Generation of Potent T cell Immunotherapy for Cancer using DAP12-based, Multichain, Chimeric Immunoreceptors

Enxiu Wang et al.

Supplemental Figure Legends

Supplemental Figure 1. Cytotoxicity induced by SS1-KIRS2, but not receptor expression at the cell surface, depends upon DAP12 co-expression.

A) Primary human T cells were simulated as in Fig 1C, transduced with either lentiviral vector expressing dsRed (Control) or a vector expressing dsRed and DAP12 separated by a T2A sequence (DAP12). Following 9 days of in vitro culture, the cells were electroporated(ECM 830, Harvard Apparatus) with in vitro transcribed mRNA encoding SS1-KIRS2(QuantiTect Reverse Transcription Kit, Qiagen) as described by Zhao Y et al(1). The expression of SS1-KIRS2 was assessed by flow cytometry as described in Figure 1C. Dotted line in the histograms show mock transfected cells. Blackand gray lines represent analysis of dsRed+ and dsRed- cells as shown in figure above each histogram. B)Antigen-specific cytotoxic activity of T cells generated as described in panel Awas assessed by a 4-hr 51Cr-release assay using Kwt or Kmeso cells.

Supplemental Figure 2. SS1-KIRS2 does not affect endogenous TCR expression

Primary human T cells were simulated and expanded as described in Figure 1C. Following 9 days of expansion, the T cells were electroporated with in vitro transcribed mRNA encoding the SS1-KIRS2 as described in Figure 1E. Flow cytometry was performed to detect the surface expression of the TCR Vβ13.1 (clone IMMU 222, Beckman Coulter).

Supplemental Figure 3. Mesothelin-specific CD3ζ CARs and KIR-CAR/Dap12 show comparable antigen-specific in vitro cytotoxicity toward EM-meso cells

Primary human T cells were stimulated with anti-CD3/CD28 stimulator beads as described in Figure 1, and transduced with a lentiviral vector expressing the indicated mesothelin-specific CAR or mock-transduced (mock). Following 9 days of culture, T cells were mixed with 51Cr-labeled EM-mesoor EMp cells at the indicated effector to target (E:T) ratio. % lysis was determined as described in the materials and methods. Results are presented of 3 independent experiments.

Supplemental Figure 4. SS1-KIRS2/DAP12 T cells mediate robust anti-tumor activity in vivo

NSG mice were injected subcutaneously with 2x106 EM-meso cells. 5x106 primary human T cells transduced with the indicated CAR were injected IV on day 20. Tumor volume was measured by caliper at the indicated times.

Supplemental Figure 5.CD19-specific KIR-CAR/Dap12 shows antigen-specific cytotoxicity in vitro

Primary human T cells were stimulated with anti-CD3/CD28 stimulator beads as described in Figure 1 and transduced with a lentiviral vector expressing either a CD19-specific KIR-CAR and DAP12 (CD19-KIRS2/Dap12), a CD19-specific CD3-based CAR with CD3 alone (CD19) or a CD19-specific CD3-based CAR with CD3 and 4-1BB cytoplasmic domains (CD19-BB). Following 9 days of culture, T cells were mixed with 51Cr-labeled Kwt, or K562 expressing CD19 (K562-CD19) at the indicated effector to target (E:T) ratio. % lysis was determined as described in the materials and methods. Results are representative of 3 independent experiments.

Supplemental Figure 6.FAP-specific KIR-CAR/Dap12 T cells induce bone marrow hypocellularityand loss of body weight

(A) Femurs were collected from mice shown in Fig 3D at necropsy on day 48. H&E sections were obtained following decalcification. Images are representative of N = 5 mice. (B) Mean ± standard deviation of weight for mice shown in Fig 3Don the indicated days following T cell injection. (C) Mean ± standard deviation of weight for mice shown in Fig 3C on the indicated days following T cell injection.

Supplemental Figure 7.Effect of TIL isolation treatment on CAR expression T cells.

An aliquot of SS1-28 CAR T cells were treated using the TIL isolation and enrichment procedure as described in the materials and methods (full enzyme). A second aliquot of CAR T cells was treated with protease digestion cocktail diluted 1:1 with PBS (1/2 enzyme) as described. Western blotting of protein lysates from theuntreated CAR T cells or those treated with the TIL isolation and enrichement procedure was then performed for CD3 (clone 6B10.2; sc-1239; Santa Cruz Biotechnology) as described in the materials and methods.

Supplemental References

1.Zhao Y, Zheng Z, Cohen CJ, Gattinoni L, Palmer DC, Restifo NP, Rosenberg SA, Morgan RA. High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Molecular therapy : the journal of the American Society of Gene Therapy. 2006 Jan;13(1):151-9. PubMed PMID: 16140584. Pubmed Central PMCID: 1473967.

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