Antibodies Directed Against the MHC-I Molecule H-2Dd Complexed with an Antigenic Peptide: Similarities to a T Cell Receptor with the Same Specificity

Poláková, K; Plaksin, Daniel; Chung, Doo Hyun; Belyakov, Igor M.; Berzofsky, Jay A.; Margulies, David H.

doi:10.4049/jimmunol.165.10.5703

Cited by 29 publications

(42 citation statements)

References 76 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Endogenous Antigen-Monoclonal antibodies have been used previously to examine the presentation of specific pMHC (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). Generation of such reagents is laborious and involves extensive screening of monoclonal antibodies, although some recent technical advances may facilitate this process (34,35).…”

Section: Tcr Multimers Can Sense Quantitative Modifications Of Antigementioning

confidence: 99%

Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition

Laugel

Boulter

Lissin³

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric ␣␤ TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virusinfected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax 11-19 epitope bound to HLA A2-HuD 87-95 (K D 120 M by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (K D 2.5 nM) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.Peptide major histocompatibility complex (pMHC) 1 antigens displayed on the surface of target cells are recognized by T cells via their specific T cell receptor (TCR) (1). The TCR coreceptors CD8 or CD4 bind to invariant domains of pMHC class I (pMHCI) or pMHC class II (pMHCII) and are known to facilitate the process of antigen recognition by T cells (2). Recent advances have enabled the generation of high quality soluble TCR, pMHCI, pMHCII, CD8, and CD4 proteins; these in turn have allowed the biophysical characterization of the interactions between these molecules. Accordingly, the TCR and CD4/8 co-receptors have been shown to have very low affinities (K D ϳ 10 Ϫ3 -10 Ϫ6 M) for cognate pMHC. Despite these low affinity interactions, however, the process of antigen engagement can initiate T cell recognition of antigen-presenting cells (APCs) bearing fewer than 10 copies of a specific pMHC complex (3, 4). The mechanisms by which these weak recognition events result in such exquisite sensitivity are not fully understood.The production of soluble recombinant ␣␤ T cell receptors has proved challenging. The main technical pitfall is heterodimeric instability in the absence of anchoring transmembrane domains and ␣/␤ pairing through an interchain disulfide bond. One of the commonest protein engineering strategies used in TCR studies to date has been the construction of singlechain TCRs. This technique, which takes advantage of the structural similarities between antibodies and TCRs, is based upon the single-chain Fv technology used to generate antibody fragments (5). In short, for the TCR it involves the cloning and expression of a unique chimerical open reading frame where the variable (V) ␣ and V␤ domains are paired with a protein linker (6, 7). Thes...

show abstract

Section: Tcr Multimers Can Sense Quantitative Modifications Of Antigementioning

confidence: 99%

Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition

Laugel

Boulter

Lissin³

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Two potent classes of reagents have been developed to bind to such complexes. Specific TCR, engineered from cloned T cells of known pMHC specificity, have been used to visualize cell surface pMHC complexes (4,5), and MHC-restricted, peptide-specific mAb, identified by various immunization and screening schemes, have been isolated and used similarly (5)(6)(7)(8)(9)(10)(11)(12). However, it has recently been suggested that in vitro selection of phage libraries displaying Ab fragments could be one of the most efficient ways to select specific pMHC binders (3).…”

mentioning

confidence: 99%

TCR-Like Human Antibodies Expressed on Human CTLs Mediate Antibody Affinity-Dependent Cytolytic Activity

Chames

Willemsen

Rojas

et al. 2002

The Journal of Immunology

View full text Add to dashboard Cite

The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes.

show abstract

“…P18-I10 complexes on the surface of infected cells were analyzed with the KP15 mAb, which specifically recognizes the H-2D d -P18-I10 peptide complex (Polakova et al, 2000), using a three-step amplification method with biotinylated secondary antibody (BD Pharmingen, San Jose, CA) and PE-labeled Streptavidin (BD Pharmingen, San Jose, CA) as previously described (Dzutsev et al, 2007).…”

Section: Antibodies Tetramer Assays and Flow Cytometrymentioning

confidence: 99%

“…The H-2D d -restricted P18-I10 epitope is expressed by the gp160-containing recombinant vaccinia virus vPE16 and can be detected (Dzutsev et al, 2007) using the KP15 antibody, which recognizes the P18-I10/H-2D d complex (Polakova et al, 2000). In the culture of P815 cells 24h post infection with vPE16 all KP15 positive cells were also gp120 positive (Fig.2b); whereas VSC8-infected cells were negative by both KP15 and anti-gp120 staining (Fig.2a).…”

Section: Estimation Of the Frequency Of Vaccinia Virus-infected P815 mentioning

confidence: 99%

“…Additionally, specific MHC-peptide complexes expressed on the surface of DCs sometimes number only a few per cell (Demotz et al, 1990), which is below the level of detection of the assays currently in use. Antibody-based detection of cells expressing specific MHC-peptide complexes is possible (Porgador et al, 1997;Polakova et al, 2000;Denkberg et al, 2002), but often presents a significant challenge due to low cellsurface expression of the specific MHC-peptide complexes. Additionally, antibodies with TCR-like specificities are difficult to generate.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Estimation of low frequency antigen-presenting cells with a novel RELISPOT assay

Dzutsev¹,

Belyakov²,

Isakov³

et al. 2008

Journal of Immunological Methods

Self Cite

View full text Add to dashboard Cite

Adequate presentation of self and foreign antigens is a key factor for efficient T-cell immunosurveillance against pathogens and tumors. Cells presenting foreign antigens usually comprise a rare population and are difficult to detect even at the peak of infection. Here we demonstrate a CD8 + T-cell-based approach that allows detection of specific antigen-presenting cells (APC) at a frequency of less than 0.0005%. When T cells are in excess, they form rosettes with rare APCs, which appear as single spots in an IFN-γ ELISPOT assay. Using this RELISPOT (Rosette ELISPOT) method we demonstrate the dynamic interplay between CD8 T cells and professional and non-professional APCs following virus challenge.

show abstract

Antibodies Directed Against the MHC-I Molecule H-2Dd Complexed with an Antigenic Peptide: Similarities to a T Cell Receptor with the Same Specificity

Cited by 29 publications

References 76 publications

Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition

Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition

TCR-Like Human Antibodies Expressed on Human CTLs Mediate Antibody Affinity-Dependent Cytolytic Activity

Estimation of low frequency antigen-presenting cells with a novel RELISPOT assay

Contact Info

Product

Resources

About