IMMUNOKINASES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 323

following ligand binding, and although its expression is
normally restricted to subpopulations of activated T- and
B-lymphocytes, it is also expressed strongly in hematopoietic
malignancies such as Hodgkin lymphoma and anaplastic
large cell lymphoma [51,52]. Several anti-CD30 immuno-
therapeutics are currently undergoing clinical trials [53].

TABLE 21.1 DAPK2 Expression and DAPK2 0 -CD30L
Activity in Three Hodgkin Lymphoma-Derived Cell Lines

L540 L1236 L428

Methylation þ

DAPK2 RT-PCR þþ

DAPK2 RT-PCR after
Decitabine treatment

þþþ

Western blot analysis of DAPK2 þ
Western blot after Decitabine treatment þþ
Toxicity after targeted DAPK2 restoration þþ

21.4 ANALYSIS OF IMMUNOKINASE EFFICACY

21.4.1 In Vitro Activity of Recombinant Immunokinase
DAPK2 0 -CD30L
The recombinant immunokinase DAPK2 0 -CD30L com-
prises a truncated DAPK2 protein retaining full catalytic
activity but lacking the CaM domain joined by a 11-aa
peptide linker to CD30L [7]. We initially tested the specific
activity of the immunokinase in proliferation and apoptosis
experiments. Specific binding to several CD30

Source: Reproduced from Reference 6.

To investigate the influence of DAPK2 0 -CD30L on overall
survival, we started anti-tumor treatment 1 day after tumor
challenge by administrating DAPK2 0 -CD30L to one group
of mice, a nonspecific immunokinase called DAPK2 0 -H22
(scFv), which targets the irrelevant antigen CD64, to a
second group, and saline solution (PBS) to a third. The
recombinant fusion proteins were administrated at the
maximum tolerable dose of 70 m g per mouse. The overall
survival rate of the PBS control and nonspecific immuno-
kinase groups was significantly lower than that of the
DAPK2 0 -CD30L group (p

target
ce lswasconfirmedbyflowcytometry, and the dis-
sociation constant (KD) values were in the 7-17 nM range.
Potent and cell-specific elimination of CD30

þ

/DAPK2 -
target cells was confirmed using proliferation and
apoptosis assays, revealing that the immunokinase had
an IC 50 value of 20 nM against CD30 þ /DAPK2-negative
L540 cells and

þ

0.028). The mean survival
times for the PBS-treated and nonspecific immunokinase
control groups (n

¼

63 nM against L1236 cells, respectively.
There was no cytotoxic effect against CD30

10) were 55 and 60 days, respectively,
whereas, 8 out of 10 mice in the DAPK2 0 -CD30L treatment
group were long-term survivors (

¼

/DAPK2 þ

þ

L428 cells (Table 21.1).

175 days) and showed no
signs of disease. Two animals died (days 60 and 100)
without developing any macroscopic signs of disease, sug-
gesting that cancer was not the cause of death (Figure 21.4).
Histological examination of the PBS-treated SCID mice
revealed the presence of disseminated tumors in the liver,
lung, spleen, and brain, as previously reported [54].

>

21.4.2 In Vivo Activity of Recombinant Immunokinase
DAPK2 0 -CD30L

In SCID mice, the intravenous injection of L540 cells
inevitably leads to the disseminated growth of tumor cells
within 5-6 weeks and ultimately to death from cancer [54].

FIGURE 21.4 Effect of DAPK2 0 -CD30L treatment on disseminated Hodgkin lymphoma-derived
tumors in SCID mice. Mice were injected intravenously with L540 tumor cells and were left
untreated (PBS control) (none, light grey line), nonspecifically treated (nonspecific, grey line), or
treated with maximum tolerable doses of DAPK2 0 -CD30L (specific, black line). Survival was plotted
using the Kaplan-Meier method. Source: Reproduced from Reference 6.