Ii-Key/MHC class II epitope peptides as helper T cell vaccines for cancer and infectious disease.

Abstract

Potent MHC class II antigenic peptide vaccines are created by covalently linking the N-terminus of a MHC class II epitope through a polymethylene bridge to the C-terminus of the Ii-Key segment of the Ii protein. Such hybrids enhance potency of presentation in vitro of the MHC class II epitope about 200 times relative to the epitope-only peptide. In vivo, as measured by IFN-gamma ELISPOT assays, the helper T cell response to vaccination is enhanced up to 8 times. The design of such hybrid vaccine peptides comes from insight into the mechanism of action of the Ii-Key motif within the Ii protein, in regulating antigenic peptide binding into the antigenic peptide binding groove of MHC class II molecules. Here we present the logic and experimental history of the development of these vaccine peptides, with particular attention to the hypothesized mechanism of action. Methods for the design and testing of these peptides are presented. Experience in developing peptide vaccines for immunotherapy of cancer is reviewed, focusing on the clinical potential of Ii-Key/MHC class II epitope hybrids.

Full text

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[Frontiers in Bioscience 11, 46-58, January 1, 2006]
46
Ii-Key/MHC class II epitope peptides as helper T cell vaccines for cancer and infectious disease
Nikoletta L. Kallinteris, Douglas Powell, Catherine E. Blackwell, MaryBeth Kim, Xueqing Lu, Shuzhen Wu, Robert E.
Humphreys, Minzhen Xu, and Eric von Hofe
Antigen Express, Inc., 100 Barber Ave., Worcester, MA 01606-2478
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Discovery of the function of the Ii-Key segment of the Ii protein to regulate tightness of binding of antigenic peptides into MHC
class II molecules
3.1. Logic and experimentation leading to potent Ii-Key/MHC class II epitope hybrid vaccine peptides
3.2. Hypothesis on the mechanism of action of Ii-Key/MHC class II epitope hybrid peptides
4. Do Ii-Key motifs occurring naturally in antigenic proteins regulate selection among their MHC class II epitopes?
5. Design of Ii-Key/melanoma gp100 (46-58) MHC class II hybrids
5.1. Algorithm and rationale for the design of Ii-Key/MHC class II epitope hybrid vaccine peptides
5.2. Preference for polymethylene chain versus residues of the native sequence N-terminal to the P1 site residue
5.3. Protease protection
5.4. Enhanced solubility
5.5. Limiting autorelease of hybrids by use of shorter spacers
6. Use of Ii-Key MHC Class II epitope hybrids as anti-cancer helper T cell vaccines
6.1. Ii-Key enhances in vivo priming of CD4+ T cells to the gp100(46-58) epitope in DR4 transgenic mice
6.2. Ii-Key enhances in vitro priming of CD4+ T cells by the Tyr(365-381) melanoma epitope in normal donor
lymphocytes
6.3. Ii-Key enhances in vitro priming of CD4+ T cells by the promiscuous HER-2/neu (776-790) epitope in normal
donor lymphocytes
6.4. Conclusions from experimental studies
7. Clinical trials with Her-2/neu MHC Class II epitope peptides
7.1. T helper stimulation and cancer immunotherapy
7.2. Active HER-2/neu immunotherapy: limitations of vaccinating with only MHC class I epitopes
7.3. Active peptide immunotherapy for melanoma
8. Summary and Perspective
9. Acknowledgement
10. References
1. ABSTRACT
Potent MHC class II antigenic peptide vaccines
are created by covalently linking the N-terminus of a MHC
class II epitope through a polymethylene bridge to the C-
terminus of the Ii-Key segment of the Ii protein. Such
hybrids enhance potency of presentation in vitro of the
MHC class II epitope about 200 times relative to the
epitope-only peptide. In vivo, as measured by IFN-γ
ELISPOT assays, the helper T cell response to vaccination
is enhanced up to 8 times. The design of such hybrid
vaccine peptides comes from insight into the mechanism of
action of the Ii-Key motif within the Ii protein, in
regulating antigenic peptide binding into the antigenic
peptide binding groove of MHC class II molecules. Here
we present the logic and experimental history of the
development of these vaccine peptides, with particular
attention to the hypothesized mechanism of action.
