Paula Henderikx

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 × 1010 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 10−4s−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. Int. J. Cancer 81:275–284, 1999.

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.

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.

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. The resulting PH1-IgG1 human antibody displays a 160-fold improved apparent kd (8.7 nmol/L) compared to the kd of the parental Fab (1.4 micromol/L). In cell-binding studies with flow cytometry and immunohistochemistry, PH1-IgG1 exhibits staining patterns typical for antibodies recognizing the tumor-associated tandem repeat region on MUC1, eg, it binds the tumor-associated glycoforms of MUC1 in breast and ovarian cancer cell lines, but not the heavily glycosylated form of MUC1 on colon carcinoma cell lines. In many tumors PH1-IgG1 binds to membranous and cytoplasmic MUC1, with often intense staining of the whole-cell membrane (eg, in adenocarcinoma). In normal tissues staining is either absent or less intense, in which case it is found mostly at the apical side of the cells. Finally, fluorescein isothiocyanate-labeled PH1-IgG1 internalizes quickly after binding to human OVCAR-3 cells, and to a lesser extent to MUC1 gene-transfected 3T3 mouse fibroblasts. The tumor-associated binding characteristics of this antibody, its efficient internalization, and its human nature, make PH1-IgG1 a valuable candidate for further studies as a cancer-targeting immunotherapeutic.

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

Abstract 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.

Abstract 3850: First-in-class cell-penetrating proteins targeting Mcl-1 induce tumor cell apoptosis and inhibition of tumor growth in vivo

We have developed Cell Penetrating Alphabodies (CPABs), a novel and unique therapeutic class of proteins engineered to efficiently enter cells. In vitro, uptake in a range of tumor and non-tumor cell types occurs rapidly with cytosol levels of up to 1 μM concentration after 2 hours of CPAB exposure. Early forms of these CPABs suffered from rapid serum clearance, thereby limiting their efficacy in vivo and amenability to drug development. The incorporation of an albumin binding region in the body of the protein has allowed extension of serum half-life in mice from a few minutes to more than one hour. These CPABs have been shown to be efficiently delivered to xenograft tumors in mice after IV bolus injection by tissue ELISA and immunohistochemistry. CPABs can be used to target and interfere with intracellular protein-protein interactions involved in tumor survival in a highly specific way. The anti-apoptotic protein Myeloid Cell Leukaemia-1 (Mcl-1) promotes through its interaction with Bak, the survival of a range of different tumor types including myeloid leukemia, breast cancer and non-small cell lung cancer. Moreover, Mcl-1 overexpression is often associated with chemotherapeutic resistance and disease relapse. Mcl-1, however, has proven difficult to target using the conventional small molecule approach. Alphabodies which bind to Mcl-1 were engineered by a combination of rational design and phage display library screening. The affinities for Mcl-1 ranged between 18 pM and 750 pM with binding to the closely related proteins Bcl-2 and Bcl-XL being below the limit of detection for the assay. In a Mammalian Two Hybrid assay, these Alphabodies inhibited Bak-Mcl-1 but not Bak-Bcl-XL interactions. Anti-Mcl-1 CPABs were shown to efficiently kill the Mcl-1 dependent multiple myeloma cell line NCI-H929 with IC50s ranging from 0.5 μM to 2 μM as monitored in cell viability assays. The dose responsive cell killing correlated with caspase-3/7 activation in NCI-H929 cells. Other Mcl-1 dependent tumor cell types including non-small cell lung cancer (NCI-H23) and Burkitt9s lymphoma (Raji) or tumor cell types with high Mcl-1 expression such as ovarian cancer (A2780) and colorectal adenocarcinoma (COLO-320DM) were also killed efficiently using anti-Mcl-1 CPABs. Despite its short half-life, daily intraperitoneal administration of a prototype Mcl-1 targeting CPAB (without half-life extension) at 30 mg/kg for 14 days resulted in tumor inhibition of 33% as compared to vehicle control. Experiments are underway in mouse models using the more optimal CPABs with extended serum half-life and tumor exposure. CPABs represent the best-in-class cell penetrating protein therapeutics both in terms of efficiency of uptake and amenability to conversion to viable drugs opening unprecedented opportunities to tackle intracellular protein-protein interactions critical to diseases with unmet medical need. Citation Format: Sabrina Deroo, Sophie Thiolloy, Johan Desmet, Franky Baatz, Stefan Loverix, Karen Vandenbroucke, Eric Lorent, Paula Henderikx, Irma Lemmens, Philippe Alard, Ignace Lasters, Yvonne McGrath. First-in-class cell-penetrating proteins targeting Mcl-1 induce tumor cell apoptosis and inhibition of tumor growth in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3850.

Selections on Biotinylated Antigens

Phage antibody library selections on peptides or proteins are usually carried out using antigens directly coated on a plastic surface (e.g. Petri dishes, microtiter plate well, immunotubes, Chapter 9). This straightforward method is easy to perform and has been shown to be very successful for a diverse set of antigens (for review see Winter et al. 1994). However, phage-antibody selections on some proteins and especially on peptides are not always successful, often due to by immobilization-associated features. The main problem observed for selection on peptides is the very poor coating efficiency of some peptides and the altered availability of epitopes on plastic-coated peptides. The direct coating of proteins on plastic is usually more efficient but can also be problematic, because the passive adsorption on plastic at pH 9.6 is a mechanism of protein denaturation. Under these conditions, 95% of adsorbed proteins are non-functional (Butler et al. 1992; Davies et al. 1994). This problem is not very important for a classical ELISA because mostly a small fraction of proteins having a native conformation is still detectable. However, this phenomenon can be very troublesome for phage antibody library selections, because phage antibodies binding to epitopes only present in denatured molecules may be selected.