and negative T cells stained positive (filled curve) for CD19-ENG in contrast to samples that were stained with secondary antibody alone (open curve). EphA2-ENG T cells were not stained by the CD19-specific scFv Id antibody, confirming specificity. (e,f) The concentration of CD19-ENG in media from CD19-ENG or EphA2-ENG T cells was determined using a coculture bioassay. (e) A standard curve was generated using recombinant CD19xCD3 protein. (f) CD19-ENG T-cell media had CD19-ENG protein concentrations ranging 39.7–223 ng/mL; (95%CI 46.6–212.3). No CD19-ENG molecules were detected in media of EphA2-ENG T cells.
Figure 2
Figure 2. In vitro characterization of CD19-ENG T cells.
(a,b) CD19-ENG or EphA2-ENG T cells were cocultured with CD19-positive (BV173, Daudi, Raji) or -negative (K562) tumor cells at a ratio of 2:1. After 48 hours, IFNγ and IL2 production was determined by ELISA (n = 3; assay performed in duplicates; *p < 0.05; **p < 0.01; ***p < 0.001; NS: not significant). (c) Cytotoxicity assays were performed using CD19-ENG, EphA2-ENG, and NT T cells as effectors and CD19-positive (BV173, Daudi, Raji) and negative (K562) tumor cells as targets at a E:T ratio of 2.5:1 (mean + SD; n = 3; assay was performed in duplicates; BV173, Raji, or Daudi: CD19-ENG vs EphA2-ENG T cells: *p < 0.001; CD19-ENG vs NT T cells: *p < 0.001; K562: CD19-ENG vs EphA2-ENG T cells: NS; CD19-ENG vs NT T cells: NS).
Figure 3
Figure 3. CD19-ENG T cells are able to recruit bystander T cells.
(a) Schematic representation of transwell assay. (b) 1 × 106 NT T-cells and 0.5 × 106 BV173.ffLuc cells were plated in the bottom well and CD19-ENG T cells in the insert well. The number of plated CD19-ENG T cells ranged from 103 to 106. EphA2-ENG T cells or media in the insert well and bottom wells without NT T cells served as controls. After 48 hours, live BV173.ffLuc cells were determined by luciferase assay (n = 3; *p < 0.05; **p < 0.01).
Figure 4
Figure 4. CD19-ENG T cells have potent antitumor activity in vivo.
Antitumor activity of CD19-ENG T cells in the i.v. BV173 leukemia NSG xenograft model. Mice received an i.v. dose of 1 × 107 CD19-ENG (n = 5) or EpHA2-ENG T cells (n = 5) and an i.p. dose of IL2 (1500 units) 7, 14, and 21 days after i.v. injection of 3 × 106 BV173.ffLuc cells. Untreated animals served as controls (n = 5). Tumor growth was followed by bioluminescence imaging. (a) Images of animals. (b) Quantitative bioluminescence imaging results for each mice (radiance = photons/sec/cm2/sr) over time (p < 0.05 starting day 6 post 1st T-cell injection for CD19-ENG vs EphA2-ENG T cells, and CD19-ENG T cells vs controls). (c) Kaplan-Meier survival curve (Control vs EphA2-ENG T cells: NS; Control vs CD19-ENG T cells: p = 0.0019; EphA2-ENG vs CD19-ENG T cells: p = 0.0021).
Figure 5
Figure 5. Safety profile of CD19-ENG T cells.
(a) Untreated (Control) and mice treated with EphA2-ENG T cells started to lose weight and died before day 40, the weight of mice treated with CD19-ENG T cells remained stable throughout the experiment. (b) Before the 1st, 2nd and 3rd T-cell infusion blood from BV173 leukemia-bearing mice treated with CD19-ENG T cells (n = 5) was collected by retro-orbital bleeding and the concentration of ENG molecules was determined using our bioassay. Only two of 5 samples pre 3rd T-cell infusion had detectable concentration of engager molecules in their blood. (c,d) At the same time points cytokines were determined using a Milliplex MAP High Sensitivity Human Cytokine Panel – Premixed 13 Plex (EMD Millipore, Billerica, MA) as per the manufacturer’s instructions. (c) GM-CSF, IFNγ, IL5, and IL7 were detectable at low levels (median: 19 pg/ml; range: 0–234 pg/ml) before the 2nd and 3rd infusion. (D) IL1b, IL2, IL4, IL6, IL8, IL10, IL12(p70), IL13, TNFα) were undetectable.
Figure 6
Figure 6. CD19-ENG T cells do not expand in the BV173 model in vivo and antitumor activity depends on T-cell dose.
(a,b) Seven days after i.v. injection of 3 × 106 BV173.ffLuc cells mice received a single i.v. dose of 1 × 106, 3 × 106, or 1 × 107 CD19-ENG T cells (n = 5 mice per group). Tumor growth was followed by bioluminescence imaging. (a) Quantitative bioluminescence imaging results for each mice (radiance = photons/sec/cm2/sr). (b) Kaplan-Meier survival curve (1 × 107 vs 3 × 106 CD19-ENG T cells: p = 0.02; 1 × 107 vs 1 × 106 CD19-ENG T cells: p = 0.02; 3 × 106 vs 1 × 106 CD19-ENG T cells: p = NS). (c,d) eGFP.ffLuc expressing CD19-ENG or EphA2-ENG T cells were injected i.v. into NSG mice 7 days after i.v. BV173 tumor-cell injection (n = 5 mice per group). Day 2 and 3 post T-cell injection there was a significant difference (*p < 0.05) between both groups.
Figure 7
Figure 7. CD19-ENG T cells expand locally in the Daudi model in vivo.
On day 14 post right lower quadrant I.P. injection of Daudi cells, mice received 3 × 106 eGFP.ffLuc-expressing CD19-ENG or EphA2-ENG T cells. Non-tumor bearing mice that received 3 × 106 eGFP.ffLuc-expressing CD19-ENG served as controls. (a) Images of animals. (b) Quantitative bioluminescence imaging results (radiance = photons/sec/cm2/sr; mean +/− SD is shown). CD19-ENG T cells + Daudi vs EphA2-ENG T cells + Daudi: p < 0.05 from day 3 to day 14; CD19-ENG T cells + Daudi vs CD19-ENG T cells: p < 0.05 starting day 3 until day 32. (c) Weight (mean +/− SD) for all three mice groups (EphA2-ENG T cells + Daudi vs CD19-ENG T cells or CD19-ENG T cells + Daudi: p < 0.05 starting day 25; CD19-ENG T cells + Daudi vs CD19-ENG T cells: p = NS).
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