Rapid antibody discovery on hard targets combined with fine-grained engineering and optimization Sarah Ives, Chelsea Jones, David Maurer, Christina Pettus, Valerie Chiou, Raymond Newland, Shahrad Dareikia, Devin Pineda, Jean-Philippe Buerckert, Jessica Salas, Austin Sanchez, Lindsay Blore, Sindy Liao, Devin Pineda, Lindsay Blore, Chris Smith, Giles Day, Sawsan Youssef, Jacob Glanville

Abstract Therapeutic candidate antibodies against challenging targets are difficult to find, and most antibody leads need multi-parameter optimization. In a field where both first-in-class and best-in-class define the victors, the time required to discover and optimize antibody leads can be problematic. Here, we review the result of ten years of progress in computational optimization of antibody discovery libraries through the lens of high-throughput data collection technologies, with specific case studies of modern repertoire design principles applied to a panel of multiple drug targets. We emphasize specific case GPCRs IO targets Anti-idiotypes studies of historically challenging drug targets, including GPCRs, Ion channels, HIV coat , functionally active IO antibodies, rare epitopes, and anti-idiotypes.

• Employing fully germline scaffolds with robust therapeutic pedigrees • Developing a computationally optimized natural CDR shuffling paradigm to maximize diversity • Constructing the library sequentially with thermal pressure and functional selection pressure at each step • Utilizing the AbGenesis sequence analysis algorithms to characterize libraries pre-and post-panning

• A unique affinity maturation strategy to explore the complete germline landscape for potential targets Broadly neutralizing HIV Allosteric inhibitors CAR-T / pMHC

Functionally active, fully human anti-GPCRs Targeting Ion Channels AntiAnti-CXCR5-CXCR5 clone P1G02 DB01_A03 Anti-Anti-CXCR5CXCR5 clone P1F03 DB01_A04 AntiAnti-CXCR5-CXCR5Anti-CXCR5 clone P2B09 DB01_A05 P1F03 Anti-CXCR5 P2B09 [Antagonist Assay] [Antagonist Assay] [Antagonist[Agonist Assay] Assay] [Agonist Assay] SelectedHEK- ClonesRepA on BackgroundHEK-RepB R4 HEK-RepC Ion-RepA Ion-RepB Ion-RepC 60000 21000 110000 HekCP_A_R4 HekCP_B_R4 HekCP_C_R4 NavCP_A_R4 NavCP_B_R4 NavCP_C_R4 0 0.01 0.01 −0.2 −0.21 −0.21

200000 HekCP_A_R4 −1

100000 0.49 0.69 0.07 0.11 0.06 HEK 20000 100000 −2 0 0 0.06 0.06 −0.1

−3 - RepA 40000 19000 150000 −4 NA NA 0.1 −0.19 0.07 90000 80000 −5 0 0.02 −0.2 −0.21 −0.21 HEK

18000 HekCP_B_R4 −1 0.66 0.12 0.12 0.09 RLU RLU RLU RLU 100000 RLU

80000 −2 - 0 0.05 0.04 −0.1 RepB 20000 17000 60000 −3 NA NA NA NA 70000 −4

16000 50000 −5 HEK 0 −0.2 −0.21 −0.21

40000 HekCP_C_R4 −1 - Anti-CXCR5 Anti-CXCR5 RepC 0 15000 60000 0.18 0.08 0.12 clone DB01_A03 clone DB01_A04 -3 -2 -1 0 1 2 −2 -3 -2 -1 0 1 2 -3 -3 -2 -2 -1 -1 0 0 1 1 2 2 -3 -2 -1 0 1 2 0.05 0.04 −0.1 10 10 10 10 10 10 10 10 10 10 10 10 10 1010 1010 10 10 10 10 10 10 10 10Over 10500 unique10 binders10 10 10 −3 CHO backbone CHO backbone −4 NA NA NA selected discovered against a sodium a both mAb [µg/mL] mAbmAb [ g/mL] [µg/mL] mAb [ g/mL] −5 mAb [µg/mL] µ µ a inSilico 0 −0.17 −0.16 a lab Tm1: 69.8°C and a potassium channel. NGS- −1 NavCP_A_R4 Tm1: 70.5°C −0.35 −0.3 aIon no −2

Tm1: 71.4°C guided discovery doubled the −0.31 −0.26 - Tagg 266: 72°C RepA Anti-CXCR5 Clone Tagg 266: 72.3°C −3 Frequency sample 2 (log10) Tagg 266: 79.8°C number of clones identified. −4 −0.25 −0.19 Control −5 Heavy V- bias observed in 0 −0.14 Ion −1 NavCP_B_R4 Jurkat backbone Jurkat backbone −0.38 - RepB successful binders compared −2 −0.33 −3 with library. Exquisite selectivity −4 −0.11 Ion −5 -

to ion channel targets. 0 RepC −1 NavCP_C_R4 −2 v −3 Parental Cell Line (CHO or Jurkat) −4 −5 CRTH2 Cell Line (off-target GPCR) −5 −4 −3 −2 −1 0 −5 −4 −3 −2 −1 0 −5 −4 −3 −2 −1 0 −5 −4 −3 −2 −1 0 −5 −4 −3 −2 −1 0 −5 −4 −3 −2 −1 0 CXCR5 Cell Line (Target GPCR) Frequency sample 1 (log10)

Exquisite specificity: therapeutic anti-idiotypes Rare epitopes: broadly neutralizing anti-HIV abs

Proof-of-Concept: Rapid De Novo Discovery of Functionally Relevant Therapeutic Leads Against Anti-HIV broadly neutralizing antibodies Tumor-Specific Epitopes in B-Cell Lymphoma Generated in 4 weeks

KD,apparent (nM) *6.2 In a collaboration with 9.7 ± 0.2 the Peter Kim lab at *3.3 ± 0.2 Anti-idiotype antibodies Stanford university, 4.0 ± 0.06 functional in-vivo Distributed Bio generated hundreds of *7.0 ± 0.05 unique, specific and *4.3 broadly binding and neutralizing anti-HIV 22 ± 0.5 antibodies against a 4.0 ± 0.05 specific epitope on the polymorphic coat of the HIV virus. Peter Kim Lab, Stanford University

Allosteric ligand ejectors Ultra-selective anti-pMHC antibodies

Total = 6,512 enriched clones to • Blocked by Growth Hormone = 3,577 clones • Partial block by Growth Hormone = 1,300 clones • Unblocked = 1,634 clones

In the 8,500 unique clones enriched GHR vs GHR GHR vs GH:GHR GH:GHR vs GH:GHR against GHR, we have discovered 3 that Anti-pMHC allosterically displace hGH upon antibodies selected v against a specific binding. pMHC complex and When split-panning with GHR 0.75 deselected against a pre-blocked by the native ligand panel of single and Ligand ejectors provide a novel GH, non-blocking clones continue double amino acid biological approach to quench an to enrich while those that 0.50 mutant variants to active signaling process. compete with the GH epitope identify clones with lower in frequency. GH/GHR ELISA OD450 0.25 superior selectivity

blocked partial−block non−block High Throughput Sequencing Binding measured on Carterra LSA

Distributed Bio antibody discovery workflow From target to hundreds of leads in 8 weeks

Monoclonal lead discovery & v development time, in months

Interested? Contact [email protected]