Methods for the design and testing of these peptides are
presented. Experience in developing peptide vaccines for
immunotherapy of cancer is reviewed, focusing on the
clinical potential of Ii-Key/MHC class II epitope hybrids.
2. INTRODUCTION
Improving immune responses of helper T cells to
tumor antigens by vaccinating with peptides from those
antigens containing MHC class II epitopes could enable
curative immunotherapies for many cancers. Stronger
helper T cell responses induce better cytotoxic T
lymphocytes (CTL), antibody
immunological memory. Today, vaccinating with peptides
containing MHC class II epitopes is not very effective
clinically compared to vaccinating with MHC class I
epitope-containing peptides. MHC class II peptides have
tight binding affinities for cell surface MHC class I
molecules, for presentation to CTL. In contrast, MHC class
II peptides much be present at much higher concentrations
to become substituted into cell surface MHC class II
molecules for presentation to helper T cells. Our discovery
centers on a method to overcome the weakness of
presentation of MHC class II epitope vaccine peptides.
responses, and
MHC class II epitopes are normally bound to
MHC class II molecules in a post-Golgi, antigenic peptide

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Ii-Key hybrid Helper T cell vaccine
47
binding compartment of antigen presenting cells (dendritic
cells, macrophages and B cells). The Ii protein, which is
coded by only one gene in all humans, forms a trimer at
synthesis in the endoplasmic reticulum. Three dimers of
MHC class II α and β chains are bound to that trimer, and
the complex is transported intracellularly to the antigenic
peptide-charging compartment. There the Ii protein is
cleaved by proteases, as are the internalized antigens, and
antigenic peptides are bound into the antigenic peptide
binding site. The CLIP (cleaved leupeptin-induced peptide)
peptide of the Ii protein remains on some MHC class II
molecules and might facilitate charging of MHC class II
molecules with antigenic peptides.
A second immunoregulatory peptide, called the
Ii-Key peptide, also regulates antigenic peptide binding and
release of antigenic peptides, even on cell surface-
expressed MHC class II molecules. Linking an Ii-Key core
sequence covalently through a simple polymethylene
spacer to an antigenic epitope creates potent Ii-Key/MHC
class II epitope hybrid peptide vaccines. For example,
linking the Ii-Key moiety LRMK to the melanoma
gp100(48-58) MHC class II epitope significantly enhances
the vaccine response to that epitope in DR4-IE transgenic
mice. In ELISPOT assays on splenic lymphocytes of those
mice both the number of responding cells and cytokine
output per cell are increased. Frequently, the most effective
vaccine hybrid is the one with a shorter linker between Ii-
Key and the epitope. Mechanistic reasons for this
observation are considered. Ii-Key/MHC class II epitope
hybrid peptide vaccines induce strong Th1 responses to
various MHC class II epitopes from HER-2/neu, melanoma
gp 100 and tyrosinase, HIV gp120, Gag and Nef, and
influenza H5 hemagglutinin. Such hybrids can be applied
to many diagnostic and therapeutic uses for cancer and
infectious diseases, and to modify allergy or autoimmune
disease.
3. DISCOVERY OF THE FUNCTION OF THE Ii-
KEY SEGMENT OF THE Ii PROTEIN TO
REGULATE TIGHTNESS
ANTIGENIC PEPTIDES INTO MHC CLASS II
MOLECULES
OF BINDING OF
The characterization of the biological function of
the Ii-Key segment of the Ii protein to regulate antigenic
peptide charging into MHC class II molecules occurred in
stages over many years. From the initial hypothesis that Ii
protein blocked the MHC class II binding site (1) many
experiments were executed to characterize the changing
structure of Ii protein in association with MHC class II
molecules (2-7). In particular, cleavage and release of Ii
fragments (8-10) led to experiments to determine whether
antigenic peptides are bound in a concerted fashion with
the release of Ii peptide from the MHC class II molecules
(11). An unusual sequence of Ii protein was evaluated as a
signal to other molecules to regulate the cleavage and/or
peptide charging events (12). Almost by accident, the
function of that peptide was discovered to enhance
charging of antigenic peptides to MHC class II molecules
(13). Recent progress has focused on optimal design and
clinical application of the hybrid, joining the core of the Ii-
Key segment through a polymethylene bridge to the N-
terminus of MHC class II antigenic epitopes (14). The
continuing questions focus on optimizing structure of such
hybrids, especially when the peptide with the antigenic
epitope contains more than one closely overlapping
epitopes. In addition, we are examining the role of the
hybrids in enhancing selectively Th1 or Th2 responses, and
approaches toward breaking tolerance in cancer patients.
