NOVEL APPLICATIONS OF BISPECIFIC DART PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 525

combining either anti-CD3 or anti-TCR b (T-cell receptor

b ) with anti-CD19 as a B-cell targeting specificity [25]. Both
of these “T-DART 1 ” proteins exhibited extremely potent
redirected T-cell mediated lysis of B-cell targets, either in an
autologous setting or using B-cell tumor lines as targets in a
cytotoxic T-lymphocyte assay. Moreover, the potency of
these agents was greater than that achieved by a dual-
scFv bispecific protein derived from identical anti-CD3
and anti-CD19 Fv sequences and differing only in the
bispecific assembly strategy utilized. The CD19

DART 1 protein was further shown to be active in vivo in a
human PBMC-reconstituted xenograft mouse model against
Raji (lymphoblastoid) cell targets.
We have also constructed several T-DART 1 proteins
against solid tumor targets including HER2. The HER2

e

TCR DART 1 was made by combining the 4D5 Fv (the
parent of trastuzumab, a mAb that interferes with the
HER2/neu receptor) with the humanized anti-TCR Fv.
The DART 1 protein was purified to homogeneity using
affinity and SEC (Figure 36.1A). Both chains of DART 1 TCR T-

FIGURE 36.1 Anti-HER2 T-DART 1 for redirectedT-cell killing against tumor cells. (A) SDS-PAGE
analysis of purified DART 1 protein under reducing conditions. Lane 1, HER2 TCRDART 1 and lane
2, molecular mass markers. Graph: Size exclusion chromatography performed on a Superdex 200HR
10/30 column demonstrates that under native conditions, a typical DART 1 protein forms a single peak
with a retention time similar to that expected for an antibody Fab fragment. (B) Cytotoxicity of DART 1
protein against HER2 positive breast cancer cell lines. Antibody-dependent cellular cytotoxicity
(ADCC) assays were performed using MCF-7 breast cancer cells with HercepTest score of 1 þ cancer
cell lines as target cells incubated with PBMC as effector cells at an effector-to-target ratio of 30:1.
Assays were performed in triplicate at each concentration with HER2 TCRDART 1 , trastuzumab and
4420 TCR DART. Cell-mediated cytotoxicity was quantified by measuring lactate dehydrogenase
(LDH) released into the culture medium. Curves were fit using nonlinear regression analysis. Error bars
represent SD. (C) The DART 1 protein functions through the Granzyme B/Perforin pathway. ADCC
assays were performed using JIMT-1 breast cancer cells withHercepTest score of 2 þ cancer cell lines as
target cells incubated with PBMC at an effector-to-target ratio of 30:1. Cytotoxicity of HER2
TCR DART 1 measured by LDH release was evaluated in the presence of either DMSO, or
Z-AAD-CMK (20 nM) or concanamycin A (1mM). Error bars represent SD. (D) In vivo antitumor
activity of DART 1 against HER2 positive (HercepTest score 1 þ )bladdercancerxenografts.
NOD-SCID mice (N ¼ 8 per treatment group) were implanted subcutaneously with a 1:1 ratio of
2.5 10 6 SW780 cells and human PBMC (5 10 6 ) that had been activated overnight with IL-2
(10 m g/mL) and mixed with matrigel. Mice were then treated with vehicle or the indicated DART 1
molecules (15 m g/animal) i.v. on days 0-4. Tumor volume was measured externally at each time
point. Error bars represent SD. P < 0.001.