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T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting.

ABSTRACT: Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8(+) T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A(27-35) (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8(+) T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8(+) T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201(+) individuals that were capable of efficient HLA A*0201(+) melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.

SUBMITTER: Ekeruche-Makinde J

PROVIDER: S-EPMC3481325 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting.

Ekeruche-Makinde Julia J Clement Mathew M Cole David K DK Edwards Emily S J ES Ladell Kristin K Miles John J JJ Matthews Katherine K KK Fuller Anna A Lloyd Katy A KA Madura Florian F Dolton Garry M GM Pentier Johanne J Lissina Anna A Gostick Emma E Baxter Tiffany K TK Baker Brian M BM Rizkallah Pierre J PJ Price David A DA Wooldridge Linda L Sewell Andrew K AK

The Journal of biological chemistry 20120905 44

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8(+) T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A(27-35) (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malign ...[more]

PMID: 22952231

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Project description:Molecular imaging and targeted radiotherapy with radiolabeled cholecystokinin-2 receptor (CCK2R) targeting peptide probes holds high promise to improve the clinical management of patients with metastatic medullary thyroid carcinoma and other CCK2R-expressing malignancies. Low stability and suboptimal targeting of currently available radiolabeled peptide analogs has prompted us to seek new stabilization strategies. In this study, we present a new minigastrin analog with site-specific C-terminal modifications showing a highly optimized targeting profile. Methods: DOTA-D-Glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2 (DOTA-MGS5) radiolabeled with 111In, 68Ga, and 177Lu was evaluated in extensive in vitro stability studies. For 177Lu-DOTA-MGS5, additional metabolic studies were performed on BALB/c mice. Receptor affinity and cell uptake were studied in A431 human epidermoid carcinoma cells transfected with human CCK2R (A431-CCK2R), as well as the same cell line transfected with the empty vector (A431-mock). A431-CCK2R/A431-mock xenografted athymic BALB/c nude mice were used for biodistribution studies and small-animal SPECT/CT. Results: DOTA-MGS5 radiolabeled with 111In and 177Lu showed a highly increased stability against enzymatic degradation in different media up to 24 h of incubation. Similar results were observed for 68Ga-DOTA-MGS5 incubated up to 4 h. In the blood of mice injected with 177Lu-DOTA-MGS5, at least 70% intact radiopeptide was detected up to 1 h after injection. The unlabeled peptide and the complexes with the natural isotopes showed retained receptor affinity, and the radiopeptides showed unexpectedly high cell uptake in A431-CCK2R cells (>60% at 4 h). Regardless of the radiometal used for labeling, impressively high uptake in A431-CCK2R xenografts was found (∼20% injected activity/g 1-4 h after injection), whereas the uptake in A431-mock xenografts was negligible. Low background activity and favorable tumor-to-kidney ratios (4-6) allowed for high image contrast in small-animal SPECT/CT. Conclusion: The excellent targeting properties of DOTA-MGS5 support future clinical studies evaluating the diagnostic and therapeutic potential in patients with progressive or metastatic medullary thyroid carcinoma, as well as other advanced-stage CCK2R-expressing malignancies.

Project description:Upon antigen recognition via their B cell receptor (BR), B cells migrate to the germinal center where they undergo somatic hypermutation (SHM) to increase their affinity for the antigen, and class switch recombination (CSR) to change the effector function of the secreted antibodies. These steps are essential to create an antigen-experienced BR repertoire that efficiently protects the body against pathogens. At the same time, the BR repertoire should be selected to protect against responses to self-antigen or harmless antigens. Insights into the processes of SHM, selection, and CSR can be obtained by studying the antigen-experienced BR repertoire. Currently, a large reference data set of healthy children and adults, which ranges from neonates to the elderly, is not available. In this study, we analyzed the antigen-experienced repertoire of 38 healthy donors (HD), ranging from cord blood to 74 years old, by sequencing IGA and IGG transcripts using next generation sequencing. This resulted in a large, freely available reference data set containing 412,890 IGA and IGG transcripts. We used this data set to study mutation levels, SHM patterns, antigenic selection, and CSR from birth to elderly HD. Only small differences were observed in SHM patterns, while the mutation levels increase in early childhood and stabilize at 6 years of age at around 7%. Furthermore, comparison of the antigen-experienced repertoire with sequences from the naive immune repertoire showed that features associated with autoimmunity such as long CDR3 length and IGHV4-34 usage are reduced in the antigen-experienced repertoire. Moreover, IGA2 and IGG2 usage was increased in HD in higher age categories, while IGG1 usage was decreased. In addition, we studied clonal relationship in the different samples. Clonally related sequences were found with different subclasses. Interestingly, we found transcripts with the same CDR1-CDR3 sequence, but different subclasses. Together, these data suggest that a single antigen can provoke a B-cell response with BR of different subclasses and that, during the course of an immune response, some B cells change their isotype without acquiring additional SHM or can directly switch to different isotypes.

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T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting.

Publications

T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting.

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