A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies.

We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10(-2) to 10(-4) s-1 and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.

EB/RP gene family encodes tubulin binding proteins.

Mutations in the adenomatous polyposis coli (APC) gene are linked to the dysplastic transformation of colorectal polyps and represent an early step in the development of colorectal tumors. Ninety-four percent of all mutations result in the expression of a truncated APC protein lacking the C-terminal region. The C-terminal region of the APC protein may have a tumor suppressor function as its absence appears to be linked to the development of dysplastic lesions. Recently, we discovered and characterized a protein called RP1 which binds specifically to the C-terminal region of the APC protein. We show now that RP1 and the other known members of the EB/RP family (EB1 and RP3) also bind directly to tubulin, both in vitro and in vivo. Immunohistochemical analyses reveal a distinct staining pattern during interphase as well as an association of RP1/EB1 with mitotic microtubule structures. The previously described puncta of the APC protein at the leading edge of membrane protrusions contact microtubule fibers that contain RP1 or EB1.

A tandem repeat of MUC1 core protein induces a weak in vitro immune response in human B cells.

We have recently described an efficient method to study the human humoral immune response in vitro and to generate isotype-switched, antigen-specific human B cells, which has allowed us to produce high-affinity IgG antibodies against different peptides. In an attempt to study the in vitro immune response against self-antigens, such as tumour-associated antigens, this protocol was used to immunise resting human peripheral blood B cells with a peptide epitope from the human-adenocarcinoma-associated antigen, MUC1. After the two-step in vitro immunisation, the secondary immunised cultures were tested for MUC-1-specific antibodies by enzyme-linked immunosorbent assay (ELISA). Phage molecular libraries were subsequently constructed, using the variable parts of Ig genes derived from cells taken from ELISA-positive wells. The libraries were selected on the MUC1 core peptide. Antigen-specific Fab fragments, specific for the self antigen MUC1, were found in the library of secondary immunised IgG+ B cells and these antibodies were evaluated by BIAcore analysis. The specific Fab fragments exhibited an unusually rapid dissociation rate constant and the overall response frequency was lower, as compared to other antibodies generated by this protocol, which might be explained by the repetitive nature of the core peptide used for immunisation.

High-affinity recombinant phage antibodies to the pan-carcinoma marker epithelial glycoprotein-2 for tumour targeting.

The tumour-associated antigen epithelial glycoprotein-2 (EGP-2) is a promising target for detection and treatment of a variety of human carcinomas. Antibodies to this antigen have been successfully used in patients for imaging of small-cell lung cancer and for adjuvant treatment of minimal residual disease of colon cancer. We describe here the isolation and complete characterization of high-affinity single-chain variable fragments (scFv) to the EGP-2 antigen. First, the binding kinetics of four murine whole antibodies directed to EGP-2 (17-1A, 323/A3, MOC-31 and MOC-161) were determined using surface plasmon resonance (SPR). The MOC-31 antibody has the lowest apparent off-rate, followed by MOC-161 and 323/A3. The V-genes of the two MOC hybridomas were cloned as scFv in a phage display vector and antigen-binding phage were selected by panning on recombinant antigen. The scFvs compete with the original hybridoma antibodies for binding to antigen and specifically bind to human carcinomas in immunohistochemistry. MOC-31 scFv has an off-rate which is better than those of the bivalent 17-1A and 323/A3 whole antibodies, providing it with an essential characteristic for tumour retention in vivo. The availability of these high-affinity anti-EGP-2 antibody fragments and of their encoding V-genes creates a variety of possibilities for their future use as tumour-targeting vehicles.

Human single-chain Fv antibodies to MUC1 core peptide selected from phage display libraries recognize unique epitopes and predominantly bind adenocarcinoma.

The tumor-associated antigen MUC1 is overexpressed and underglycosylated in human adenocarcinomas of diverse origins, such as breast, ovary, and colon. We isolated and describe five human single-chain (sc) Fv antibodies specific for the MUC1 variable number of tandem repeats region isolated by in vitro selection from a large naive phage antibody library containing over 6 x 10(9) different scFv antibodies. A synthetic biotinylated 100-mer peptide corresponding to five tandem repeats of the MUC1 peptide core was used for selection. Two of the antibodies were highly specific for MUC1 as judged by ELISA and flow cytometry. In immunohistochemistry, antibody clone 10A stained MUC1 in the cytoplasm and membrane of adenocarcinoma cells of breast and ovary, whereas in normal epithelium, only cytoplasmic or no staining was observed. With antibody clone 10B, staining was less pronounced and was not always membrane associated in adenocarcinoma. Determination of the fine specificity of 10A and 10B using a novel "indirect epitope fingerprinting" ELISA showed that both antibodies recognize unique epitopes that have not been described for hybridoma-derived anti-mucin antibodies of mouse origin. The selected human antibodies, like many of the murine MUC1 antibodies, recognize epitopes on the protein core of MUC1 that are abundantly present in the underglycosylated form of cell surface mucin on adenocarcinoma. The best human scFv, clone 10A, appears to distinguish normal cells from adenocarcinoma cells, which makes it an attractive candidate for use in antibody-based tumor targeting.

