Non-Natural and Photo-Reactive Amino Acids as
Biochemical Probes of Immune Function
Marta Go ´mez-Nun ˜ez1., Kurtis J. Haro1,2., Tao Dao1, Deming Chau1,2, Annie Won1,2, Sindy Escobar-
Alvarez1,2, Victoriya Zakhaleva1, Tatyana Korontsvit1, David Y. Gin1, David A. Scheinberg1*
1Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 2Weill Cornell Graduate
School of Medical Sciences, New York, New York, United States of America
Wilms tumor protein (WT1) is a transcription factor selectively overexpressed in leukemias and cancers; clinical trials are
underway that use altered WT1 peptide sequences as vaccines. Here we report a strategy to study peptide-MHC interactions
by incorporating non-natural and photo-reactive amino acids into the sequence of WT1 peptides. Thirteen WT1 peptides
sequences were synthesized with chemically modified amino acids (via fluorination and photo-reactive group additions) at
MHC and T cell receptor binding positions. Certain new non-natural peptide analogs could stabilize MHC class I molecules
better than the native sequences and were also able to elicit specific T-cell responses and sometimes cytotoxicity to
leukemia cells. Two photo-reactive peptides, also modified with a biotin handle for pull-down studies, formed covalent
interactions with MHC molecules on live cells and provided kinetic data showing the rapid clearance of the peptide-MHC
complex. Despite ‘‘infinite affinity’’ provided by the covalent peptide bonding to the MHC, immunogenicity was not
enhanced by these peptides because the peptide presentation on the surface was dominated by catabolism of the complex
and only a small percentage of peptide molecules covalently bound to the MHC molecules. This study shows that non-
natural amino acids can be successfully incorporated into T cell epitopes to provide novel immunological, biochemical and
Citation: Go ´mez-Nun ˜ez M, Haro KJ, Dao T, Chau D, Won A, et al. (2008) Non-Natural and Photo-Reactive Amino Acids as Biochemical Probes of Immune
Function. PLoS ONE 3(12): e3938. doi:10.1371/journal.pone.0003938
Editor: Jacques Zimmer, Centre de Recherche Public-Sante ´, Luxembourg
Received October 31, 2008; Accepted November 16, 2008; Published December 15, 2008
Copyright: ? 2008 Go ´mez-Nun ˜ez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH PO1 23766, RO1 55349, RO1 58833, the Ellen and Sandy Levin fund, the Lymphoma Foundation, The Glades fund, the
Experimental Therapeutics Center, and the Geoffrey Beene Center. This work was further supported by training grant #GM73546. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Specific T cell mediated immune responses involve T-
lymphocytes that respond to linear peptide epitopes, typically
between 8 and 20 amino acids in length. The peptides recognized
by CD8+ T cells are 8–10 amino acids in length and are presented
by class I major histocompatibility complex (MHC) molecules on
the target cells. Class I MHC molecules have an affinity preference
for peptides with particular major anchor residues, usually at
amino acid positions 2 and 9. The stability of the peptide/MHC
(pMHC) complexes correlates generally with the strength of the T
cell response to the epitope.
Most cancer antigens are ‘‘self-antigens’’ expressed on normal
cells and sometimes overexpressed on cancer cells. Immunogenic
peptides derived from these tumor-associated proteins have been
used in therapeutic vaccination protocols. Advances in the
understanding of the cellular immune response to peptide antigens
and structural studies of the pMHC have led to different strategies
for improving cancer vaccines. One frequently studied antigen is
the Wilms tumor protein (WT1), a zinc-finger transcription factor
expressed during normal ontogenesis [1,2,3]. In adults, WT1
expression is limited to low levels in the nuclei of normal CD34+
hematopoietic stem cells, myoepithelial progenitor cells, renal
podocytes and some cells in the testis and ovaries [4,5,6]. The
WT1 gene product is over-expressed in hematological [7,8,9] and
solid malignancies , making it an attractive target for
immunotherapy. Short peptides derived from WT1 protein have
been identified that generate a WT1-specific cytotoxic response
The feasibility of targeting WT1 has resulted in different clinical
trials using four peptides with different adjuvants in the context of
HLA-A0201 and HLA-A2402. Several trials are currently testing
the WT1 235–243 natural sequence and modified sequence
[17,18,19] in patients who are HLA-A2402; another set of studies
tested the WT1 126–134 natural sequence  and modified
peptides in patients who are HLA-A0201 .
Overcoming the often weak immunogenicity of and tolerance to
tumor antigens may require appropriate modifications in the
peptide sequence to increase pMHC or the T cell receptor (TcR)
TcR-pMHC interactions; such changes should induce a more
robust immune response if cross-reactivity to the native sequence
was produced. One strategy used by our group and others is the
design of synthetic analog peptides with natural amino acid
substitutions at the anchor positions in the MHC molecule
[18,19,21] that increase affinity, stimulate greater T cell
recognition or break tolerance. Here we propose a new strategy
PLoS ONE | www.plosone.org1 December 2008 | Volume 3 | Issue 12 | e3938
to incorporate non-natural amino acids and photo-reactive amino acids
into the CD8 peptide antigen. Incorporation of photo-reactive
amino acids into the peptide sequences may provide unique
opportunities to manipulate peptide-MHC binding interactions.
The added modification of a biotin handle on the peptide allows
kinetic studies of the peptide-MHC complex. Such non-natural
amino acids could also serve to improve affinity at the pMHC or
TcR recognition sites, break tolerance, or reduce catabolism of the
peptide. In the present study, the incorporated non-natural amino
acids into the sequence of WT1 peptides allowed us to determine if
these alterations would increase avidity of binding to MHC,
improve immunogenicity and result in cross-reactivity and
cytolytic activity against WT1 expressing cancer cells, and study
the MHC-peptide complex.
