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1. Three main methods for bispecific antibody: hybrid hybridoma

chemical conjugation methods recombinant BsAb fragment 2. How to get?

Inoculation of animals with antigen

High-throughput screening of hybridoma clones

Synthetic and semi-synthetic libraries(combine with phage display and so on)

3. What is the mechanisms?

1) antibody binds antigen and prevents it from interacting with other molecules

2) act as a receptor antagonist

Antibody binds the receptor and inactive it by blocking the binding site of an activating ligand

3) act as agonist. Function of a natural ligand and activating the receptor 4) act as an effector function activator

Antibody dependent cellular cytotoxicity(ADCC) and CMC 5) act as a chemotherapy or radiotherapy adjunct

6) As a way to redirect cytotoxica agent or immune effector cells to target sites.

4. The meaning of Bispecific antibodies(BsAb) Simultaneously bind two separate and distinct antigens Or different epitopes of the same antigen Example: cross-linking tumor and effector cells

One arm binds an antigen expressed on the cell targeted for destruction and the other arm binds a chemotherapeutic drug, radioisotope or toxin

5. What is the obstacle of BsAb?

Low yield and poor quality after many step”s purification

Expressing antibody A and antibody B in a single cell through hybridoma technique or DNA co-transfection

there will be a minor prosuct of the target antibody in the ten molecules 6. What is the main requirement of effective BsAb? 1) a novel structural format

2) An effective expression and high level production 3) Smaller for penetration but have a short in vivo half life Or do not need glycosylation and have higher yield in bacterial. 7. Recombinant approaches 1) increasing heterodimer

A: HC homodimerization problem

Knobs-into-holes BsAb IgG (T->W Y->A) B: LC-HC mispairing problem

Common light chain BsAb IgG

(using two antibodies of different specificities that share an identical LC)

Single chain Fv-Fc “ knobs-into-holes” BsAb

IgG-scFv fusion

(direct addition of a new antigen binding specificity to a fully functional antibody or an antibody-like molecule)

C-terminal scFv fusion

(VLA-CL+VHA-CH1-CH2-CH3-scFVB)2

(VLA-CL+VHA-CH1-scFVB)2

N-terminal scFv fusion

ScFVA-CL+scFVB-CH1-CH2-CH3 ScFVB-CL+scFVA-CH1-CH2-CH3

(Drawback low expression level for its large size and complexity structure)

Diabody- Fc fusion

Replace the Fab fragment with a bispecific diabody (VHA-VLB+VHB-VLA)

Single chain diabody-Fc Fusion

(VHA-VLB-VHB-VLA-CH2-CH3), then assembles into IgG-like dimers through the Fc domains, low levels of expression

Di-diabody

Heavier chain: VLA-VHB-CH2-CH3 fusion to the Fc domain Lighter chain: VLB-VHA