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1.
Sci Immunol ; 6(62)2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417258

RESUMO

Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)-like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4+ T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença Celíaca/imunologia , Glutens/imunologia , Antígenos HLA-DQ/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Epitopos de Linfócito T/imunologia , Glutens/química , Antígenos HLA-DQ/química , Humanos , Ativação Linfocitária/imunologia , Camundongos , Modelos Moleculares , Peptídeos/química , Receptores de Antígenos de Linfócitos T/química
2.
Vaccine ; 39(11): 1583-1592, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33612340

RESUMO

Targeted delivery of antigen to antigen-presenting cells (APCs) enhances antigen presentation and thus, is a potent strategy for making more efficacious vaccines. This can be achieved by use of antibodies with specificity for endocytic surface molecules expressed on the APC. We aimed to compare two different antibody-antigen fusion modes in their ability to induce T-cell responses; first, exchange of immunoglobulin (Ig) constant domain loops with a T-cell epitope (Troybody), and second, fusion of T-cell epitope or whole antigen to the antibody C-terminus. Although both strategies are well-established, they have not previously been compared using the same system. We found that both antibody-antigen fusion modes led to presentation of the T-cell epitope. The strength of the T-cell responses varied, however, with the most efficient Troybody inducing CD4 T-cell proliferation and cytokine secretion at 10-100-fold lower concentration than the antibodies carrying antigen fused to the C-terminus, both in vitro and after intravenous injection in mice. Furthermore, we exchanged this loop with an MHCI-restricted T-cell epitope, and the resulting antibody enabled efficient cross-presentation to CD8 T cells in vivo. Targeting of antigen to APCs by use of such antibody-antigen fusions is thus an attractive vaccination strategy for increased activation of both CD4 and CD8 peptide-specific T cells.


Assuntos
Linfócitos T CD4-Positivos , Epitopos de Linfócito T , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos , Linfócitos T CD8-Positivos , Camundongos
3.
JCI Insight ; 2(17)2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28878121

RESUMO

Selection of biased T cell receptor (TCR) repertoires across individuals is seen in both infectious diseases and autoimmunity, but the underlying molecular basis leading to these shared repertoires remains unclear. Celiac disease (CD) occurs primarily in HLA-DQ2.5+ individuals and is characterized by a CD4+ T cell response against gluten epitopes dominated by DQ2.5-glia-α1a and DQ2.5-glia-α2. The DQ2.5-glia-α2 response recruits a highly biased TCR repertoire composed of TRAV26-1 paired with TRBV7-2 harboring a semipublic CDR3ß loop. We aimed to unravel the molecular basis for this signature. By variable gene segment exchange, directed mutagenesis, and cellular T cell activation studies, we found that TRBV7-3 can substitute for TRBV7-2, as both can contain the canonical CDR3ß loop. Furthermore, we identified a pivotal germline-encoded MHC recognition motif centered on framework residue Y40 in TRAV26-1 engaging both DQB1*02 and the canonical CDR3ß. This allowed prediction of expanded DQ2.5-glia-α2-reactive TCR repertoires, which were confirmed by single-cell sorting and TCR sequencing from CD patient samples. Our data refine our understanding of how HLA-dependent biased TCR repertoires are selected in the periphery due to germline-encoded residues.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Códon , Regiões Determinantes de Complementaridade/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Doença Celíaca/imunologia , Células Clonais , Clonagem Molecular , Epitopos de Linfócito T/imunologia , Glutens/imunologia , Antígenos HLA-DQ/imunologia , Humanos , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T alfa-beta/genética
4.
Front Oncol ; 4: 378, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25629004

