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 CAR VY D D Y G D D Y F N I 61 >B CAR ES G Y Y A D Y A GY G YAY GMDP 52 >C CAR AE G Y D D WH L S L 8 >G CAR DS Y D D Y G D WGG MDP 6 >E CAR DI Y D D Y G D P TR S DL 5 >I CAR EDE Y A E Y Y F DL 4 >D CAR AK N D D Y G D P DS L DL 3 >H CAR EAD G A Y T G Y GY S YGMDP 3 >K CAR YNA D D Y G D Y YG L DP 3 >Q CAR EGD WS Y S L D L 3 >F CAR DS S G WG R L D L 2 >J CAR S NY G A L D L 2 >R CAR GT G Y A G Y G D AT G GF N I 2 >S CAR PL V S G WV F G GMD L 2 >L CAR AL Y G D H T F D P 1 >M CAR AY G S N G G Y N PG R L DL 1 >N CAR DDWF Y F D L 1 >O CAR DS L G G F D L 1 >P CAR EF N Y D A Y S D YY A L DP 1 >T CVR DP A Y S Y V M 1 >U CVR GY P G G S V G G DL 1 Figure 6. 21 CDR-H3 groups identified Figure 7. Imprint of in vivo affinity maturation detectable in CDR-H3 sequences: >3SO_31 CARESGYYPDYAGYGYAYGMDP >3P_9 CARESGYYPDYAGYGYAYGMDP >3PO_37 CARESGYYADYAGYGYAYGMDP >2P1_1 CARESDYYADYAGYGYAYGMDP >2B1_18 CARESDYYS DYAGYGYAYGMDP >2B1_31 CARESGYYADYAGLGYAYGMDP >3S_37 CARESGYYADYGGYGYAFGMDP >2S1_17 CARESGYYADYGGYGYAYGMDP >Consensus CARESGYYADYAGYGYAYGMDP