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)

Antibody cloning strategies

Development of tailor-made vccines

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

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

Antibody cloning strategies

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?

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

Development of tailor-made vccines

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

Development of tailor-made vccines

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

AgPURE ANTIGEN RELEASED INTO

THE STOMAC

pepsin

pepsin

Development of tailor-made vccines

x

LIGTH CHAIN HEAVY CHAIN

WHOLE ANTIBODY

DM3

Ag

Ag

Ag

Introducing V regions on a human C chain from pre-existing mAb (murine)

Development of tailor-made vccines

DM3

IMMUNOCOMPLEX

Development of tailor-made vccines

CH2 CH3

Ag

CH1

DM1 + +CL

Sc library technology

DM4

1. Select anti Ag ScAb

2. attach VH and VL to the corresponding constant domains

3. Produce the whole Ab-AG protein in plant

Moreover, a more ambitous strategy could be to couple the chiameric approach to the scAb libraries that can be creted in vitro

Development of tailor-made vccines

DM4

Library

A GENERAL SYSTEM TO GENERATETAILOR 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

Development of tailor-made vccines

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

Selected links

ENEA www.enea.it

http://ideas.enea.it/ideas-in-biotechnology-agricolture-and-helth%20prevention/molecular-farming

St. George Hospital Medical SchoolProf. Julian K-C. Ma

Hotung Chair of Molecular Immunologyhttp://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