The document summarizes antibody cloning strategies for producing antibodies and vaccines in plants. It discusses producing full-length antibodies, single-chain antibodies, and using antibody heavy chains to carry antigen proteins. Specifically, it proposes using antibody heavy chains as carriers to stably express vaccines in plants since heavy chains express well but light chains do not. It also suggests designing chimeric proteins by fusing antigen proteins to antibody domains and expressing them as monoclonal immune complexes in plants for low-cost vaccine production.

1. Antibody cloning strategies A summary of the mainstream strategies I studied during a post-doc at S.George Hospital Medical School (UK) and at ENEA Casaccia Reserach Centre (IT) Development of tailor-made v  ccines

2. Antibody cloning strategies x LIGTH CHAIN HEAVY CHAIN WHOLE ANTIBODY A conceptually simple approach to antibody cloning consist in transferring the cDNA encoding for the respective light and heavy chain into separate plants and then cross them to select the progeny that express the whole antibody

3. Antibody cloning strategies Yet, not always a full antibody is needed , some immunology studies focused only on the binding domain of the antibody that could be derived ina tailor made single protein named single-chain antibody Sc Ab

4. Fairly good expression levels in plants Whole Ab Sc Ab Low expression levels in plants VH CH1 CH2 CH3 Several studies highligthed that while is relatively difficult to obtain high level of production of scAb proteins, the full sized antibody is usually nicely produced in plant WHY?

5. WHY? The Ab heavy chain is recognised by (a) plant chaperone(s) that stabilize synthesis Heavy chain: good expr. levels Ligth chain: low expr..levels Whole Ab (H+L): good expr. levels Therefore, if the heavy chain is stably producde in plant it could then become a general carrier of other protein of interest, such as for example, vaccines. This suggest several chimaeric protein design as suggested in the next slides

6. Development of tailor-made v  ccines Ab heavy chain is a good “carrier” for the expression of eterologous proteins in plants CH2 CH3 Ag Fc guarantee good levels of expression in plant 1) 2) CH2 CH3 RetroViral Antibody guarantee good levels of expression in plant Fc is recognised by mucosal receptor and “sticks” the retroviral molecule to the mucosal tissues

7. Development of tailor-made v  ccines CH2 CH3 Ag CH1 FAVOUR Good expr. levels in plants AGAINST Anti Heavychain reaction in mouse DM1 CH2 CH3 Ag CH1 FAVOUR Good expr. levels in plants AGAINST Anti Heavychain reaction in mouse is MINIMIZED if ORAL IMMUNISATION DM2 Pepsin cleavage site Ag PURE ANTIGEN RELEASED INTO THE STOMAC pepsin pepsin

8. Development of tailor-made v  ccines x LIGTH CHAIN HEAVY CHAIN WHOLE ANTIBODY DM3 Ag Ag Ag Introducing V regions on a human C chain from pre-existing mAb (murine)

9. Development of tailor-made v  ccines DM3 IMMUNOCOMPLEX

11. Development of tailor-made v  ccines DM4 Library A GENERAL SYSTEM TO GENERATE TAILOR MADE MONOCLONAL IMMUNOCOMPLEX ALL HUMAN In sinthesys, that approach would potentially enable to produce a designed monoclonal immunocomplex to be produced in plant (with then relatively low cost of production) and without the need of immunisation

12. Development of tailor-made v  ccines CH2 CH3 Ag CH1 DM1 CH2 CH3 Ag CH1 DM2 Ag Ag (DM3) DM4 Higly plant-produced Vaccine Higly plant-produced CLEAVABLE Vaccine Human semi-synthetic monoclonal immunocomplexl Human synthetic monoclonal immunocomplexes

13. Selected links ENEA www.enea.it http://ideas.enea.it/ideas-in-biotechnology-agricolture-and-helth%20prevention/molecular-farming St. George Hospital Medical School Prof. Julian K-C. Ma Hotung Chair of Molecular Immunology http://www.sgul.ac.uk/about-st-georges/divisions/faculty-of-medicine-and-biomedical-sciences/cellular-molecular-medicine/staff-1/prof.-julian-k-c.-ma EC FP6 pharmaplanta project www.pharma-planta.org