Phage-displayed Fab Research Articles

Human recombinant Fab antibodies with T-cell receptor-like specificities generated from phage display libraries.

In this chapter we describe efficient procedures for the construction, expression and screening of comprehensive libraries of human Fab antibody fragments displayed on the surface of filamentous phage. Phagemid vectors are used for placing randomly paired light (L) and heavy (H) chain coding regions under transcriptional control of Plac. The H chain coding region is fused in-frame with the phage gene, ΔgIII, coding for a truncated version of the phage surface protein pIII (ΔpIII). After superinfection with helper phage and induction of Plac, Fd (composed of VH and CH1 domains) and κ-or α-L chains assemble into Fab fragments in the periplasm of the Escherichia coli host strain, and the Fab-ΔpIII protein complex is displayed at one end of the phage by displacing one (or more) of the wild-type pIII proteins. Enrichment of Fab phages with affinity for a selected “antigen” is then carried out by successive rounds of affinity purification using “antigen”-coated immunotubes or plastic beads followed by reinfection of E. coli cells with the eluted bound phages (see refs. , , , , , , , , , ). An outline of the method is illustrated in Fig. 1. Open image in new window Fig. 1. Overview of the steps involved in the generation of human antibody Fab libraries. Messenger RNA is isolated from peripheral blood of donors and copied into cDNA. This material is then used as template for PCR amplification of immunoglobulin gene fragments, which in turn are cloned and expressed as functional Fab molecules in fusion with a phage surface protein (pIII) as mentioned in the text. The entire mixture of phage-displayed Fab molecules are then submitted to affinity purification methods to obtain specific binders to a selected “antigen.”

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.

Sequence Plasticity in the Antigen-binding Site of a Therapeutic Anti-HER2 Antibody

We have examined the plasticity of the antigen-combining site of a high-affinity antibody. In phage-displayed Fab libraries, selected CDR positions and one FR position of the humanized anti-Her2 antibody hu4D5 were substituted with all 20 amino acids. Antigen-binding selections were used to enrich for high-affinity variants, and a large number of sequences were obtained prior to convergence of the selected pool to a small set of clones. As expected, sequence variability of the antigen-binding site is overall diminished compared to known IgG sequences; however, certain positions retain much higher variability than others. The sequence variability map of the hu4D5 binding site is compared with a map derived from previous alanine-scanning of the antibody. Affinities of soluble Fab fragments for antigen confirm that multiple variants were selected with high affinity for antigen, including one variant with a single point mutation that was about threefold improved in affinity compared to the parental hu4D5. Interestingly, this mutation is one of the most radical in terms of changing side-chain chemistry (Trp for Asp) and occurs at the most plastic site as calculated by the Wu–Kabat variability coefficient. Thus variability mapping yields information about the antibody–antigen interaction that is useful and complementary to that obtained by alanine scanning mutagenesis.

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.

Restricted VH3 gene usage in phage-displayed Fab that are selected by intravenous immunoglobulin.

To perform a comparative analysis of 1) intravenous Ig (IVIG)-bound Fab fragments from a patient with autoimmune thrombocytopenia that had progressed to systemic lupus erythematosus (SLE) and 2) IVIG-selected Fabs from an SLE patient without thrombocytopenia. IVIG preparations have been successfully used to treat certain cases of autoimmune thrombocytopenia and SLE. Specific interactions of IVIG with the components of the immune system are not well characterized. To investigate these, we had previously cloned a large number of phage-displayed IgG Fab fragments, derived from 3 patients with autoimmune thrombocytopenia, that were specifically bound by IVIG molecules during panning. Many of these Fabs reacted with platelets. Sequencing revealed that the most frequently used VH germline gene segments of all IVIG-bound Fabs were 3-23 and 3-30/3-30.5. One patient's autoimmune thrombocytopenia had progressed to SLE. Using the same cloning and panning procedures, we performed a comparative analysis of this patient's IVIG-bound Fab fragments and the IVIG-selected Fabs from an SLE patient without thrombocytopenia. We observed an exclusive selection of antibodies derived from 3-23 and 3-30/3-30.5 germline segments. In contrast to the Fab fragments from the autoimmune thrombocytopenia patient who developed SLE, none of the IVIG-selected Fabs from the SLE patient without thrombocytopenia bound to thrombocytes. Our results suggest a preferential interaction of a subfraction of IVIG-representative of normal Ig repertoires-with antibodies and B cell receptors derived from these 2 gene segments. Importantly, these are the most frequently rearranged VH germline genes among human B cells. This kind of interaction is characteristic of a B cell superantigen, since light chains, antigen specificity, and the high variation in the third complementarity-determining region 3 showed little influence on the selection of 3-23- or 3-30/3-30.5-derived Fabs by IVIG. However, at least some of the contact residues on Fabs for IVIG appear to be different from those for staphylococcal protein A and human immunodeficiency virus gp 120. The IVIG-selected Fabs may now be used to clone antibodies representative of this IVIG subfraction to study their possible regulatory influence on the B cell repertoire during normal development and disease.

