Objectives:The objectives of this proposal are partially based on our previous results from the EC Biotechnology (1992-1994) project "In vitro immunization of human B lymphocytes" where we have already shown the potential of in-vitro immunization to generate affinity-maturated and isotype-switched human monoclonal antibodies which potentially could be applied in a therapeutic context. Based on this progress, the objectives have been refocused to be more directly applied and complies with the policies of the 4th Framework EC Biotechnology programme (1994-98).The project involves two conceptually different but complementary approaches to obtain develop human high affinity antibodies of therapeutic interest. These approaches use cell biology to mimick the germinal center environment and molecular biology approaches based on antibody libraries and phage display. Our objectives include the following elements:
(1) Optimised in-vitro immunization protocols generating specific immunization and somatic mutation of human B cells.
(2) Improved methods for the rescue of V regions from B cells. (3) Novel design of V-gene repertoires.
(4) Analysis and characterization of anti-tumour antibodies with respect to specificity and effector functions.
(5) Improvement of antibodies by in-vitro evolution of affinity and specificity.
(6) Engineering and production of molecules (antibodies and fusion proteins) for therapy.
Outline of proposal content: Mimicking the GC micro-environment. The production of high affinity human antibodies requires that B cells first undergo somatic hypermutation of Ig V-region genes and that the high-affinity mutations are selected and expanded. In-vivo, these somatic mutations take place within the germinal centers (GC) of B cell follicles in secondary lymphoid organs. Peripheral blood provides a more accessible pool of B cells than lymphoid organs, but in order to use blood lymphocytes as the starting material the signals which drive B cells activated by antigen need to be analysed. Different approaches to in-vitro immunization for the generation of somatically mutated antibodies will be applied to selected antigenic systems of high relevance for applications in cancer therapy, i.e. MUC-l (a breast carcinoma associated antigen), other mucin epitopes and gangliosides.
Mutational analysis. Single B cell culture systems supporting mutations will be developed on the basis of results obtained from bulk cultures. Single cell cultures are the only way that will permit definite conclusions as to whether base differences between clonally related Ig-genes indeed have been generated in-vitro and are not the result of selection of in-vivo mutated B cells. Rescue of V-regions and novel design and selection of repertoires. Here, we will attempt a conceptually different route to the generation of high affinity therapeutic human antibodies as compared to above. The antibody phage display technology will be applied to generate human high-affinity anti-mucin or ganglioside antibodies. In the process of making these binding pairs, we seek to improve the existing phage technology, to improve vectors and improve the quality of display and selection efficiencies.
Engineering and production of molecules for therapy. For engineering of antibodies (Fab) construction of fusion proteins between a high-affinity anti-tumour antibody and effector molecules capable of biological response modification, such as human IL-12, will be attempted, and the fusion proteins will be expressed by eukaryotic host cells.
Analysis of antibodies. Human, high-affinity antibodies will be produced using a combination of the technologies described. Antibodies against the above antigens will be tested for biological relevance by immunohistology and flow cytometry, as well as by in-vitro cellular assays and in animal models. Tumor-transplanted SCID mice have been chosen as a preclinical model for therapy experiments. The reason for this is that this system can be adapted to studies of the immunotherapeutic effects of e.g.
antibody-IL-12 fusion proteins using human lymphocytes injected into tumour-bearing experimental animals.
Funding SchemeCSC - Cost-sharing contracts
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