Phage display generated antigen mimicry and associated vaccination strategies. 

International context and working hypothesis 

Two consecutive student summer jobs (1998-99) in an R&D department of Novo Nordic A/S (Copenhagen, Denmark) specialized in generating highly stable enzymes by directed evolution inspired me to specialize in the technical field of protein engineering. I therefore joined the laboratory headed by Pr. Clark within the team of Dr. Kristensen (Aarhus University, Denmark). My research project was to isolate and characterize immunogenic targets for vaccination therapy of epithelial tumours. The Mucin 1 (encoded by the MUC1 gene) surface protein expressed on epithelial cells is heavily glycosylated. Interestingly, MUC1 is overexpressed and its glycosylation inhibited in many epithelial cancers. Indeed, unmodified Tn and Thomsen-Friedenreich (TF) bound to Mucin 1 is a hallmark for 90% of epithelial cancers, including breast, colon, lung and prostate cancers. Whereas this feature renders Mucin 1 bound Tn- and TF-antigens highly relevant for cancer targeting the inherent immunological inertia of carbohydrates complicates their applicability. Indeed, natural antibody responses to Tn- and TF-antigens are generally T-independent low affinity non-class switched IgM responses that consequently lack sufficient potency to block tumour growth. My project was to generate T-dependent high affinity class switched IgG responses to the TF-antigen by vaccination with immunogenic protein mimics of the carbohydrate structure.   

Objectives

My PhD was divided in an initial work aiming at generation of antigenic entities by antibody phage display and protein engineering conducted in the laboratory of Dr. Kristensen, followed by immunological evaluation of the isolated antigenic entities conducted by me in the laboratory of Nemod GmbH (Berlin, Germany).

Results 

The first part of the project aimed at generating protein mimics of Mucin 1 carbohydrate antigens. To achieve this I panned a scFv antibody phage library against monoclonal antibodies specific for Mucin 1 bound Tn- or TF-antigen. This approach led to the generation of a panel of anti-idiotypic scFvs specific for the idiotype of the target monoclonal antibody and thus potentially mimicking the corresponding Mucin 1 Tn- or TF- antigen.¹ To further characterize the selected anti-idiotypic scFvs, I subcloned, expressed and purified them as soluble protein. A number of scFvs lost their function when detached from the phage coat-protein, a phenomenon observed by numerous laboratories.² As scFvs are selected as active fusion proteins with the phage coat protein III, I hypothesized that retaining this fusion would rescue the inactive scFvs. I therefore developed and patented a scFv-protein III N-terminal domain fusion system, which was successfully applied to the anti-idiotypic antibodies and a number of other antibody phage display projects running in the laboratory.^3-6 To properly evaluate if the selected scFvs were indeed structural mimics of Mucin 1 bound Tn- or TF-antigen I performed i.p. mouse vaccinations with purified anti-idiotypic scFvs fused to the N-terminal domain of phage coat protein III. Serum from vaccinated animals had high anti-scFv titers, including anti-idiotypic antibodies, however the generated antibodies bound neither Mucin 1 glycoprotein nor Mucin 1 expressing tumour cells. I therefore concluded that the anti-idiotypic scFvs were not structural mimics of the Mucin 1 glycoepitope. However, I was able to show that the phage coat protein III fusion system was an effective adjuvant system that induces potent antibody responses to the fusion partner without the need of additional adjuvants.^{7, 8} The natural presence of filamentous phages and the extensive use of these viruses as “natural antibiotics” for human therapy make it an attractive adjuvant system with likely no or little side effects.  

The work conducted during my thesis has resulted in the following publications:

• Larsen M^*, Jensen KB^* et al. Biochem Biophys Res Commun. 2002; 298 : 566-573.
• Larsen M et al. Patent. 2002.
• Ravn P, Danielczyk A, Jensen KB, Kristensen P, Christensen PA, Larsen M et al. JMB. 2004; 343 : 985-996.
• Cuesta AM, Suarez E,Larsen M et al. Immunology. 2006; 117 : 502-506.
• Schilling R, Heil A, Langner K, Pohlmeyer K, Larsen M et al. Vaccine. 2006. 24. 4648-4650
• Larsen M et al. JIM. 2008; 339 : 220-227.
• Christensen PA, Danielczyk A, Ravn P, Larsen M et al. Scand J Immunol. 2009; 69 : 1-10.

^* Contributed equally