3.1. Logic and experimentation leading to potent Ii-
Key/MHC class II epitope hybrid vaccine peptides
A novel mechanism for boosting responses to
MHC class II epitope vaccine peptides exploits a MHC
class II molecule regulatory allosteric site, which governs
tightness of binding of MHC class II epitope peptides. The
normal process of MHC class II antigen charging and
presentation is highly controlled, in order to assure fidelity
in presentation of selected peptides. The Ii protein
associates with MHC class II molecules at synthesis in the
endoplasmic reticulum and prevents their charging with
endogenous peptides, which are otherwise destined for
binding to MHC class I molecules. After the MHC class II
molecule complexes have been transported into a post-
Golgi antigenic peptide-charging compartment, the Ii
protein is digested away allowing exogenous peptides to
bind into the antigenic peptide-binding site of MHC class II
molecules (10, 11, 15, 16).
A critical role of the Ii-Key motif in the Ii protein
is during the process of Ii protein digestion and in the
concerted release of fragments from the MHC class II
molecules and antigenic peptide charging. The binding of
radiolabeled, photo-crosslinking, antigenic peptides to
MHC class II molecules is more efficient during the
cleavage and release of the Ii protein from MHC class II
alpha and beta chains in the presence of cathepsin B but not
cathepsin D (11). Mutants of putative cleavage sites in the
Ii protein confirm the role of residues in the R78-K80-K83-
K86 region (within the sequence of the Ii-Key peptide), in
the final cleavage and release of the avidin-labeled Ii
fragments that are still immunoprecipitated with MHC
class II alpha and beta chains using anti-MHC class II
antibodies (15). MHC class II protein adherence of the Ii-
Key motif segment of the Ii protein (murine Ii(77-92)
sequence: LRMKLPKSAKPVQMR; termed “Ii-Key”),
appears to regulate lability of the antigenic peptide binding
site on MHC class II molecules to accept antigenic
peptides. Proteolytic cleavage in or around R78-K80-K83-K86
in the Ii-Key motif of Ii protein or cleaved fragments which
still adhere to MHC class II molecules, terminates the
effect on binding site lability. Nevertheless, the Ii-Key
allosteric site on the MHC class II molecules remains
intact, even on the cell surface as judged by the activity of
Ii-Key peptides on paraformaldehyde-fixed antigen
presenting cells (13). However, there are few ambient
peptides capable of acting at the Ii-Key allosteric site on
cell surface MHC class II molecules to release antigenic
peptides for two reasons. First, such sequences
(hydrophobic-cationic side chains motifs) are targeted for
proteases. Secondly, the motif in the Ii-Key regulatory
segment of the Ii protein, XOXOX where X is a member of
the group LIVFM and O is any other amino acid, is highly

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Ii-Key hybrid Helper T cell vaccine
48
Table 1. Alignment of Ii-Key peptide based on crystallographic images with Ii-CLIP and HA(306-318) antigenic peptide in
HLA-DR4
Binding groove positions 123456789
M91
Human Ii CLIP(Ii81-99)
LPKPPKPVSK MRMATPLLM
Human Ii-Key (Ii77-92)
LRMKLPKPPKPVSK MR
Murine Ii CLIP(Ii81-98)
LPKPPKPVSK MRMATPLLM
Murine Ii-Key (Ii77-92)
LRMKLPKSAKPVSQ MR
Core Ii-Key (77-83)
LRMKLPK
Influenza HA (306-318)
PK YVKQNTLKLAT
The linearly aligned relationships among Ii-Key, CLIP and MHC class II epitope (HA306-318) sequences, relative to MHC class
II binding groove, as deduced from X-ray crystallographic images (19).
suppressed (P < 0.005) in proteins in general (17). That is,
the Ii-Key regulatory motif evolved from a sequence which
is very rarely found in natural proteins. That is, the chance
that a sequence would act on cell surface MHC class II
molecules to release bound antigenic peptides is very low.
(Table 1).
