pCDCAR1 GD2 h(BBζ) (CAR-ZP4260)

CAR Construction : MB3.6 scfv-CD28-CD3ζ-IL2

Fig.1 Clonal proliferation of GD3 CAR+T cells after tumor contact.

CAR-modified and untransduced T cells were plated at 1e6/mL at ratios of 1:0.125, 1:0.25, and 1:0.5 with irradiated GD3+ tumor M21 and GD3- tumor M24, with weekly tumor stimulation. T-cell number was counted every 3 or 4 d in triplicate from two separate wells.

Lo, A. S., Ma, Q., Liu, D. L., & Junghans, R. P. (2010). Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal TumorsSecond-Generation Anti-GD3 Designer T Cells. Clinical Cancer Research, 16(10), 2769-2780.

CAR Construction : MB3.6 scfv-CD28-CD3ζ-IL2

Fig.2 Clonal expansion of CAR+ T cells after tumor contact.

Cultures from CAR-modified and untransduced T cells, shown here with the E:T ratio of 1:0.5, were assayed on days 0, 7, and 14 by flow
cytometry for expression of CAR for CD4+ T-cell subset.

Lo, A. S., Ma, Q., Liu, D. L., & Junghans, R. P. (2010). Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal TumorsSecond-Generation Anti-GD3 Designer T Cells. Clinical Cancer Research, 16(10), 2769-2780.

CAR Construction : MB3.6 scfv-CD28-CD3ζ-IL2

Fig.3 Clonal expansion summary of CAR+ T cells after tumor contact.

Profiles of percentage for CAR on days 7 and 14 as in Bi are averaged from three different experiments versus CAR+ T cells without antigen.
stimulation as control.

Lo, A. S., Ma, Q., Liu, D. L., & Junghans, R. P. (2010). Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal TumorsSecond-Generation Anti-GD3 Designer T Cells. Clinical Cancer Research, 16(10), 2769-2780.

CAR Construction : MB3.6 scfv-CD28-CD3ζ-IL2

Fig.4 IL2 and IFNγ release analysis of Transduced T cells were stimulated with tumor cells.

Transduced T cells were stimulated overnight with GD3+ M21 tumor cells or with B7.1+ M21 tumor cells at E:T ratio of 1:1.
IL2 and IFNγ in medium are presented as the average of two wells (pg/mL).

Lo, A. S., Ma, Q., Liu, D. L., & Junghans, R. P. (2010). Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal TumorsSecond-Generation Anti-GD3 Designer T Cells. Clinical Cancer Research, 16(10), 2769-2780.

CAR Construction : MB3.6 scfv-CD28-CD3ζ-IL2

Fig.5 specific antitumor cytolysis of Transduced T cells were stimulated with tumor cells.

Transduced T cells were assayed in a 4-h killing assay at different E:T ratios with 51Cr-labeled GD3+ M21 target cells. The transduced fractions for IgTCR and Tandem were 42% and 37%, respectively.

Lo, A. S., Ma, Q., Liu, D. L., & Junghans, R. P. (2010). Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal TumorsSecond-Generation Anti-GD3 Designer T Cells. Clinical Cancer Research, 16(10), 2769-2780.

CAR Construction : MB3.6 scfv-CD28-CD3ζ-IL2

Fig.6 Inhibition of established human tumor xenografts by designer T cells.

Nude mice were s.c. implanted with 5e6 GD3+ M21 melanoma on the right flank. On day 7, the mice received i.v. injection of 5e7 transduced T cells. A parallel set of mice also received continuous IL2 via micro-osmotic pump placed s.c. Tumor was measured.

Lo, A. S., Ma, Q., Liu, D. L., & Junghans, R. P. (2010). Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal TumorsSecond-Generation Anti-GD3 Designer T Cells. Clinical Cancer Research, 16(10), 2769-2780.

CAR Construction : MB3.6 scfv-CD28-CD3ζ-IL2

Fig.7 Inhibition of established human tumor xenografts by designer T cells.

Tumor size for each group of mice was compared on day 16 to assess the effect of IL2 and T-cell modification on tumor suppression

Lo, A. S., Ma, Q., Liu, D. L., & Junghans, R. P. (2010). Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal TumorsSecond-Generation Anti-GD3 Designer T Cells. Clinical Cancer Research, 16(10), 2769-2780.

CAR Construction : MB3.6 scfv-CD28-CD3ζ-IL2

Fig.8 Inhibition of established human tumor xenografts by designer T cells.

Tumor necrosis. Histology of cells in progressing and regressing tumors by H&E staining.

Lo, A. S., Ma, Q., Liu, D. L., & Junghans, R. P. (2010). Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal TumorsSecond-Generation Anti-GD3 Designer T Cells. Clinical Cancer Research, 16(10), 2769-2780.

CAR Construction : MB3.6 scfv-CD3ζ

Fig.9 Cell conjugation of IgTCR + T cells with GD3+ melanoma cells.

GD3+IgTCR + modified T cells were tightly bound to M21 CELL.

Yun, C. O., Nolan, K. F., Beecham, E. J., Reisfeld, R. A., & Junghans, P. (2000). Targeting of T lymphocytes to melanoma cells through chimeric anti-GD3 immunoglobulin T-cell receptors. Neoplasia, 2(5), 449-459.

CAR Construction : MB3.6 scfv-CD3ζ

Fig.10 Cell conjugation of IgTCR + T cells with GD3+ melanoma cells.

A single giant melanoma tumor cell target ( orange; apparently in mitosis ) is bound by 19 effector T cells (green).

Yun, C. O., Nolan, K. F., Beecham, E. J., Reisfeld, R. A., & Junghans, P. (2000). Targeting of T lymphocytes to melanoma cells through chimeric anti-GD3 immunoglobulin T-cell receptors. Neoplasia, 2(5), 449-459.

CAR Construction : MB3.6 scfv-CD3ζ

Fig.11 IL2 secretion of IgTCR transduced cells after stimulation withimmobilized proteins and melanoma cell lines.

Unmodified gd3-CAR Jurkat T cells were coincubated with various stimuli, and thesupernatants were harvested and tested for IL2 concentration.

Yun, C. O., Nolan, K. F., Beecham, E. J., Reisfeld, R. A., & Junghans, P. (2000). Targeting of T lymphocytes to melanoma cells through chimeric anti-GD3 immunoglobulin T-cell receptors. Neoplasia, 2(5), 449-459.

CAR Construction : MB3.6 scfv-CD3ζ

Fig.12 IgTCR expression on primary T lymphocytes.

T lymphocytes werepurified from PBMCs, transduced with the gd2-CAR encoding retroviralvector, and analyzed for surface expression.

Yun, C. O., Nolan, K. F., Beecham, E. J., Reisfeld, R. A., & Junghans, P. (2000). Targeting of T lymphocytes to melanoma cells through chimeric anti-GD3 immunoglobulin T-cell receptors. Neoplasia, 2(5), 449-459.

CAR Construction : MB3.6 scfv-CD3ζ

Fig.13 Cytotoxicity of IgTCR + CTLs against GD3 + melanoma cells

GD3+ M21 tumor targets were seeded at 2E5cells per well, and then anti - GD3 IgTCR + CD8 + CTLs were added at different E:T ratios, ranging from 0.2:1 to 2:1. 0:1 indicates growth oftumor cells without added effector CTLs.

Yun, C. O., Nolan, K. F., Beecham, E. J., Reisfeld, R. A., & Junghans, P. (2000). Targeting of T lymphocytes to melanoma cells through chimeric anti-GD3 immunoglobulin T-cell receptors. Neoplasia, 2(5), 449-459.