Using phage display in neurobiology.

In this unit some of the basic protocols involved in the manipulation of phage display libraries are described, including the rescue and amplification of such libraries, selection and screening from them and testing of derived clones.

Identification and characterization of ErbB-3-binding protein-1 as a target for immunotherapy.

Based on immune reactivity in response to a whole-cell colon tumor vaccine and using serological identification of Ags by recombinant cDNA expression cloning, we here describe the molecular and functional identification of a novel human tumor Ag. By screening a cDNA expression library derived from the coloncarcinoma cell line HT-29 with pooled colorectal cancer patients' sera, 26 clones reactive with IgG Abs could be identified. Characterization of these cDNA clones by sequence analysis and alignment, and detailed serological analysis revealed cancer-related immunoreactivity for the ErbB-3-binding protein-1 (Ebp1).

MHC-peptide-specific antibodies reveal inefficient presentation of an HLA-A*0201-restricted, Melan-A-derived peptide after active intracellular processing.

MHC-peptide-specific Fab antibodies binding to HLA-A*0201 complexes presenting the wild-type EAAGIGILTV (EAA) or analogue Melan-A 10-mer ELAGIGILTV (ELA) peptide were generated to study efficacy of peptide processing and presentation. None of the selected Fab antibodies detected the naturally processed EAA/HLA-A*0201 complex on melanoma tumor cells, confirming the known low peptide number on the cell surface. To study the effect of peptide presentation and processing in more detail, genes coding for the A27L-mutated Melan-A protein or the processed ELA peptide were introduced into HLA-A*0201(+) B cells by infection with the respective recombinant vaccinia virus construct producing equimolar amounts of GFP-ubiquitin directly linked to the fragment of interest.

Selecting and screening recombinant antibody libraries.

During the past decade several display methods and other library screening techniques have been developed for isolating monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are now able to identify mAb leads with virtually any specificity.

Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter.

Optimizing the exogenous antigen loading of monocyte-derived dendritic cells.

Dendritic cell (DC) vaccination, i.e. the adoptive transfer of antigen-loaded DC, is still at an early stage and requires standardization.

Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity.

Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found.

A major histocompatibility complex-peptide-restricted antibody and t cell receptor molecules recognize their target by distinct binding modes: crystal structure of human leukocyte antigen (HLA)-A1-MAGE-A1 in complex with FAB-HYB3.

Antibodies with T cell receptor-like specificity possess a considerable diagnostic and therapeutic potential, but the structural basis of the interaction between an antibody and an histocompatibility antigen has so far not been determined. We present here the crystal structure (at 2.15 A resolution) of the recombinant, affinity-matured human antibody fragment Fab-Hyb3 bound to the tumor-associated human leukocyte antigen (HLA)/peptide complex HLA-A1.

Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments.

Yeast display is a powerful technology for the affinity maturation of human antibody fragments. However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10(6) and 10(7) are typically possible. We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae [van den Beucken, T.

Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR.

Dissecting cytotoxic T cell responses towards the NY-ESO-1 protein by peptide/MHC-specific antibody fragments.

NY-ESO-1 is a germ cell antigen aberrantly expressed by different tumor types that elicits strong humoral and cellular immune responses, representing one of the most promising candidates for vaccination of cancer patients. A detailed analysis of CD8(+) T cells generated in vaccine trials using NY-ESO-1-derived peptides (157-165 and 157-167) revealed that the dominant immune response was directed against a cryptic epitope (159-167) diverting the immune response from tumor recognition. Only CTL reactivity to the NY-ESO-1(157-165) peptide appeared to be capable of lysing NY-ESO-1/HLA-A0201-expressing tumor cells.

Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries.

We introduce a procedure for the rapid generation of fully human antibodies derived from "Fab-on-phage" display libraries. The technology is based on the compatibility of display vectors and IgG expression constructs, and allows reformatting of individual Fab clones to IgG, as well as reformatting of antibody repertoires. Examples of batch reformatting of an uncharacterized Fab repertoire and of a pool of Fabs, previously analyzed at the phage level, are presented.

Isolation of human antibodies to tumor-associated endothelial cell markers by in vitro human endothelial cell selection with phage display libraries.

Human antibodies selectively targeting angiogenic vessels hold great promise for the immunotherapy of human malignancies and can help to elucidate the molecular mechanisms regulating angiogenesis. By selecting a large antibody phage display library on proliferating stimulated HUVEC, we have isolated 15 human antibodies that bind to HUVEC in flow cytometric analysis, 11 of which target the vasculature of colorectal carcinomas as demonstrated by immunohistochemical analysis. The four most specific antibodies, TEM-A, TEM-C, TEM-M and TEM-Q, also stain the vasculature of a series of carcinomas derived from liver, ovary, kidney, bladder, lung and breast, and either react weakly or not all with the vasculature of corresponding normal tissues.

Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries.

We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy.

Direct detection and quantitation of a distinct T-cell epitope derived from tumor-specific epithelial cell-associated mucin using human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells.

The recent characterization of MHC-displayed tumor-associated antigens that recognize effector cells of the immune system has created new perspectives for cancer therapy. Antibodies that recognize these tumor-associated MHC-peptide complexes with the same specificity as the T-cell antigen receptor will therefore be valuable tools for immunotherapy, as well as for studying antigen presentation in human cancers. Most tumor-associated antigens are expressed in only one or a few tumor types; however, specific T-cell epitopes derived from the Mucin-1 tumor-associated antigen (MUC1) that are widely expressed in many cancers were identified and shown to be recognized by CTLs.

Target validation for genomics using peptide-specific phage antibodies: a study of five gene products overexpressed in colorectal cancer.

Genomic approaches are providing a wealth of information on differential gene expression in cancer. To identify the most interesting genes amongst the many identified, high-throughput methods for analysis of genes at the translational level are required. We have used a rapid method for the in vitro selection of antibodies to peptide antigens for the generation of probes to 5 gene products that we have found to be overexpressed in colorectal cancer.

A panel of candidate tumor antigens in colorectal cancer revealed by the serological selection of a phage displayed cDNA expression library.

In the last few years it has been shown that the humoral immune response in cancer patients is a rich source of putative cancer vaccine candidates. To fully explore the complex information present within the Ab repertoire of cancer patients, we have applied a method, serological Ag selection, to molecularly define tumor Ags recognized by the humoral immune response in colorectal cancer (CRC). First, we built a cDNA display library by cloning a cDNA library from CRC cell line HT-29 for expression as a fusion protein with a filamentous phage minor coat protein, pVI.

TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity.

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.

Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC- restricted T cell receptor-like specificity.

Specificity in the cellular immune system is controlled and regulated by the T cell antigen receptor (TCR), which specifically recognizes peptide/major histocompatibility complex (MHC) molecules. In recent years many cancer-associated MHC-restricted peptides have been isolated and because of their highly restricted fine specificity, they are desirable targets for novel approaches in immunotherapy. Antibodies that would recognize tumor-associated MHC-peptide complexes with the same specificity as the TCR would be valuable reagents for studying antigen presentation by tumor cells, for visualizing MHC-peptide complexes on cells, and eventually for monitoring the expression of specific complexes during immunotherapy.

Isolation and characterization of human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells directed toward the widely expressed tumor T-cell epitopes of the telomerase catalytic subunit.

The recent characterization of MHC-displayed tumor-associated antigensthat recognize effector cells of the immune system has created new perspectives for cancer therapy. Antibodies that recognize these tumor-associated MHC-peptide complexes with the same specificity as the T-cell antigen receptor will therefore be valuable tools for immunotherapy as well as for studying antigen presentation in human cancers. Most tumor-associated antigens are expressed in only one or a few tumor types; however, recently specific T-cell epitopes derived from the telomerase catalytic subunit (hTERT) that are widely expressed in many cancers were identified and shown to be recognized by CTLs derived from cancer patients.

A human immunoglobulin G1 antibody originating from an in vitro-selected Fab phage antibody binds avidly to tumor-associated MUC1 and is efficiently internalized.

We describe the engineering and characterization of a whole human antibody directed toward the tumor-associated protein core of human MUC1. The antibody PH1 originated from the in vitro selection on MUC1 of a nonimmune human Fab phage library. The PH1 variable genes were reformatted for expression as a fully human IgG1.

Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein.

The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses.

A phage display selected fab fragment with MHC class I-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes.

The clinical benefit of adoptive transfer of MHC-restricted cytotoxic T lymphocytes(CTL) for the treatment of cancer is hampered by the low success rate to generate antitumor CTLs. To bypass the need for tumor-specific CTL, we developed a strategy that allows for grafting of human T lymphocytes with MHC-restricted antigen specificity using in vitro selected human Fab fragments fused to the Fc(epsilon)RI-gamma signaling molecule. Retroviral introduction of a Fab-based chimeric receptor specific for MAGE-A1/HLA-A1 into primary human T lymphocytes resulted in binding of relevant peptide/MHC complexes.