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. 2015 Feb;3(2):125-35.
doi: 10.1158/2326-6066.CIR-14-0127. Epub 2014 Sep 11.

The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity

Michael Hudecek  1 , Daniel Sommermeyer  2 , Paula L Kosasih  2 , Anne Silva-Benedict  3 , Lingfeng Liu  2 , Christoph Rader  4 , Michael C Jensen  5 , Stanley R Riddell  6
Affiliations

The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity

Michael Hudecek et al. Cancer Immunol Res. 2015 Feb.

Abstract

The use of synthetic chimeric antigen receptors (CAR) to redirect T cells to recognize tumor provides a powerful new approach to cancer immunotherapy; however, the attributes of CARs that ensure optimal in vivo tumor recognition remain to be defined. Here, we analyze the influence of length and composition of IgG-derived extracellular spacer domains on the function of CARs. Our studies demonstrate that CD19-CARs with a long spacer from IgG4 hinge-CH2-CH3 are functional in vitro but lack antitumor activity in vivo due to interaction between the Fc domain within the spacer and the Fc receptor-bearing myeloid cells, leading to activation-induced T-cell death. We demonstrate that in vivo persistence and antitumor effects of CAR-T cells with a long spacer can be restored by modifying distinct regions in the CH2 domain that are essential for Fc receptor binding VSports手机版. Our studies demonstrate that modifications that abrogate binding to Fc receptors are crucial for CARs in which a long spacer is obligatory for tumor recognition as shown here for a ROR1-specific CAR. These results demonstrate that the length and composition of the extracellular spacer domain that lacks intrinsic signaling function can be decisive in the design of CARs for optimal in vivo activity. .

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Conflict of interest statement (VSports app下载)

Conflicts of interest: M. H. , M. C. J. , and S. R. R. are inventors on a patent application (PCT/US2013/055862) related to this work that has been filed by the Fred Hutchinson Cancer Research Center and licensed by Juno Therapeutics. M. C V体育ios版. J. and S. R. R. are founders and shareholders of Juno Therapeutics. C. R. is inventor on a patent application (PCT/US2011/062670) that claims anti-ROR1 mAb R11 and has been filed by the National Institutes of Health.

