abcheck s.r.o* plzen czech republic · abcheck s.r.o* plzen czech republic mass humanization of...
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AbCheck s.r.o* Plzen Czech Republic
Mass Humanization of Rabbit Antibodies
Introduction: Since decades rabbits are used for generation of antibodies and are known to produce a great diversity of high affinity antibodies even against difficult targets. Humanization of rabbit antibodies, however, is considered to be problematic due to several structural constraints.
We developed a general approach that allows humanization of rabbit derived antibodies and enables mass humanization of entire rabbit immune repertoires.
www.abcheck.eu [email protected] +420 378 051 500
• Mass humanization of rabbit antibodies delivers multiple humanized antigen-specific hits
• Highly potent method to isolate humanized antibodies from rabbit immune repertoires
Conclusion:
Jacob Glanville (Distributed Bio), Peter Milovník (AbCheck), Remko Griep (AbCheck), Ahmad Trad (AbCheck) and Vera Molkenthin (AbCheck)
Figure 4. Workflow:
Immunize rabbits
PCR amplify SDRs
Clone rabbit SDRs in humanization landscape
Phage Panning: 2-3 cycles
Hit identification: ELISA
Sequence analysis of hits
*AbCheck is a 100% subsidiary of Affimed GmbH
Figure 2. Mass humanization landscapes represent the intermediate average of all possible humanizations:
Figure 1. Humanization landscapes are prepared to accept rabbit-derived SDRs:
Transfer principle specificity determining elements
Rabbit candidate antibodies
Humanization landscape: Selected human framework
regions with diversified CDRs
Figure 3. Each H3-defined clone can undergo tens of thousands of successful humanizations
ELISA with soluble scFv after 2 panning rounds
Figure 5. Method delivered many hits after 2 panning rounds
# of sequences within group
>A C A R V Y D D Y G D D Y F N I 61
>B C A R E S G Y Y A D Y A G Y G Y A Y G M D P 52
>C C A R A E G Y D D W H L S L 8
>G C A R D S Y D D Y G D W G G M D P 6
>E C A R D I Y D D Y G D P T R S D L 5
>I C A R E D E Y A E Y Y F D L 4
>D C A R A K N D D Y G D P D S L D L 3
>H C A R E A D G A Y T G Y G Y S Y G M D P 3
>K C A R Y N A D D Y G D Y Y G L D P 3
>Q C A R E G D W S Y S L D L 3
>F C A R D S S G W G R L D L 2
>J C A R S N Y G A L D L 2
>R C A R G T G Y A G Y G D A T G G F N I 2
>S C A R P L V S G W V F G G M D L 2
>L C A R A L Y G D H T F D P 1
>M C A R A Y G S N G G Y N P G R L D L 1
>N C A R D D W F Y F D L 1
>O C A R D S L G G F D L 1
>P C A R E F N Y D A Y S D Y Y A L D P 1
>T C V R D P A Y S Y V M 1
>U C V R G Y P G G S V G G D L 1
Figure 6. 21 CDR-H3 groups identified
Figure 7. Imprint of in vivo affinity maturation detectable in CDR-H3 sequences:
>3SO_31 C A R E S G Y Y P D Y A G Y G Y A Y G M D P
>3P_9 C A R E S G Y Y P D Y A G Y G Y A Y G M D P
>3PO_37 C A R E S G Y Y A D Y A G Y G Y A Y G M D P
>2P1_1 C A R E S D Y Y A D Y A G Y G Y A Y G M D P
>2B1_18 C A R E S D Y Y S D Y A G Y G Y A Y G M D P
>2B1_31 C A R E S G Y Y A D Y A G L G Y A Y G M D P
>3S_37 C A R E S G Y Y A D Y G G Y G Y A F G M D P
>2S1_17 C A R E S G Y Y A D Y G G Y G Y A Y G M D P
>Consensus C A R E S G Y Y A D Y A G Y G Y A Y G M D P