Download File - JOHN J. HADDAD, Ph.D.

Peptide-Based Active Immunotherapy in Cancer 119
skin and in peripheral blood lymphocytes in a HLA-A2þ melanoma patient. The
patient showed major regression of metastatic melanoma under continued
immunization with peptides derived from the antigens MART-1, tyrosinase, and
gp100 (66). The authors demonstrated that i.d. immunization with peptides
alone leads to oligoclonal expansion of MART-1-specific CTL. These findings
provide strong evidence for the effective induction of specific T-cell responses to
MART-1 by i.d. immunization with peptide alone, which accounts for specific
cytotoxicity against MART-1-expressing melanoma cells and clinical tumor
regression. Brinckerhoff et al. evaluated the stability of the same peptide—
MART-127–35—in fresh normal human plasma and possible peptide modifications
that convey protection against enzymatic destruction without loss of
immunogenicity (67). When this peptide was incubated in plasma prior to pulsing
on target cells, CTL reactivity was lost within three hours. The stability of
MART-127–35 was markedly prolonged by C-terminal amidation and/or N-terminal
acetylation, or by polyethylene-glycol modification of the C-terminus. These
modified peptides were recognized by CTL. This study suggests that the immunogenicity
of the peptide vaccines might be enhanced by creating modifications
that increase their stability.
We investigated the effectiveness of several synthetic insertion signal
sequences in enhancing the presentation of the HLA-A2.1-restricted melanoma
epitope MART-127–35 (68). An important step in presentation of the class
I-restricted antigens is the translocation of processed proteins from the cytosol
across the ER membrane mediated by TAP proteins, or as an alternative, by ERinsertion
signal sequences located at the NH 2-terminus of the precursor molecules
(69). Using a technique known as osmotic lysis of pinocytic vesicles (70),
we loaded several synthetic peptide constructs into the cytosol of antigen
processing deficient T2 cells, TAP-expressing human melanoma cells, and
dendritic cells. We examined whether the natural signal sequences ES (derived
from the adenovirus E3/19K glycoprotein) (71) and IS (derived from IFN-b) (72)
could enhance and prolong presentation of MART-127–35. We found that the
addition of signal sequence at the N-terminus, but not at the C-terminus, of
MART-1 27–35 greatly enhanced its presentation in both TAP-deficient and TAPexpressing
cells. A newly designed peptide construct, composed of the epitope
replacing the hydrophobic part of a natural signal sequence, was also effective.
Interestingly, an artificial signal sequence containing the epitope was the most
efficient construct for enhancing its presentation. These peptide constructs
facilitated epitope presentation in a TAP-independent manner when loaded into
the cytosol of TAP-deficient T2 cells. In addition, loading of these constructs
into TAP-expressing melanoma cells also led to a more efficient presentation
than loading of the minimal peptide. Most importantly, loading of human dendritic
cells with the same constructs resulted in a prolonged presentation of this
melanoma epitope (68). The efficient presentation of MART-127–35, loaded into
TAP-expressing tumor cells and dendritic cells, may be explained by the
availability of intact TAP transporters in these cells. In this case, some of the