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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 v.lang@abcheck.eu +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