TARGETED AND UNTARGETED FUSION PROTEINS: CURRENT APPROACHES TO CANCER IMMUNOTHERAPY - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 287

the patient to immunodeficiency states or tolerance [25].
Most recently, it has become recognized that in addition to
the induction of an effective immunologic response to
tumor, it is also important to produce a lasting response
by the development of immunologic memory [26]. Placed in
the correct context, the immune system is capable of massive
cell destruction as witnessed by graft versus host responses
and certain autoimmune states. On the basis of these obser-
vations, it is highly feasible that once harnessed, the immune
system will be capable of destroying deep-seated primary
tumors and metastatic lesions in cancer patients.
In general, there is a variety of antibody fusion proteins
targeting various tumor antigens in which different immu-
noregulatory ligands, toxins, or enzymes have been fused to
full-length antibodies or their derivatives. Immune activa-
tors, including cytokines, chemokines, and costimulatory
fusion proteins, can serve as one class of these potent
modulators and have been shown to generate antitumor
responses in animal models. A second class of fusion
proteins has been developed by combining antibody mole-
cules with truncated tissue factor (tTF) that target the
thrombogenic capacity of the tumor vasculature, thereby
enabling the initiation of coagulation and occlusion of blood
flow within the tumor [27,28]. In addition, attempts have
been made to engineer a third class fusion molecules
combining antibody and human enzyme together as a single
fusion protein [29-31]. This approach is called antibody
directed enzyme prodrug therapy (ADEPT), and specifically
aims at causing bystander effects by targeting enzymes to
the tumor cell and delivering a prodrug that is converted to a
chemotherapeutic by the targeted enzyme [32]. Overall,
these multiple fusion protein constructs have demonstrated
significant antitumor activity as direct therapeutics in pre-
clinical studies, paving the way for their clinical evaluation.

Targeted Antibody Fusion Proteins

N-Terminal
B7.1
LEC

C-Terminal
OX40L
CD137L
GITRL

C-Terminal
OX40L
CD137L
GITRL

N-Terminal
B7.1
LEC

Untargeted Fc-Fusion Proteins

FIGURE 19.1 Schematic of targeted and untargeted fusion
proteins.

reagents that can provide an enhanced immune stimulation
and treatment of cancer, investigators [34-36] have devel-
oped a novel mAb targeting approach, designated tumor
necrosis therapy (TNT), which targets single stranded DNA
exposed in degenerating cells. Because of these attributes,
TNT has been used to deliver radionuclides [37,38] to treat
experimental and human tumors [39,40].
To ensure the clinical potential of this approach, a variety
of ways have been investigated to enhance the antitumor
response of the host at the tumor site, thereby minimizing
systemic toxicity. To do so, TNT mAb linked to immune
effector or ligands molecules was produced by genetic
engineering as fusion proteins. These fusion proteins have
diverse effects including ability to target solid tumors in
immunocompetent mice, overcome peripheral tolerance,
and incite an effective antitumor immune response that
brings about tumor regression in solid tumor models. As
a first approach, a number of TNT fusion proteins were
successfully generated with cytokines IL-2 and IL-12, gran-
ulocyte macrophage-colony stimulating factor (GM-CSF),
tumor necrosis factor- a (TNF- a ), and interferon- g (IFN- g ),
which elicited significant immune responses in tumor-bear-
ing hosts. Secondarily, fusion proteins consisting of the
cysteine-cysteine class of chemokines (CC) liver-expression
chemokine (LEC, CCL16) were generated to chemoattract
polymorphonuclear leukocytes (PMNs), T cells, macro-
phages, B-cells, and dendritic cells into the tumor micro-
environment [41]. These studies confirmed the ability of
LEC/chTNT-3 to induce a multiarmed immune response
against solid tumors by attracting antigen-presenting cells
(APC) and effector cells into the tumor to induce significant
tumor regression [14]. More recently, it was found that
tumor regression can be achieved through the use of fusions
proteins consisting of co-stimulatory molecules such as B7.1

19.3 IMMUNOTHERAPEUTIC APPLICATIONS OF
ANTIBODY-TARGETED AND UNTARGETED Fc
FUSION PROTEINS

The development and applications of molecular engineering
technology led to the design of several new fusion proteins
targeting various tumors antigens in which different immune
activators/ligands have been fused to full-length antibodies,
single chain Fv (scFv), Fab, F(ab 0 ) 2 , or their Fc derivatives
(Figure 19.1). A broad variety of these molecules resulted in
improved functions to enhance the tumoricidal activity of
the antibodies and/or activate a secondary antitumor
immune response; all with promising results, particularly
from both academic institutions and biotechnology compa-
nies [33]. Each immune activator or ligand can be fused at
the amino- (N-terminal) or carboxy-terminus (C-terminal)
of the antibody or Fc derivatives (Figure 19.1) to conserve its
biological activity. For instance, in an attempt to identify