Hamster Anti-AMICA1 Recombinant Antibody (clone HL4E10) (CAT#: PABL-252)

Figure 1 HL4E10 and CAR interaction with JAML as observed by SPR reveal distinct binding affinities and kinetics.

SPR binding data of HL4E10 Fab and CAR to immobilized JAML. Injection start and end points are indicated with red arrows. (A) HL4E10 Fab (2–500 nM) binding to JAML. Data (colored lines) are overlaid with a global fitting using a standard 1:1 Langmuir binding isotherm (black lines). The interaction is characterized by a fast on-rate and slow off-rate, apparent by the long half-life in the order of ~10 min. (B) CAR (0.3–20 µM) binding to JAML. Data are overlaid with a global fitting using a complex binding model accounting for heterogeneity of the ligand. The JAML-CAR interaction exhibits rapid on-and off-rates and a half-life of ~1 s.

Verdino, P., Witherden, D. A., Ferguson, M. S., Corper, A. L., Schiefner, A., Havran, W. L., & Wilson, I. A. (2011). Molecular insights into γδ T cell costimulation by an anti-JAML antibody.Structure, 19(1), 80-89.

Figure 2 PI3K recruitment and induction of DETC proliferation by HL4E10 binding to JAML.

(A) Anti-PI3K Western blots of JAML immunoprecipitated from epithelial γδ T cell lysates at various time points after stimulation with HL4E10 IgG. After 1 min, PI3K association to JAML is significantly elevated over the basal level. (B) Costimulation of γδ T-cell activation through HL4E10 IgG or Fab-binding to JAML. Proliferation of DETC to immobilized anti-CD3 IgG either alone (shaded bars) or in combination with anti-JAML HL4E10 IgG (solid bars), control IgG 1F4 (open bars), anti-JAML HL4E10 Fab fragment (striped bars) or control Fab (dotted bars) was assessed by 3H-thymidine incorporation. HL4E10 IgG and its Fab fragment both induce comparable activation of DETC when surface immobilized.

Verdino, P., Witherden, D. A., Ferguson, M. S., Corper, A. L., Schiefner, A., Havran, W. L., & Wilson, I. A. (2011). Molecular insights into γδ T cell costimulation by an anti-JAML antibody.Structure, 19(1), 80-89.

Figure 3 7-17 cells by flow cytometry for HL4E10 or control mAb staining.

Witherden, D. A., Verdino, P., Rieder, S. E., Garijo, O., Mills, R. E., Teyton, L.,... & Havran, W. L. (2010). The junctional adhesion molecule JAML is a costimulatory receptor for epithelial γδ T cell activation. Science, 329(5996), 1205-1210.

Figure 4 γδ TCR and HL4E10 fluorescent staining (magnification ×600) of 7-17 cells grown on coverslips. The nucleus is stained with 4′,6′-diamidino-2-phenylindole (DAPI). Scale bar represents 50 μm.

Witherden, D. A., Verdino, P., Rieder, S. E., Garijo, O., Mills, R. E., Teyton, L.,... & Havran, W. L. (2010). The junctional adhesion molecule JAML is a costimulatory receptor for epithelial γδ T cell activation. Science, 329(5996), 1205-1210.

Figure 5 Western blot analysis of 7-17 lysates immunoprecipitated with mAb HL4E10 (lane 1) or a control hamster mAb, 1F4, (lane 2). Protein identification by liquid chromatography and electrospray ionization mass spectrometry gave a high-scoring match with mouse JAML (Mascot score 151).

Witherden, D. A., Verdino, P., Rieder, S. E., Garijo, O., Mills, R. E., Teyton, L.,... & Havran, W. L. (2010). The junctional adhesion molecule JAML is a costimulatory receptor for epithelial γδ T cell activation. Science, 329(5996), 1205-1210.

Figure 6 Proliferation of short-term DETC cell lines to immobilized CD3-specific antibody (anti-CD3) either alone or in combination with JAML-specific (anti-JAML) mAb, HL4E10, or a control mAb, 1F4.

Witherden, D. A., Verdino, P., Rieder, S. E., Garijo, O., Mills, R. E., Teyton, L.,... & Havran, W. L. (2010). The junctional adhesion molecule JAML is a costimulatory receptor for epithelial γδ T cell activation. Science, 329(5996), 1205-1210.