Selection of phage-displayed Fab antibodies on the active conformation of Ras yields a high affinity conformation-specific antibody preventing the binding of c-Raf kinase to Ras

The Ras proteins cycle in the cell between an inactive state and an active state. In the active state, Ras signals via the switch I region to effectors like c-Raf kinase, leading to cell growth. Since Ras mutations in cancer are often associated with the presence of permanently active Ras, molecules that prevent downstream signaling may be of interest. Here, we show that by selection on the active conformation of Ras, using a recently described large phage antibody repertoire [de Haard et al. (1999) J. Biol. Chem. 274, 18218–18230], a Fab antibody (Fab H2) was identified that exclusively binds to active Ras, and not to inactive Ras. Using surface plasmon resonance (SPR) analysis, the interaction was demonstrated to be of high affinity (7.2 nM). In addition, the interaction with Ras is specific, since binding to the homologous Rap1A protein in BIAcore analysis is at least three orders of magnitude lower, and undetectable in an enzyme-linked immunosorbent assay. The antibody fragment prevents the binding of active Ras to the immobilized Ras-binding domain of c-Raf kinase (Raf-RBD) at an IC 50 value of 135 nM. This value compares well to the K D of active Ras-binding to immobilized Raf-RBD using SPR, suggesting identical binding sites. Like the IgG Y13-259, which does not demonstrate preferential binding to either inactive or active Ras, Fab H2 inhibits intrinsic GTPase activity of Ras in vitro. Mapping studies using SPR analysis demonstrate that the binding sites for the antibodies are non-identical. This antibody could be used for dissecting functional differences between Ras effectors. Due to its specificity for active Ras, Fab H2 may well be more selective than previously used anti-Ras antibodies, and thus could be used for gene therapy of cancer with intracellular antibodies.

Contribution of Antibody Heavy Chain CDR1 to Digoxin Binding Analyzed by Random Mutagenesis of Phage-displayed Fab 26-10

We constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 of the anti-digoxin antibody 26-10 Fab to investigate sequence constraints necessary for high affinity binding in an antibody of known crystal structure. Phage were selected by panning against digoxin and three C-16-substituted analogues. All antigen-positive mutants selected using other analogues also bound digoxin. Among 73 antigen-positive clones, 26 different nucleotide sequences were found. The majority of Fabs had high affinity for digoxin (Ka 3.4 x 10(9) M-1) despite wide sequence diversity. Two mutants displayed affinities 2- and 4-fold higher than the parental antibody. Analysis of the statistical distribution of sequences showed that highest affinity binding occurred with a restricted set of amino acid substitutions at positions H33-35. All clones save two retained the parental Asn-H35, which contacts hapten and hydrogen bonds to other binding site residues in the parental structure. Positions H30-32 display remarkable diversity, with 10-14 different substitutions for each residue, consistent with high affinity binding. Thus complementarity can be retained and even improved despite diversity in the conformation of the N-terminal portion of the H-CDR1 loop.

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.