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

14.2 CRs with Antitumor Specificity
that participate in the receptor-proximal stage of the chain of events leading to T cell
activation [9]. In order to bypass these defective steps, we tried to directly linksoluble,
cytoplasmic PTKs of the ZAP-70 family such as ZAP-70 and Syk, known to be
recruited to the phosphorylated immuno receptor tyrosine activation motifs (ITAMs)
of lymphocyte receptors. After testing different combinations of these PTKs with various
transmembranal and extracellular spacer domains [10, 11], we found that Sykis
the preferable cytoplasmic PTK. Whether Syk-based CR performs better in cancer
patients' lymphocytes has still to be shown.
14.2.1.3 Combining stimulatory and co-stimulatory signals
Redirection of the specificity of effector lymphocytes, using single-chain CR composed
of an antibody linked to a lymphocyte triggering receptor subunit, has become
a valid option for adoptive cancer therapy. Although the CR can stimulate effector
functions in pre-activated T cells, it has been shown that this CR, lacking the capacity
to provide critical co-stimulatory signaling, cannot activate resting lymphocytes,
such as T cells derived from genetically modified stem cells or from CR transgenic
mice [12]. It is well established that, in the absence of co-stimulatory signaling by
CD28, resting T lymphocytes typically undergo anergy or apoptosis [13] and pre-activated
T lymphocytes may also display suboptimal effector responses in response to
CR engagement [14].
To overcome these obstacles, several groups have constructed CRs made of scFv
linked to the intracellular part of CD28 and have shown that co-expression of two CR
genes, each made of the same scFv linked to CD3z in the one and CD28 in the second,
could provide both stimulatory and co-stimulatory signals for T cell activation
[15±17]. Nevertheless, this approach required co-expression of two genes. A better design
included the two signaling moieties on a single CR [18]. We have designed a novel
tripartite CR composed of a scFv recognition moiety fused to the non-ligand binding
part of the extracellular and the entire transmembrane and intracellular domains
of the CD28 co-stimulatory molecule and the intracellular domain of FcRg (scFv±
CD28±g) [19]. Human peripheral blood lymphocytes (PBLs) transduced with such a
CR gene demonstrated specific stimulation of IL-2 production and target cell killing
[19]. This was dependent on CD28 co-stimulatory activity as evidenced by IL-2 secretion
profiles in the presence of the certain signal transduction inhibitors (A. Bendavid
et al., unpublished). Moreover, we have recently generated lines of transgenic
mice expressing CR under the control of T cell-specific regulatory sequences. Unprimed
naive T lymphocytes from mice transgenic for scFv±CD28±g undergo high
levels of proliferation, IL-2 secretion and rescue from apoptosis as a result of stimulation
by plastic-bound cognate antigen (A. Bendavid et al., unpublished).
In these studies we have demonstrated for the first time that FcRg and CD28 can cooperate
in providing co-stimulatory signaling to human T lymphocytes and activation
of unprimed naive T lymphocytes via an engineered single-chain receptor of
predefined specificity. For clinical application, these results point to a critical advantage
for the tripartite configuration of CR in the generation of persistent and functional
redirected T cells.
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