In studying the role of fragments of the Ii protein
generated during the staged cleavage and release of Ii
during antigenic peptide binding to MHC class II
molecules, we synthesized the murine Ii-Key peptide (18).
This peptide was synthesized because it contains six
positively charged side chains, no negatively charged side
chains, many alternating hydrophobic residues, and four
prolines. This cluster of residue suggested a tightly kinked,
positively charged knob which might attract a protease or
“exchangease” to the MHC class II alpha,beta,Ii trimer.
Antibodies to the peptide recognized the peptide and
denatured Ii protein, but did not recognize the Ii protein in
nonionic detergent-solubilized trimers.
Subsequently, this peptide was found to enhance
greatly in vitro presentation of I-E-restricted antigenic
peptides to responding T hybridomas (1). This “Ii-Key”
peptide enhanced in vitro presentation of antigenic peptides
by living or paraformaldehyde-fixed antigen presenting
cells to murine T-cell hybridomas (20). Nested C-terminal
deletions of Ii-Key showed maximal activity in the N-
terminal Ac-LRMKLPK-NH2, with half-maximal activity
being retained with Ac-LRMK-NH2. Structure-activity
relationship studies of 160 homologs revealed many
homologs with significantly greater activity than the
original 15-amino acid peptide (21). The mechanism was
explored further by assaying binding or release of
biotinylated hMBP(90-102) from purified exomembranal
human MHC class II molecules. With purified soluble
exomembranal HLA-DR1 protein, the existence of an
allosteric site was indicated by competitive binding
experiments with biotinylated Ii-Key peptides or antigenic
peptides (22). These experiments support the model that
acting through an allosteric site on HLA-DR1 molecules,
Ii-Key regulates the binding of a second, epitope-only
hMBP(90-102) peptide to HLA-DR1 molecules (Table 2).
The potency of presentation of an antigenic
epitope from pigeon cytochrome C, PGCC(95-104), was
enhanced about 200 times in vitro when the N-terminus of
the antigenic peptide was linked covalently through a
simple chemical bridge to the C-terminus of the Ii-Key
peptide, forming an Ii-Key/antigenic epitope hybrid (14). In
mouse immunizations, the Ii-Key/HIV Gag(46-59) hybrid
significantly enhanced the potency of the Gag epitope as
ELISPOT-measured T-cell IFN-gamma responses (23). In
addition, an Ii-Key/HER-2/neu MHC class II epitope
peptide induced much greater IFN-gamma release
fromperipheral blood mononuclear cells of breast cancer
patients than did the comparable HER-2/neu MHC class II
epitope-only peptide (24) (Table 3, Figure 1).
3.2. Hypothesis on the mechanism of action of Ii-
Key/MHC class II epitope hybrid peptides
Our experiments support the view that Ii-Key
peptides act at an allosteric site on MHC class II molecules
to facilitate charging of vaccine peptides into the antigenic
peptide binding site and their presentation. The allosteric
site appears to be located just a few amino acids away from
the end of the groove holding the N-terminus of the
antigenic peptide. Binding of a ligand to the allosteric site
induces a conformational change in the antigenic peptide-
binding groove such that it adopts a more accessible
conformation. The allosteric-site ligand, with a lesser
binding affinity than the antigenic peptide for the antigenic
peptide-binding groove (25), dissociates to allow
stabilization of the MHC class II/epitope complex (“the
clamshell closes”). Our results are consistent with those of
others. Hammerling’s group (26) has demonstrated that the
N-terminal segment of CLIP (81-91) loosens the binding
groove and releases the core CLIP (81-105) and other self
epitopes from the groove. Without CLIP (81-91), the core
CLIP (91-105) cannot be released from the groove even at
acidic conditions. Their results indicate that CLIP (81-91)
may bind at a site, which is adjacent to the N-terminus of
the binding groove. They have also shown that core CLIP
(91-105) binds to the groove and competes with antigenic
peptides. This was also predicted by Rammensee et al.from
a complete list of MHC class I and class II epitopes known
at the time (27). X-ray crystallographic images of the Ii-
derived peptide CLIP in the antigenic peptide binding
groove of MHC class II molecules, indicates that
methionine91 (M91) binds to the P1 site of the groove (21).