RESUMO

Cancer immunotherapy has finally come of age, demonstrated by recent progress in strategies that engage the endogenous adaptive immune response in tumor killing. Occasionally, significant and durable tumor regression has been achieved. A giant leap forward was the demonstration that the pre-existing polyclonal T cell repertoire could be re-directed by use of cloned T cell receptors (TCRs), to obtain a defined tumor-specific pool of T cells. However, the procedure must be performed with caution to avoid deleterious cross-reactivity. Here, the use of engineered soluble TCRs may represent a safer, yet powerful, alternative. There is also a need for deeper understanding of the processes that underlie antigen presentation in disease and homeostasis, how tumor-specific peptides are generated, and how epitope spreading evolves during tumor development. Due to its plasticity, the pivotal interaction where a TCR engages a peptide/MHC (pMHC) also requires closer attention. For this purpose, phage display as a tool to evolve cloned TCRs represents an attractive avenue to generate suitable reagents allowing the study of defined pMHC presentation, TCR engagement, as well as for the discovery of novel therapeutic leads. Here, we highlight important aspects of the current status in this field.

5.
Sci Rep ; 3: 1162, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23362461

RESUMO

We here report a novel phage display selection strategy enabling fast and easy selection of thermostabilized proteins. The approach is illustrated with stabilization of an aggregation-prone soluble single chain T cell receptor (scTCR) characteristic of the murine MOPC315 myeloma model. Random mutation scTCR phage libraries were prepared in E. coli over-expressing the periplasmic chaperone FkpA, and such over-expression during library preparation proved crucial for successful downstream selection. The thermostabilized scTCR(mut) variants selected were produced in high yields and isolated as monomers. Thus, the purified scTCRs could be studied with regard to specificity and equilibrium binding kinetics to pMHC using surface plasmon resonance (SPR). The results demonstrate a difference in affinity for pMHCs that display germ line or tumor-specific peptides which explains the tumor-specific reactivity of the TCR. This FkpA-assisted thermostabilization strategy extends the utility of recombinant TCRs and furthermore, may be of general use for efficient evolution of proteins.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Biblioteca de Peptídeos , Peptidilprolil Isomerase/metabolismo , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Camundongos , Chaperonas Moleculares , Mieloma Múltiplo/genética , Peptidilprolil Isomerase/genética , Receptores de Antígenos de Linfócitos T/genética
6.
Nucleic Acids Res ; 40(16): e120, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22539265

RESUMO

Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems.


Assuntos
Bacteriófagos/genética , Vírus Auxiliares/genética , Biblioteca de Peptídeos , Animais , Antígenos/análise , Bacteriófagos/fisiologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Vírus Auxiliares/fisiologia , Camundongos , Receptores de Antígenos de Linfócitos T/análise
7.
J Immunol ; 181(10): 7062-72, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18981126

RESUMO

Targeting of T cell epitopes to APC enhances T cell responses. We used an APC-specific Ab (anti-IgD) and substituted either of 18 loops connecting beta strands in human IgG constant H (C(H)) domains with a characterized T cell peptide epitope. All Ab-epitope fusion molecules were secreted from producing cells except IgG-loop 2(BC)C(H)1, and comparing levels, a hierarchy appeared with fusions involving C(H)2 > or = C(H)1 > C(H)3. Within each domain, fusion at loop 6(FG) showed best secretion, while low secretion correlated with the substitution of native loops that contain conserved amino acids buried within the folded molecule. Comparing the APC-specific rAb molecules for their ability to induce T cell activation in vitro, the six mutants with epitope in C(H)2 were the most effective, with loop 4C(H)2 ranking on top. The C(H)1 mutants were more resistant to processing, and the loop 6C(H)1 mutant only induced detectable activation. The efficiency of the C(H)3 mutants varied, with loop 6C(H)3 being the least effective and equal to loop 6 C(H)1. Considering both rAb secretion level and T cell activation efficiency, a total of eight loops may carry T cell epitopes to APC for processing and presentation to T cells, namely, all in C(H)2 in addition to loop 6 in C(H)1 and C(H)3. Comparing loop 4C(H)2 with loop 6C(H)1 mutants after injection of Ab in BALB/c mice, the former was by far the most efficient and induced specific T cell activation at concentrations at least 100-fold lower than loop 6C(H)1.


Assuntos
Apresentação de Antígeno/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Regiões Constantes de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Ativação Linfocitária/imunologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Humanos , Regiões Constantes de Imunoglobulina/química , Imunoglobulina G/química , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia
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