Figures

Figure 1
Figure 1. CD19-CAR-T-cells with short and long spacers show specific in vitro function
(A) Design of lentiviral transgene inserts encoding CD19-specific CARs with different extracellular spacer lengths. (B) Analysis of EGFRt expression on CD8+ TCM-derived T cells transduced with the lentiviral vectors encoding the CD19-CARs with short or long spacer domains before enrichment (pre) and after enrichment and expansion (post). (C) Cytolytic activity, (D) IFNγ production, and (E) proliferation of CD19-CAR-T-cells after co-culture with CD19+ (K562/CD19, Raji) and control (K562) target cells. Proliferation after stimulation with CD19− K562 cells is shown as a comparison in each histogram (gray). Numbers above each histogram indicate the number of cell divisions the proliferating subset underwent, and the fraction of T cells in each gate that underwent ≥4/3/2/1 cell divisions is provided in the upper left of each plot. Data in B–E are representative of experiments with CAR-T-cells derived from at least three different donors.
Figure 2
Figure 2. CD19-CAR-T-cells with a short spacer domain eradicate Raji tumors in NSG mice
(A) NSG mice were inoculated with Raji-ffluc cells, and seven days later treated with 2.5×106 T cells expressing short or long spacer CD19-CARs containing a 4-1BB/CD3ζ signaling module or with T cells expressing only EGFRt (4–5 mice per group). Arrows mark the day of T-cell transfer. Tumor growth was analyzed by bioluminescence imaging and results from individual mice are plotted for short and long spacer CARs. Red triangles show the mean tumor burden in untreated mice at each time point. Images from one day before T-cell transfer (d6) and eleven days after transfer (d18) are shown for mice that had received short or long spacer CART-cells. (B) Survival of mice treated with short and long spacer 4-1BB/CD3ζ-CAR-T-cells was compared to mice that had received control T cells (EGFRt) or no T cells (untreated). (C) Frequency of CD8+/EGFRt+ T cells in the peripheral blood obtained at days three and ten after T-cell transfer. Asterisk indicates significant differences between groups. (D) Survival of Raji-ffluc-bearing NSG mice treated with a high dose (1×107) of CD19-CAR-T-cells with long spacers (long/CD28, long/CD28_4-1BB), with a short spacer (short/CD28), or with control T cells (4 mice per group). (E) Frequency of CD8+/EGFRt+ T cells in the blood obtained at days three and ten after T-cell transfer in mice treated in (D).
Figure 3
Figure 3. CD19-CAR-T-cells with a long spacer are activated in vivo but fail to increase in cell number
(A) Expression of activation markers CD69 and CD25 on CAR-T-cells prior to transfer into NSG/Raji mice compared to isotype controls (gray). (B,C) Expression of activation markers and proliferation of CD19/4-1BB/CD3ζ-CAR-T-cells after adoptive transfer. Flow cytometric analysis of bone marrow obtained (B) 24 hours and (C) 72 hours after T-cell transfer. Dot plots show anti-CD3 and anti-CD45 staining after gating on PI− cells. The CD3−/CD45+ gate contains Raji tumor cells. Expression of CD25 and CD69 is shown for live (PI−) CD3+/CD45+ T cells in comparison to isotype controls (gray). Proliferation of transferred T cells was analyzed by CFSE dye dilution and cell death by PI staining. (D) Frequency and PI staining of CD3+/CD45+ T cells in spleens obtained 72 hours after T-cell transfer. (E) Tumor growth in mice treated with T cells expressing CD19-CARs with a short or long spacer. Arrows mark the day of T-cell transfer. Data are representative of two independent experiments with 2–3 mice per group.
Figure 4
Figure 4. CD19-CAR-T-cells with a long spacer are activated in the lung
(A) Frequency of transferred T cells (CD45+/CD8+) and the expression of CD25 and CD69 in the bone marrow 24 hours after transfer of 1×107 CD19/4-1BB/CD3ζ-CAR-T-cells into tumor-free NSG mice. (B) T cells were transduced with ffluc/eGFP and short or long spacer CD19-CARs and enriched for eGFP+/EGFRt+ cells. 1×107 T cells were injected into tumor-free NSG mice and localization of T cells was examined by bioluminescence imaging 1 and 24 hours later. Lungs and spleens were isolated, imaged separately, and analyzed for the presence of eGFP+ T cells and the expression of CD25 and CD69 on eGFP+ T cells. (C) Staining of a single-cell suspension of lung cells from NSG mice with IgG4 protein and anti-Ly6C mAb. (D) Analysis of CD25 expression on short and long spacer CD19-CAR-T-cells after 24 hours of co-culture with Ly6C+ and Ly6C− cells isolated from spleens of NSG mice. (E) Staining of K562/CD64 cells with anti-CD64 mAb and IgG4 protein. Untransduced K562 cells were used as a control (white). (F) CD25 and CD69 expression and (G) cytolytic activity and IFNγ release of CD19-CAR-T-cells with short and long spacers after co-culture with CD64+ and CD64− K562 cells. Data are representative of at least two independent experiments.
Figure 5
Figure 5. CD19-CAR-T-cells with a CH2-deleted spacer are functional in vitro and in vivo
(A) Cytolytic activity and (B) IFNγ release of CD19/4-1BB/CD3ζ-CAR-T-cells was analyzed after co-culture with Raji, K562, K562/CD19, and K562/CD64 cells. (C) 2.5×106 CAR-T-cells were injected into Raji-ffluc-bearing NSG mice (4 mice per group). Survival of mice was compared to mice receiving control EGFRt-T-cells. (D) Persistence of T cells in blood was analyzed three days after T-cells transfer. Asterisk indicates significant differences between groups. Data are representative of experiments with CAR-T-cells derived from three different donors.
Figure 6
Figure 6. CD19-CAR-T-cells with long spacers comprised of a modified Fc domain persist in vivo and eliminate tumor cells
(A) The amino acid sequence of the hinge and parts of the CH2 region for short, long and long variants (4/2 and 4/2NQ). (B) CD25 expression on CD8+ TCM-derived CD19/4-1BB/CD3ζ-CART-cells after 24 hour co-culture with CD64+ and CD64− K562 cells. (C) Cytolytic activity, (D) IFNγ production, and (E) proliferation of CD19-CAR-T-cells with different spacers after stimulation with K562, K562/CD19, and Raji cells. Proliferation after stimulation with K562 cells is shown as a comparison in each histogram (gray). (F) Antitumor activity of 2.5×106 CART-cells with different spacers in Raji-ffluc-bearing NSG mice (4 mice per group). Results obtained one day before (day 6) and one week after (day 14) T-cell injection are shown. (G) Survival of mice treated with CD19-CAR-T-cells, control T cells (EGFRt), and untreated mice. Statistical analyses were performed by log-rank test and the asterisk indicates significant differences (p<0.01) between groups. (H) Persistence of T cells in blood three days after T-cells transfer. The asterisk indicates significant differences between groups. Data in B–E are representative of experiments with CAR-T-cells derived from two different donors.
Figure 7
Figure 7. T-cells with a ROR1-specific CAR eliminate tumor cells in vivo only with a modified long spacer
(A) Cytotoxicity, (B) IFNγ release, and (C) proliferation of CD8+ TCM transduced with ROR1-specific R11-CARs with short, intermediate, long, and long 4/2NQ spacers after co-culture with K562 and K562/ROR1 cells. (D) Antitumor activity of 5×106 R11-CAR-T-cells with different spacers in NSG mice engrafted with 5×105 JeKo-1-ffluc seven days before T-cell infusion (3 mice per group). Images obtained one day before (day 6) and three days after (day 10) T-cell injection are shown. (E) Persistence of ROR1-CAR-T-cells in blood three days after T-cell transfer. Asterisk indicates significant differences between groups. Data in A–C are representative of experiments with CAR-T-cells derived from at least two different donors.
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