If Ii-Key, Ii protein and CLIP all maintain their registry
with respect to the primary amino acid sequence, Ii-Key
can be expected to lay outside the antigenic peptide binding
groove. The relative alignments among Ii-Key, CLIP and
the crystallographic placement
hemagglutinin antigenic peptide (HA) (19) in the peptide
binding groove of MHC class II molecules is illustrated in
Figure 2.
of an influenza

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Ii-Key hybrid Helper T cell vaccine
49
Table 2. Activities of Ii-Key homologs
SequenceResponse
Ed
1.0
13.6
13.3
3.4
2.6
4.5
16.9
21.7
32.0
39.3
47.1
39.2
42.8
36.3
39.8
19.9
7.1
2.3
1.0
Ek
1.0
1.0
1.0
0.7
0.8
0.7
2.0
1.0
1.2
6.9
7.6
7.2
15.3
15.5
15.9
18.6
15.5
14.6
5.6
YRMKLPKSAKPVSQMR
RMKLPKSAKPVSQMR
KLPKSAKPVSQMR
PKSAKPVSQMR
SAKPVSQMR
YRMKLPKSAKPVSQ
YRMKLPKSAKPV
YRMKLPKSAK
Ac-YRMKLPKSAK-NH2
Ac-LRMKLPKSAK-NH2
Ac-YRMKLPKSA-NH2
Ac-YRMKLPKS-NH2
Ac-YRMKLPK-NH2
Ac-LRMKLPK-NH2
Ac-YRMKLP-NH2
Ac-YRMKL-NH2
Ac-YRMK-NH2
Ac-YRM-NH2
Ac-YRMKLP-NH2 (Ed) and Ac-YRMK-NH2 (Ek) were the shortest, most active peptides in the truncation series. N- and C-terminally
blocked peptides were more active than their unblocked counterparts. For these assays, MHC class II-positive APC, treated with
mitomycin C, were incubated for 24 h with antigenic peptide-specific T cell hybridomas, at a submaximal dose of the respective
antigenic peptide, and different concentrations of the series of Ii peptides. The concentrations of antigenic peptides used were 0.4 µmol/L
of HEL(l06-116) for Ed and 0.075 µmol/L THMCC(82-103) for Ek. The presented data were generated using 64 µmol/L of each Ii
peptide. Interleukin released by the T hybridoma cells was quantified by [3H]TdR incorporation by interleukin-dependent HT-2 cells,
using aliquots of culture supernatant. The values presented, 'Times Baseline Response', equaled the CPM of (T + APC + Ag peptide +
hybrid series peptide)/ CPM of (T + APC + epitope-only peptide). The means of triplicate wells had an average SEM of 10%. The T cell
response to antigenic peptide alone ('None') was designated as the baseline value 1.0. These data led to the choice of Ii-Key “core” motifs
of 4 or 7 amino acids for assay in the Ii-Key/MHC class II hybrids of Table 3. Figure and table with permission from 20,21.
Table 3. Biological activities of Ii-Key/MHC class II epitope hybrid peptides with variable spacers between the Ii-Key core motif
and an antigenic epitope
Figure 1. Potency of Ii-Key Hybrids. The immunological response to the antigenic epitope measured by tritiated thymidine uptake (y
axis in thousands of counts/min), is presented as a function of the dilution factor of the hybrid (1:4 serial dilution from a 3 uM stock
solution). The symbols for the respective hybrids and their structures are presented in Table 3. These results show that a simple 4-
amino-acid Ii-Key motif with one of the shortest spacers has potent activity in this assay. Adapted with permission from 1.

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Ii-Key hybrid Helper T cell vaccine
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Figure 2. Direct charging of MHC class II molecules with Ii-Key hybrid vaccine. A: Ii-Key, a small domain on the Ii protein
(black), interacts with Ii lock, an allosteric site distal to the antigen binding groove of MHC class II molecules. B: Ii-Key hybrids
consist of an Ii-Key peptide linked to a therapeutic antigenic peptide ; when they bind to previously charged MHC class II
molecules, the resident antigen ischarged and the antigenic end of the hybrid occupies the antigen binding groove.
4. DO Ii-KEY MOTIFS OCCURRING NATURALLY
IN ANTIGENIC PROTEINS
SELECTION AMONG POTENTIAL MHC CLASS II
EPITOPES?
