Phage-displayed Fab Library Research Articles

Selection of an affinity-matured antibody against a defined epitope by phage display of an immune antibody library

In a previous study, we generated a murine hepatitis B virus (HBV)-neutralizing monoclonal antibody (mAb), KR127, that binds to an epitope (amino acids 37–45, NSNNPDWDF) of the preS1 antigen. Furthermore, an epitope tag, S1 (NANNPDWDF), was developed for protein tagging. The aim of the present study was to develop a high-affinity antibody to the same preS1 epitope. Mice were immunized with the N-terminal domain of human thrombopoietin fused to the S1 tag (nTPO-S1), and a phage-displayed chimeric Fab library was constructed and screened by panning against nTPO-S1. A high-affinity antibody (3–34) was selected that binds to the preS1 antigen. The IgG molecules of 3–34 showed approximately nine-fold higher affinity (KD 1.2 nM) for preS1 compared with KR127 (KD 10.4 nM), competed with KR127 for binding to the epitope, and bound to HBV particles. This study provides a simple and efficient way to develop a high-affinity antibody to a defined epitope by phage display of an immune antibody library.

Antigen-driven oligoclonal expansion of tumor-infiltrating B cells in infiltrating ductal carcinoma of the breast.

The objective of this study was to determine whether tumor-infiltrating B cells (TIL-B) of infiltrating ductal carcinoma (IDC) of the breast represent a tumor-specific humoral immune response. Immunohistochemical analysis of three Her-2/neu-negative IDC tumors from geriatric patients showed that TIL-B cluster in structures similar to germinal centers containing CD20(+) B lymphocyte and CD3(+) T lymphocyte zones with interdigitating CD21(+) follicular dendritic cells, suggesting an in situ immune response. A total of 29, 31, and 58 IgG1 H chain clones was sequenced from the three IDC tumors, respectively. Intratumoral oligoclonal expansion of TIL-B was demonstrated by a preponderance (45-68%) of clonal B cells. In contrast, only 7% of tumor-draining lymph node and 0% of healthy donor PBL IgG H chains were clonal, consistent with the larger repertoires of node and peripheral populations. Patterns and levels of TIL-B IgG H chain somatic hypermutation suggested affinity maturation in intratumoral germinal centers. To examine the specificity of TIL-B Ig, a phage-displayed Fab library was generated from the TIL-B of one IDC tumor. Panning with an allogeneic breast cancer cell line enriched Fab binding to breast cancer cells, but not nonmalignant cell lines tested. However, panning with autologous tumor tissue lysate increased binding of Fab to both tumor tissue lysate and healthy breast tissue lysate. These data suggest an in situ Ag-driven oligoclonal B cell response to a variety of tumor- and breast-associated Ags.

Progressive epitope-blocked panning of a phage library for isolation of human RSV antibodies

Epitope-blocked panning is an approach to mining antigen-specific diversity from phage display antibody libraries. Previously, we developed and used this method to recover a neutralizing antibody to respiratory syncytial virus (RSV) by blocking a dominant response to a nonneutralizing epitope on a recombinant derivative of the viral F antigen. We have extended this approach to the blocking of multiple epitopes simultaneously, which led to the recovery of new antibodies of different specificity, including one new neutralizing activity. A phage display Fab library was selected on recombinant F antigen in the presence of three representative antibodies recovered in the unblocked and subsequent single-blocked panning procedures. Restriction endonuclease fingerprinting of 13 F+ clones revealed seven unique Fabs. DNA sequence analysis of five of these clones revealed five new light chains in combination with different heavy chains, three of which were very similar or identical to Fabs previously isolated from this library. The blocking antibodies did not compete with the new Fabs, demonstrating effective masking of their binding sites in the panning procedure. Conversely, these Fabs did show variable inhibition of two of the blocking antibodies suggesting a close proximity or interdependence of their epitopes. One of the antibodies did inhibit virus infection, albeit with modest potency. These results demonstrate that epitope-blocked panning is a self-progressing approach to retrieving diverse antibodies from phage libraries.

