Biomedical Engineering Reference
In-Depth Information
TABLE 19.1 Summary of Pharmacokinetic Clearance and
Tumor Uptake Studies of Antibody/Cytokine Fusion Proteins
cell interaction establishes it as a prime candidate for
targeted immunotherapy.
Li et al. [14] first described the generation and testing of
an antibody/chemokine fusion protein consisting of chTNT-
3 and LEC that targets central necrotic areas of tumor to
chemoattract and amplify responding lymphoid and den-
dritic cells capable of inducing an effective antitumor
immune response. Since the N-terminus of chemokines is
essential for bioactivity, we fused the C-terminus of the LEC
gene to the N-terminus of the chTNT-3 HC gene with a five
amino acid universal linker (Gly4Ser). The biological activ-
ity of the LEC fusion protein was demonstrated by measur-
ing the migration of human leukemia TPH-1-0 cells in a
microchemotaxis chamber in a dose-dependent manner or
by binding to receptor positive TPH-1-0 cells by flow
cytometry. Biodistribution studies demonstrated that
25 I-labeled LEC/chTNT-3 specifically bound to tumor
with excellent retention at the tumor site (2.4% injected
dose per gram at both 12 and 24 h postinjection). The
immunotherapeutic potential of LEC/chTNT-3 was also
demonstrated by treating different experimental solid
tumors of the mouse. As shown in Figure 19.2,
LEC/chTNT-3 treatment produced a 37% reduction in the
MAD109 lung tumor model, as compared to untreated
controls. These observations fit with other solid tumors
that produced a 55% tumor growth reduction in the COLON
26 tumor model and a 42% reduction in the RENCA tumor
model. Recently, a nontargeted LEC fusion protein consist-
ing of human LEC and soluble Fc (LEC-Fc) was constructed
and showed similar tumor regression as the targeted fusion
protein (Figure 19.2). This indicates that sufficient amount
of LEC reached and bound to tumor to reverse tolerance.
% Injected
Dose Per
Gram (%ID/g)
of Tumor at
3 Days
Antibody/Cytokine
Fusion Protein
Half-Life
(h)
Tumor
Model
chTNT-3/muIFN- g
46 (134) MAD 109
2.4 (13.5)
chTNT-3/huIFN- g
19.1 (134)
LS 174T
1.2 (7.1)
chTNT-3/huIL-2
12 (134)
LS 174 T
1.7 (7.1)
chTNT-3/huTNF- a
8 (134)
LS 174 T
1.3 (7.1)
muTNT-3/muGM-CSF
15 (150) COLON 26
1.27 (5.3)
Values in parentheses refer to the representative naked antibody.
In vivo, the fusion proteins were found to have a substan-
tially shorter whole body half-life than parental antibodies,
yet were able to target tumor as shown by biodistribution
analyses. Because of their retention in tumor and rapid
clearance from normal tissues, the fusion proteins were
found to have equivalent or higher normal tissue/tumor
ratios than parental antibody. In particular, different treat-
ment studies have broadly explored the potential of using
cytokine fusion proteins combinations. These observations
are presented in Section 4.1 and open the door to superior
tumor therapeutic efficacy for these reagents.
19.3.2 Chemokine Fusion Proteins
Chemokines are small (7-15 kDa), secreted, and structurally
related soluble proteins that are involved in leukocyte and
dendritic cell chemotaxis, PMN degranulation, and angio-
genesis [55]. Because of their ability to recruit leukocytes
into tumors, alter tumor vasculature structure, and stimulate
host anti-cancer immune responses, chemokines are prom-
ising reagents in cancer therapy. The human chemokine LEC
(CCL16, also known as NCC-4, LMC, and HCC-4) was
originally found in an expression sequence tag library and
was also found to chemoattract monocytes, lymphocytes,
and PMNs on binding to chemokine receptors [56]. LEC is
unique because unlike any other chemokine, it is the first
chemokine whose mRNA expression is strongly increased
and stabilized by the presence of IL-10. The potential
therapeutic applications of LEC were first studied by
Giovarelli et al. [41], who showed that mammary carcinoma
TSA cells engineered to express LEC inhibit the metastatic
spread of tumor and induce tumor rejection due to an
impressive infiltration of macrophages, dendritic cells, T
cells, and PMNs, and the production of IFN- g and IL-12.
Furthermore, LEC is a potent chemotactic factor for both
human monocytes and dendritic cells (APC cells) [57]. The
ability of LEC to improve markedly the recognition of
poorly immunogenic tumor cells by promoting APC-T
19.3.3 Co-Stimulatory Fusion Proteins
Most tumors express antigens that can be recognized to a
variable extent by the host immune system, but in many
cases, an inadequate immune response is elicited because of
the ineffective activation of effector T cells via the immune
synapse. Studies showed that the weak immunogenicity of
tumor antigens might be due to inappropriate or absent
expression of co-stimulatory molecules on tumor cells
[58]. For most T cells, proliferation and IL-2 production
will not occur unless a co-stimulatory signal is also pro-
vided. In the absence of co-stimulatory signals during T cell
receptor (TCR) engagement, T cells enter a functionally
unresponsive state, referred to as clonal anergy. One of the
major signaling pathways involved in delivering the co-
stimulatory signal is mediated by interaction between CD28
on T cells and the B7 family members including B7.1
(CD80) and B7.2 (CD86) on APC [59]. T cell-mediated
rejection of tumors is achieved by presenting co-stimulatory
signals directly on the tumor cells surface. Transfection of
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