REGULATE
The presence of Ii-Key motifs N-terminal to a
potential MHC class II epitope in a protein antigen, might
enhance selection of such an epitope, relative to other MHC
class II epitopes without N-terminal Ii-Key motifs. In order
to evaluate this possibility, we counted the frequency of Ii-
Key motifs N-terminal and C-terminal to known MHC
class II epitopes, as a function of spacer lengths of 3 to 6
amino acids, in a series of classical antigens. In this study
the motif was defined arbitrarily to comprise a segment of 5
contiguous amino acids containing at least two amino acids
of the group comprising Leu, Ile, Val, Phe, and Met, and at
least one of the group comprising His, Lys, and Arg, where
that contiguous 5 amino acid segment is separated by 3 to 6
amino acids from the N-terminal residue of a MHC class II-
presented epitope. While a preliminary study indicated a
greater frequency of such appropriately spaced motifs N-
terminally to MHC class II epitopes than C-terminally, this
hypothesis is subject to further experimental testing.
There are several immediate practical values to
proving that naturally occurring Ii-Key motifs contribute to
the selection of MHC class II epitopes in an antigen. One
use is in prioritizing, and thus often reducing, the number
of candidate peptides selected for synthesis and testing.
Another is the ability to either reduce the immunogenicity
of a therapeutic protein or to enhance the immunogenicity
of an antigen. For example in the case of allergens, one
might actually wish to rebalance the immune response to a
Th1 phenotype by enhancing immunogenicity (28).
5. DESIGN OF Ii-KEY/MELENOMA gp100 (46-58)
MHC CLASS II HYBRIDS
A principal antigen for studying immunogenicity
is human gp100, which has been a good source of MHC
class I and MHC class II epitopes for therapeutic
vaccination trials (29-32). CD4+ lymphocytes from more
than 75 % of melanoma patients recognize gp100 (33), and
CD8+ tumor infiltrating T cells also recognize gp100 (34).
Some gp100 peptides stimulate lymphocytes of melanoma
patients who are disease-free after therapeutic intervention,
but not lymphocytes from healthy donors (32).
Immunization with both MHC class I- and MHC class II-
restricted epitopes from the same or different melanoma-
related antigens might increase the therapeutic efficacy of
CTL by activating intermediary dendritic cells (35).
We focused on a DR4 transgenic mouse model to
study the effect of Ii-Key/MHC class II epitope
hybridization on enhancement of vaccination with h-
gp100(46-58). Others had already found that immunization
of HLA-DR4-IE transgenic (Tg) mice with recombinant h-
gp100 protein followed by screening of candidate epitopes
(identified with a computer-assisted algorithm for HLA-
DRB1*0401-presented epitopes) identified h-gp100(46-58)
and its clinical relevance (29). We tested a homologous
series of Ii-Key/MHC class II hybrids with that epitope,
varying systematically the structure and length of the
spacer connecting the Ii-Key moiety and the MHC class II
epitope in order to identify optimal Ii-Key/melanoma
gp100(46-58) homologs for clinical trials. We found that
the Ii-Key moiety enhances the potency of the gp100(48-
58) MHC class II epitope in vivo in terms of epitope-
specific CD4+ T cell activation.
5.1 Algorithm and rationale for the design of Ii-
Key/MHC class II epitope hybrid vaccine peptides
Analysis of the gp100(46-58; RQLYPEWTEAQRL)
peptide using two computer epitope prediction programs,
(http://syfpeithi.bmiheidelberg.com/scripts/MHCServer.dll/home.html) and
(http://www.imtech.res.in/raghava/propred/index.html) indicated the
HLA-DR4-presented epitope to be LYPEWTEAQ (amino
acid L48 occupies the P1 site of MHC class II molecules). A
primary objective in the design of Ii-key/MHC class II
epitope hybrids was to determine the effects of spacer
length and requirements for natural sequence residues N-
terminal to the P1 site residue of the HLA-DR4-presented
epitope in gp100(46-58). Prior studies by others of MHC
class II epitope peptides generally have found a
requirement for non-epitope or “flanking” residues at both
the N- and C-terminal of the MHC class II epitope (36). In
order to evaluate the role of such additional “epitope
flanking” amino acids N-terminal to the P1 residue, we
synthesized hybrids extending the natural sequence at the