Creating and engineering human antibodies for immunotherapy.

Targeting in immunotherapy has traditionally been achieved by using monoclonal rodent antibodies. Despite gene-engineering, there are many problems and limitations associated with the non-human origin, the targeting specificity and the binding strength of these molecules. Now these issues may be addressed in a more rational way, by designing and then shaping, in vitro, the desired human antibodies. This review addresses how this may be achieved by the selection of monoclonal human antibodies from phage display libraries and the engineering of affinity and specificity thereafter. Phage display of antibody fragments has allowed access to large collections of different phage antibodies, created by cloning antibody V-genes from B-cells. Antibodies against any type of antigen may be derived from such repertoires, by rounds of enrichment on antigen and re-amplification. This review presents the state of the art in rational antibody design and creation. It will highlight the strengths of this increasingly important field, which will aid in the generation of tailor-made targeting entities for immunotherapy.

Human CD8+ T cell clone regulates autologous CD4+ myelin basic protein specific T cells.

Normal human CD8+ T cell clones were co-isolated from the same culture wells as CD4+ T effector cell clones specific for myelin basic protein (MBP). Microcultures from which the CD8+ clones were isolated initially proliferated weakly to whole MBP and to an MBP peptide spanning residues 90-170. This pattern of response was similar to strongly proliferating wells that yielded CD4+ T cell clones specific for the 90-170 peptide. After repeated stimulation, however, no response to MBP or MBP 90-170 was detected, even though the number of cells increased after stimulation. Phenotyping and TCR analyses revealed the presence of two CD8+, CD4-, IL-2R+ T cell isolates that expressed a single V beta gene (V beta 17) that differed from the CD4+ isolates that uniformly expressed V beta 14. One of these CD8+ clones (C9) inhibited the antigen-driven proliferation of an autologous MBP 90-170 reactive clone but not an autologous clone specific for Herpes simplex virus (HSV), without affecting MHC non-restricted mitogen responses of the same clones. Moreover, C9 did not inhibit heterologous CD4+ T cell clones specific for MBP 1-38 or 90-170. A culture supernatant of the CD8+ clone showed the same pattern but lower levels of inhibition. C9 had mild cytolytic activity when incubated at high ratios with an autologous MBP-specific CD4+ clone. Lysis was blocked completely by anti-MHC class I antibodies, but not by anti-MHC II antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity of human T cell clones reactive to immunodominant epitopes of myelin basic protein.

Several recently discovered lines of evidence support the involvement of myelin basic protein (BP)-specific T cells in multiple sclerosis (MS). To identify potentially relevant immunodominant T cell epitopes, human BP (Hu-BP)-reactive T cell lines were selected from MS and normal donors and tested for reactivity to cleavage fragments and synthetic peptides of Hu-BP. The MS T cell lines responded to more Hu-BP epitopes than did normal lines, showing biased recognition of the N terminal half of the molecule, and one region in the C terminal half, suggesting increased sensitization to BP. The MS lines also differed from normal lines in their decreased percentage of CD8+ T cells. One hundred nine T cell clones isolated from these lines confirmed the reactivity pattern of the lines but did not reflect the mixed phenotype, since all but three clones tested were CD4+. T cell clones from HLA-DR2 homozygous donors responded to a variety of epitopes, indicating that this molecule was permissive in its ability to restrict T cell responses. Other epitopes, including the immunodominant 149-170 sequence, were restricted by several different major histocompatibility complex (MHC) molecules from both MS and normal donors. T cell receptor (TCR) V gene products could be identified on six of 38 clones tested using monoclonal antibodies. From one HLA-DR2 homozygous donor, four of eight clones utilized V beta 5.2 in response to different BP epitopes, providing initial support for the preferential use of a limited set of V region genes in the human response to BP. Preferential TCR V gene use in MS patients would provide the rationale to regulate selectively BP-reactive T cells through immunity directed at the TCR and thus test for the first time the hypothesis that BP-reactive T cells play a critical role in the pathogenesis of MS.

Antibodies to myelin basic protein and measles virus in multiple sclerosis: precursor frequency analysis of the antibody producing B cells.

Antibody-producing B lymphocytes were polyclonally activated and transformed, by Epstein-Barr virus (EBV), into multiple B lymphoblastoid cell lines in a microculture system. The frequencies of B precursor cells producing antibodies to myelin basic protein (MBP) and measles virus were analyzed in peripheral blood of patients with multiple sclerosis (MS) and control subjects. Measles virus-specific B cells were detected at a significantly higher frequency in MS patients (n = 10, P less than 0.005) than patients with other neurological diseases (n = 10) and normal subjects (n = 10). In contrast, the frequencies of B cells producing anti-MBP antibodies and natural antibodies did not differ statistically among the three groups tested (P greater than 0.05). In addition, the anti-MBP antibodies produced by a panel of stable B cell lines obtained were found to react selectively with an epitope(s) within the C-terminal half fragment 90-171 of the human MBP molecule. In our experiments, no antibody cross-reactivity between MBP and measles virus could be detected in a total of 2760 B cell cultures.