Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

312 16 Immunocytokines: Versatile Molecules for Biotherapy of Malignant Disease
tentiating immunogenicity of syngeneic tumors, followed in some cases by activation
and expansion of T cells, and a subsequent memory immune response. Importantly,
immunocytokines are not limited by either a patient-specific modus operandi nor by
heterogeneity of TAAs, since only a relatively limited number of antigen sites are required
as their docking sites. Immunocytokines placed in the tumor microenvironment
can activate and expand such immune effectors as T lymphocytes, natural killer
cells, macrophages and granuloctyes, and thereby eradicate tumor cells and their metastases.
This same effect can amplify insufficient Tcell immune responses previously
induced by cytokine gene therapy or DNA-based cancer vaccines and lead to effective
tumor eradication with subsequent long-lasting protective immunity.
16.1.2
Construction of Immunocytokines
The immunocytokines used in the preclinical evaluations of our laboratories described
here were constructed by following one common strategy. Thus, Gillies et al.
[3, 4] generated the coding sequences for the cytokines by reverse transcriptase polymerase
chain reaction (RT-PCR) with primers that include designated restriction
sites used for cloning purposes. Once generated, these cytokines were fused with the
human Cg1 gene at the Cl end of the heavy chain of an antibody. Specifically, Gillies
et al. [5, 6] inserted the fused gene of a chimeric human/mouse antiganglioside GD2
antibody (ch14.18) and recombinant human interleukin (rhIL)-2 into the vector
pdHL2, which also encodes the dihydrofolate reductase gene. The same vector carried
the gene encoding for the light chain of the ch14.18 antibody in a separate expression
unit. Both expression units were driven by a metallothionine promotor. The
expression plasmid was transduced into the immunoglobulin-non-producer murine
hybridoma cell line Sp2/0Ag14 cells by protoplast fusion and selected in the presence
of increasing concentrations of methotrexate (100 nM to 5mM). The same strategy
was employed for the construction of an antibody±cytokine fusion protein that
specifically recognizes a well-characterized human epithelial cell adhesion molecule
Ep-CAM/KS antigen (KSA) [7], which is extensively expressed on epithelial cell-derived
carcinomas including colon, breast, prostate and non-small cell lung carcinoma.
In contrast to ch14.18, a humanized antibody (huKS1/4) directed against
KSA was produced by grafting of the CDR-binding regions of the murine KS1/4
antibody onto a human IgG1 or IgG4 framework. The heavy chain was subsequently
fused to IL-2. In this case, the expression vector pdhL7s was used in which the cytomegalovirus
promotor drives the expression of each antibody chain [7].
All the fusion proteins described were purified by making use of the Fc portion of
the antibody molecule, which selectively binds Protein A±Sepharose. Following elution
at low pH, pure preparations of the antibody±cytokine fusion protein were obtained
and used for further characterization. This article focuses mainly on antibody±IL-2
and lymphotoxin (LT)-a fusion proteins and the preclinical in vivo results
obtained in the authors' laboratories. Functional characterizations are described
mainly for ch14.18±IL-2 and huKS1/4±IL-2 immunocytokines, which were used to
critically evaluate antitumor responses in syngeneic and SCID mouse models.