Antibody Engineering

The problems : How can I make large amounts of monoclonal antibodies in a safe, reproducible and efficient manner? How can I circumvent the immunization of animals, i.e. how can I create new antibody specificities in vitro ? How can I select the desired antibody specificities from large synthetic repertoires? Is it always necessary to make a full size antibody or can I use small fragments for certain applications , like !"I#$?
%hich e&pression systems are suitable for the various antibody formats?
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Antibody Engineering
5lassical antibody production .unctions and $pplications of antibodies #tructure function relationships of antibodies 'odern techni6ues for the generation and selection of new antibodies $lternatives to antibodies engineered binding proteins

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Antibody Applications
8H!/$9: 2. ;I#!$#!# I'$<I=< 2. ;I#!$#!# !=>:'! "I=3!; I''4=2#2/(!=8 $##$: ?!"I#$@ 9/28!I= ?%!#8!/=@ ("288I=< I''4=2HI#825H!'I#8/: 1 I''4=25:825H!'I#8/: I''4=29/!5I9I8$8I2= '$=: '2/! ...
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Ig<
How can I engineer this molecule...

papain cleavage site

pepsin cleavage site

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8he antigen binding site of an antibody is formed by C 5;/ regions in the variable domain

2nline 'acromolecular 'useum:

http:11www.clunet.edu1(io;ev1omm1gallery.htm
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num na

$ntibody diversity

8he human genome is presently estimated to contain ca. ,-.--- 7-.--thousand ) genes 8he number of different kinds of antibody molecules is probably about ,.B & +-

How could 7 & +)

genes encode ,.B & +-

different antibody

molecules ?
8he answer: each antibody chain is encoded by several different gene segments 8he genome contains a pool of gene segments for each type of chain. /andom assortment of these segments and mutations make the largest contribution to receptor diversity.

%e can learn

engineering principles from nature #tructure of a heavy chain D gene

"eader

framework +
5;/+

framework ,

framework 7
5;/, 5;/7

5;/ regions: c omplementarity d etermining r egions or hypervariable regions

5;/ +, , and 7 of the light chain

"D

framework light chain

4nrearranged DH genes

/earr ange ment of a DH gene

; ;

5h H +

5H,

D <ene ;atabase: http:11www.vbase,.org1

5H A- D & B E B+ D & ,B ; & C E Functional diversity D1 E D1;1E = segments

k k
H

5H+

,-)CB7 ca. AG +-

B+ DH

,B ;

CE

rearrangement
;

5H+

heavy and light chain combinations G +- C plus Functional diversity plus somatic mutations G +- H
http:11www.bio.davidson.edu1courses1Immunology1.lash1somaticrecomb.htmlc

HereIs an animation
of D" rearrangement
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<ene segments that contribute to antibody diversity .or the heavy ?H@ chains of human antibodies, the gene segments are:
J B+ D segments . !ach of these encodes most of the = terminal of
H

the antibody, including the first two ?but not the third@ hypervariable region.
J ,B ; ?KLdiversityL@ gene segments . 8hese encode part of the third
H

hypervariable region.
J C E ?KLFoiningL@ gene segments . 8hese encode the remainder of the
H

D region, including the remainder of the third hypervariable region.

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<ene segment usage for human antibody diversity

J on chromosome , for kappa light chain gene segments
J on chromosome ,, for lambda light chain gene segments J on chromosome +A for heavy chain gene segments

$ntibodies
DM

EM
DN

<ene #egments AB 7+
A B+ ,B C

5ombinations
,-- M chains
+,A N chains

EN
D ;
H H

),CB- H chains

$ny H chain ?)CB-@ with any " chain ?7,A@: ,.B & +- C possibilities (ut the true number is probably virtually limitless because of J variation in the e&act splice point and J the introduction of = nucleotides

;ifferent ways to produce antibodies polyclonal antibodies

by immunization of animals :

mice, rabbits, goats preparation from the blood of these animals

monoclonal antibodies
by immortalization of antibody producing ( cells

from immunized animals, usually miceO LhybridomasL

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Making Hybridomas
'yeloma cells have to #pleen cells are primary cells thus they are not able to grow in culture and: they supply an intact H<9/8 gene to the

be mutants: they lack the

enzyme H<9/8 thus they cannot grow in the presence of aminopterin

myeloma cell H$8 medium: contains

Hypo&anthine $minopterine <uanine

7 types of cells in the mi&ture after cell fusion: unfused spleen cells: will die naturally unfused myeloma cells: cannot survive in H$8 medium ?because they are H<9/8 negative@ hybridoma cells: will survive in H$8 medium ?H<9/8 positive, transformed cells@
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5ell fusion

three clones of hybrid cells

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'ethods for cell fusion

cells can be fused by treatment with polyethylenegylcol ?9!<@: binding of water by 9!< allows very close contact by membranes membranes fuse spontaneously 9!< is to&ic to cells, concentration and time of treatment are very important P electric pulses ?LelectrofusionL@: cells are centrifuged together in electroporation chamber low concentration of 9!< sometimes added for closer cell to cell contact application of pulse regeneration of cells plating after fusion: heterokaryon nuclei must fuse spontaneously for the generation of a hybrid cell sometimes chromosomes get lost during cell division hybridomas therefore tend to be unstable
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electroporation and electrofusion

>immermann 4, Dienken E, Halfmann E, !meis 55. !lectrofusion: a novel hybridization techni6ue. $dv (iotechnol 9rocesses. +*HBOA:)* +B-. <abriFel ', 3reft ', >orec /. 'onitoring lysosomal fusion in electrofused hybridoma cells. (iochim (iophys $cta. ,--) 2ct ,7O .
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