Recombinant human single-chain MHC-peptide complexes made fromE. coli byin vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens

Denkberg, Galit; Cohen, Cyril J.; Segal, Dina; Kirkin, Alexei F.; Reiter, Yoram

doi:10.1002/1521-4141(2000012)30:12<3522::aid-immu3522>3.0.co;2-d

Cited by 49 publications

(51 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Single-chain MHC (scMHC)/peptide complexes were produced by in vitro refolding of inclusion bodies produced in Escherichia coli, as described (30). Briefly, a single-chain ␤ 2 -microglobulin (␤ 2 m)-HLA/A2 (scMHC) construct, in which the ␤ 2 m and HLA-A2 genes are connected to each other by a flexible peptide linker, was engineered to contain the BirA recognition sequence for site-specific biotinylation at the C terminus (sc-MHC-BirA).…”

Section: Production Of Biotinylated Single-chain Mhc/peptide Complexesmentioning

confidence: 99%

Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies

Cohen

Sarig

Yamano

et al. 2003

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

The advent in recent years of the application of tetrameric arrays of class I peptide-MHC complexes now enables us to detect and study rare populations of Ag-specific CD8+ T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells nor detect APCs in tissues. In this study, we describe for the first time studies of human class I peptide-MHC ligand presentation. These studies were facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant Abs directed toward a viral epitope derived from human T cell lymphotropic virus type I. Using a large human Ab phage display library, we isolated a large panel of recombinant Fab Abs that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner. We used these Abs to visualize the specific complex on APCs and virus-infected cells by flow cytometry, to quantify the number of, and visualize in situ, a particular complex on the surface of APCs bearing complexes formed by naturally occurring active intracellular processing of the cognate viral Ag. These findings demonstrate our ability to transform the unique fine specificity, but low intrinsic affinity of TCRs into high affinity soluble Ab molecules endowed with a TCR-like specificity toward human viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I Ag presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.

show abstract

Section: Production Of Biotinylated Single-chain Mhc/peptide Complexesmentioning

confidence: 99%

Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies

Cohen

Sarig

Yamano

et al. 2003

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recombinant peptide-HLA-A2 complexes that present the TARP 29-37-derived peptide were generated using a single-chain (sc) MHC construct that was described previously [35]. In this construct, the extracellular domains of HLA-A2 are connected into a single-chain molecule with b2m using a 15-aa flexible linker.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…Production of biotinylated scMHC/peptide complexes scMHC/peptide complexes were produced by in vitro refolding of inclusion bodies produced in E. coli, as described [35,36]. Biotinylation was performed using the BirA enzyme (Avidity, Denver, CO) as described [67].…”

Section: Discussionmentioning

confidence: 99%

“…The refolded scHLA-A2/TARP complexes were found to be highly purified, homogenous, and monomeric by SDS-PAGE (data not shown). Recombinant scMHC-peptide complexes generated by this strategy had been previously characterized in detail for their biochemical, biophysical and biological properties, and were found to be correctly folded and functional [35,36].We have previously used the scHLA-A2/TARP complexes in the form of tetramers in order to detect TARP-specific CTL from patients' PBMC [17].To select TCR-like antibodies, we used a large naive Fab antibody phage library consisting of a repertoire of 3.7 Â 10 10 independent Fab clones [37]. In our selection strategy (see Materials and methods), we first depleted the library of streptavidin binders and subsequently applied the library to panning in solution on soluble recombinant scHLA-A2-peptide complexes containing the native TARP 29-37 peptide.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Targeting TARP, a novel breast and prostate tumor‐associated antigen, with T cell receptor‐like human recombinant antibodies

et al. 2008

Self Cite

View full text Add to dashboard Cite

MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/ peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptidespecific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRc alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/ TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes. Key words: Immunotoxin Á MHC Á Recombinant antibody Á T-cell receptor IntroductionMost patients with metastatic prostate and breast cancers are provided with the limited benefits from standard chemo-and hormone-based therapies. During the recent years, much effort has been invested in developing new approaches, such as immunotherapy, to improve the therapeutic abilities, by combining the tumor specificity of cell-mediated immunity with the freedom from toxic chemotherapies.Recent immunotherapy approaches employ the principle that CD8 + CTL recognize and kill tumor cells that display peptides from tumor-associated antigens presented by MHC class I molecules. Several tumor antigens and HLA allele-specific peptides from prostate cancer-associated antigens have been identified as CD8 + T cell epitopes, including HLA-A2-binding peptides derived from prostate-specific antigen (PSA) [1,2], prostate-specific membrane antigen (PSMA) [3], prostate stem cell antigen (PSCA) [4,5], and prostate acid phosphatase 6, which are all now components of current vaccine trials [5][6][7][8][9][10][11].Identification of new tumor-specific antigens is an essential step for the successful development of immunotherapy ap- [12][13][14]. One of these, T cell receptor gamma alternate reading frame protein (TARP), is expressed on breast and prostate cancer cells [15,16]. It was shown that TARP was expressed on >90% of cancer specimens examined [9,15]. In order to define new breast and prostate CD8 + T cell tumor antigens, two wild-type HLA-A2 epitopes from TARP were identified [17]. The wild-type sequences were also improved by sequence modifications to produce epitopeenha...

show abstract

HLA class II tetramers: Tools for direct analysis of antigen-specific CD4+ T cells

Nepom

Buckner

Novak

et al. 2002

Arthritis & Rheumatism

View full text Add to dashboard Cite

Recombinant human single-chain MHC-peptide complexes made fromE. coli byin vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens

Cited by 49 publications

References 43 publications

Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies

Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies

Targeting TARP, a novel breast and prostate tumor‐associated antigen, with T cell receptor‐like human recombinant antibodies

HLA class II tetramers: Tools for direct analysis of antigen-specific CD4+ T cells

Contact Info

Product

Resources

About