SOLUBLE T-CELL ANTIGEN RECEPTORS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 461

FIGURE 31.2 Detection of p53 (aa264-272)/HLA-A 0201 complexes on formalin-fixed paraffin-
embedded tissue microarrays (TMAs) from normal or cancerous human tissues with STARMultimer.
(A) TMAs from normal colorectal or breast tissues were stained with HRP-conjugated 264scTCR
multimer. (B) TMAs from cancerous colorectal and breast tissues stained with HRP-conjugated
264scTCR multimer, negative control HRP-conjugated CVMscTCR multimer, anti-HLA-A2 Ab, or
anti-p53 Ab (magnification 200). The results show differential staining of p53 þ tumor specimens
compared to normal tissue by the p53-specific STAR Multimer. Source: Reproduced with the
publisher's permission from Reference [15].

31.3.3 Diagnostic and Research Applications

cells on a per cell basis during the course of HIV infection.
The results of these studies in conjunction with evolving
knowledge of CD8 þ T-cell responses and HIV escape from
T-cell immunity should aid in the selection of the most
relevant HIV targets for vaccine development. Successful
application of this approach could also be used for other
viruses and infectious agents to more fully understand the
antigen presentation in regard to optimization of vaccine
design.
We have found that STAR reagents are capable of
detecting as little as 300 pMHC complexes per cell by
flow cytometry and can distinguish peptide antigens that
differ by a single amino acid [15]. Thus, STAR Multimers
have the specificity and sensitivity to characterize peptide
antigen presentation in different experimental settings in
much the same way pMHC tetramers have been used to
characterize antigen-specific T-cell populations. We have
also found that these regents can be readily used to evaluate
pMHC complexes on the surface of APCs and dendritic cells
and could have utility in characterizing autologous cellular
immunotherapies, such as the recently approved prostate
cancer vaccine, Provenge 1 . Current methods to characterize
the potency of such cell-based therapies have relied on T-cell
response assays or surrogate assessment of APC activation
[39]. With sensitivity comparable to T cell-based methods,
STAR Multimer staining could provide a more direct and
quantitative approach to optimizing and validating the pro-
duction of APC-based vaccines.
Based on the unique binding capabilities, we have been
offering STAR Multimers to the research community to
expand the utility and application of these reagents in other
areas of interest. For example, we have created a STAR
Multimer reagent capable of detecting adeno-associated
virus 2 (AAV) caspid peptide/HLA-B 0702 complexes on

In addition to their potential as targeted therapeutics, STAR
molecules have utility as diagnostic or research reagents. As
described earlier, we have found that multimeric forms of
STAR molecules can be used to specifically detect endog-
enous peptide antigens displayed in the context of MHC
molecules on antigen-presenting cells (APCs); cancer cells
and tissues; and virally infected cells, including formalin-
fixed, paraffin-embedded tissue, and microarray specimens.
Additionally, we have developed simple and sensitive stain-
ing procedures using STAR reagents to differentiate human
tumors from normal tissues (Figure 31.2) [15]. These
methods are being employed in the clinical development
of ALT-801 to identify patients whose tumor biopsies are
positive for p53 (aa264-272)/HLA-A 0201 complexes.
Thus, STAR reagents can be used to select cancer patients
who would most likely to receive benefit from targeted
therapies. This approach is commonly used with anti-
body-based diagnostic methods, such as the HercepTest TM
for determination of HER2 protein overexpression in breast
cancer patients prior to selection of Herceptin TM treatment.
We are also exploring use of STAR reagents as tools for
viral vaccine development. Current methods to examine
presentation of peptide antigens derived from HIVand other
viruses are not fully quantitative and cannot evaluate events
in real time or on an individual cell basis. As a result, there is
no clear consensus as to which viral peptide antigens are
actually presented to the immune system at different stages
of infection. With our panel of viral peptide-specific STAR
reagents and simple cytometry methods, we can directly
quantitate peptide antigen presentation on HIV-infected
cells. Efforts are underway to use these methods to fully
evaluate the levels and timing of HIVantigen presentation in