Materials and Methods
Potentially immunogenic peptides were selected from the
Wilms’ tumor (WT1) protein  (Table 1). Structures of the
non-natural amino acids are shown in Figure 1. Peptides WT1 B,
WT1B-S1Y, WT1B-S1V, WT1 B 8mer, WT1B-L2LF3, WT1 J,
WT1J-C1Y and WT1J-M2Y were synthesized by Genemed
Synthesis Inc. (CA, USA) using fluorenylmethoxycarbonyl chem-
istry, solid phase synthesis and purified by high-pressure liquid
chromatography. The quality of the peptides was assessed by high-
performance liquid chromatography analysis, and the expected
molecular weight was observed using matrix-assisted laser
desorption mass spectrometry. Peptides were sterile and greater
than 70% pure. The peptides were dissolved in DMSO and
diluted in phosphate-buffered saline (PBS; pH 7.4) or saline to give
a concentration of 5 mg/ml and were stored at 280uC.
We synthesized the peptides: WT1B-S1VF3, WT1B-S1FF2,
WT1B-S1FA-bio, WT1B-L2FF3,WT1B-S1Fbz-bio, WT1J–W4WF,
WT1J-C1Fbz(Table 1) using solid phase synthesis, on a Wang
resin via automated synthesizer (PS3TMPeptide Synthesizer,
Protein Technologies, Inc) using a single-coupling protocol. After
cleavage from the resin using Reagent R (TFA:thioanisole:ED-
T:anisole, 90:5:3:2), the peptides were purified by high-pressure
liquid chromatography (HPLC) on a reverse-phase C18 column
(ZORBAX 300SB-C189.4 mm625 cm, Agilent Technologies)
with a 0 to 98% gradient of acetonitrile containing 0.1%
trifluoroacetic acid (TFA). Peptide identity was verified by mass
spectrometry. Biotinylation was achieved by using a commercially
available Fmoc-L-glutamine amino acid containing biotin by way
of a polyethylene glycol linker (Calbiochem) during peptide
synthesis. Purities were greater than 90%.
Peptides WT1B-S1FA, and WT1J-C1FA (Table 1) were
synthesized by coupling the WT1B and WT1J 8-mers LGEQ-
QYSV and MTWNQMNL, respectively, to the F-moc-4-azido-L-
phenylalanine, using 0.4 M NMM/DMF as a coupling solution.
After removal of F-moc by adding 20% piperidine/DMF, final
deprotection and cleavage from the resin was done using Reagent
R. Peptide identity was verified by mass spectrometry.
TAP-deficient T2 cells were obtained from the American Type
Culture Collection (Rockville, MD). 697 CML cells (WT1+) and
SKLy-16 lymphoma cells (WT1-) were obtained from Sloan-
Kettering stocks. Cells were grown in RPMI 1640 medium
supplemented with 5% fetal bovine serum, 2 mM L-glutamine, b-
mercaptoethanol, penicillin (100 U/ml) and streptomycin (100 mg/
ml) ina humidified atmosphere containing 5%CO2inan incubator.
Peptide binding assay
Peptide binding to HLA-A0201 was assessed using a flow
cytometry-based MHC stabilization assay , on the T2 cell line,
which has HLA-A0201 expression and is TAP deficient in
Figure 1. Chemical structures of the non-natural amino acids
used in this study.
Table 1. Name and sequence* of the peptides.
Classification Of peptide Peptide name: ‘‘B’’ series Sequence: ‘‘B’’ seriesPeptide name: ‘‘J’’ series Sequence: ‘‘J’’ series
Native peptidesWT1BSLGEQQYSV WT1 J CMTWNQMNL
WT1B 8merLGEQQYSV WT1J 8mer MTWNQMNL
Heteroclitic peptidesWT1B-S1Y YLGEQQYSV WT1J-C1YYMTWNQMNL
Unnatural and photo-reactive
*FA=4-azido-L-phenylalanine; LF3=5,5,5-trifluoro-DL-leucine; VF3=4,4,4-trifluoro-DL-valine; FF2=3,4-difluoro-L-phenylalanine; WF=5-fluoro-DL-tryptophan; bio=biotin;
Non-Natural WT1 Epitopes
PLoS ONE | www.plosone.org2 December 2008 | Volume 3 | Issue 12 | e3938
WT1J. The assay was conducted as described in Material and
Methods, with each panel representing a different experiment.
Sequences of the peptides are shown in Table 1. The Y axis shows
the mean fluorescence or the binding Index, that is the ratio
between the median fluorescence with the peptide tested divided
by median fluorescence with irrelevant peptide. Mean fluorescence
was used for time course studies as the indices became low at the
later time points with loss of MHC. The X axis show the time-
points of incubation of the peptide tested or the different
concentrations of the peptide tested. 1 min refers to UV
irradiation of the peptide for 1 min after adding it to the cells.
T2 stabilization assay using peptides derived from
Found at: doi:10.1371/journal.pone.0003938.s001 (5.71 MB TIF)
WT1B. The assay was conducted as described in Fig. 1, with each
panel representing a different experiment. Sequences of the
peptides are shown in Table 1.
Found at: doi:10.1371/journal.pone.0003938.s002 (5.61 MB TIF)
T2 stabilization assay using peptides derived from
Conceived and designed the experiments: MGN KJH DYG DAS.
Performed the experiments: MGN KJH AW SEA VZ TK. Analyzed the
data: MGN KJH DAS. Contributed reagents/materials/analysis tools:
MGN KJH DC AW SEA. Wrote the paper: MGN KJH TD DYG DAS.
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Non-Natural WT1 Epitopes
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