| dc.description.abstracteng | Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a poor prognosis and
limited treatment options. In recent years, chimeric antigen receptor (CAR) T cells have
achieved remarkable success in the treatment of liquid malignancies but efficacy in solid
malignancies is limited to date. A major hurdle is the lack of tumor specific targets. We aimed
to address this issue in the case of PDAC by analyzing the expression of almost 400 cell-surface
antigens on tumor cells of different patient derived xenograft (PDX) mouse models. Identified
candidates were prioritized with respect to their off-tumor expression bioinformatically by
using RNA and protein expression databases. The specificity of these target candidates was
further validated on primary PDAC specimens using flow cytometry and a newly developed
cyclic immunofluorescence (cyclic IF) microscopy technology. We identified Cutaneous
Lymphocyte Antigen (CLA), CEACAM6 (CD66c), CDCP1 (CD318) and Tetraspanin-8
(TSPAN8) as having high specificity to tumor cells and restricted off-tumor expression. In
addition, we investigated the expression of these targets using flow cytometry and cyclic IF
microscopy on healthy tissue arrays. Target expression on healthy tissues was very restricted,
which further underlined the quality of the novel target candidates. We generated CAR
constructs differing in spacer length (XS, S, M, L) and scFv orientation (Vh-Vl, Vl-Vh) for each
target candidate and thoroughly validated these CARs in co-culture assays that utilized
cytotoxicity, activation marker expression and cytokine release as readouts. All CLA CARs
were dysfunctional due to self-antigenicity on activated T cells. The most promising constructs
for the other target candidates were evaluated in two pre-clinical PDAC mouse models. This
revealed CD318 Vh-Vl XS, CD66c Vl-Vh XS and TSPAN8 Vh-Vl S as the most effective CAR
constructs of the remaining targets. During CAR development we observed inferior in vivo
functionality of long IgG-based spacers compared to their in vitro performance. It has been
shown that these spacers convey unspecific binding in murine models. However, long spacers
are regarded as beneficial when targeting membrane proximal epitopes. Thus, we evaluated a
novel class of long spacers, derived from the SIGLEC family, with similar structural attributes
as the IgG family but lack the natural unspecific binding sites. In non-solid and solid tumor
settings, a novel SIGLEC-4 based spacer demonstrated superior functionality over long IgG
spacers in vitro and in vivo with favorable activation marker expression and cytokine release
for putative subsequent clinical applications. In summary, this project led to the identification
of novel target candidates for PDAC and establishment of a set of highly functional targetspecific
CARs that may pave the road for later clinical application and hopefully will help
PDAC patients by establishing more efficient treatment options in the future. | de |