Isolation of an IgG anti-B from a human Fab-phage display library.

ABO incompatibility is a common cause for mild hemolysis in the newborn, ranging from 1 in 30 to 1 in 150 births. Fortunately, hemolysis requiring transfusion is rare and restricted to blood group O mothers, because blood group A and B individuals make poor IgG anti-B and anti-A responses. No human IgG ABO antibody sequences have been reported, in part because of the difficulty in obtaining human IgG hybridomas. Phage-display technology may be able to circumvent these difficulties, but its application to carbohydrate antigens is poorly studied. A human IgG1 phage-display Fab library was constructed from splenocytes derived from a nonhyperimmunized blood group O person, and panned against group B RBCs. After five rounds of panning, essentially all phage bound to group B RBCs. Nucleotide sequence analysis of a single monoclonal IgG1lambda phage, FB5.7, revealed a highly mutated VH4 family heavy chain, and a nearly germline VL7 family lambda light chain. The Fab agglutinated group B, but not group A, random-donor RBCs. However, group B ELISA reactivity could be inhibited by soluble B-trisaccharide, soluble A-trisaccharide, galactose, and N-acetyl galactosamine. Similarly, galactose and N-acetyl galactosamine were able to inhibit group B RBC agglutination. FB5.7 is the first human IgG ABO MoAb described. Alhough it behaves serologically like a group B-specific antibody, it demonstrates interaction with both the A and B epitopes. Phage-display technology can be used to better define the relationship between antibody genotype and phenotype in anti-carbohydrate responses in nonhyperimmunized hosts, and thus to improve our understanding of the composition of the antibody repertoire.

Analysis of the binding of pro-urokinase and urokinase-plasminogen activator inhibitor-1 complex to the low density lipoprotein receptor-related protein using a Fab fragment selected from a phage-displayed Fab library.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) mediates endocytosis of a number of structurally unrelated ligands, including complexes of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA), free t-PA, single-chain urokinase (pro-u-PA), alpha 2-macroglobulin-protease complexes, and lipoprotein lipase. So far, all ligands have in common the fact that they bind to the receptor in a Ca(2+)-dependent way and the fact that binding to the receptor can be inhibited by a 39-40-kDa protein, termed the receptor-associated protein. To obtain inhibitory antibodies for the analysis of the structure and function of the receptor we applied the combinatorial immunoglobulin repertoire cloning technique in order to specifically select monoclonal Fab fragments directed against Ca(2+)-dependent epitopes. In this report we describe the isolation of a Fab fragment (Fab A8) showing a high relative affinity for the receptor (0.5 nM). The binding of this Fab fragment to purified LRP is inhibited in the presence of 5 mM EDTA, receptor-associated protein, and lipoprotein lipase (IC50 values of 1.4 and 31 nM, respectively). By immunoblotting of CNBr-digested LRP it is shown that Fab A8 binds to a fragment that harbors the second cluster of cysteine-rich complement-type repeats flanked by epidermal growth factor repeats. Binding studies using 125I-labeled ligands and immobilized receptor show that Fab A8 partially inhibits the binding of [125I]u-PA.PAI-1 complexes (IC50 = 1.1 nM) and completely inhibits the binding of [125I]pro-u-PA to the receptor (IC50 = 2.2 nM). No inhibition was observed for the binding of 125I-labeled methylamine-activated alpha 2-macroglobulin or [125I]t-PA.PAI-1 to LRP. Degradation of [125I]u-PA.PAI-1 complexes by COS-1 cells was also partially (43%) inhibited by Fab A8. Our results provide evidence for the presence of an interaction site for pro-u-PA localized in the second cluster of cysteine-rich repeats that is unrelated to the t-PA.PAI-1 or methylamine-activated alpha 2-macroglobulin interaction sites.