EX-99.2 3 d805445dex992.htm EX-99.2 EX-99.2

Exhibit 99.2

 

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Using a Novel Synthetic Biology Platform to Generate a Homogeneous Dipegylated IL-10 With Native Potency

Carolina E. Caffaro; Jerod L. Ptacin; Nicole Acuff; Yelena Pavlova; Jasmine Nguyen; Kristine M. San Jose; Gavin Hong; Lina Ma; Taylor Ismaili; Kelsea Loescher; Ingrid B. Joseph; Jill Mooney; Joseph A. Leveque; Marcos E. Milla

Synthorx, Inc., La Jolla, CA

 


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INTRODUCTION Synthorx Expanded Genetic Code Platform For Optimized Biologics Production System for Synthorins in E. Coli " dX-Y containing plasmid encodes gene product with X-Y codon and matching X-Y tRNA gene " (d)X-YTPs imported via transporter " Novel amino acid (UAA) diffuses into cells; orthogonal aminoacyl tRNA synthetase charges it onto X-Y tRNA " Translation machinery decodes X-Y codon to introduce UAA into Synthorin proteins Site-Specific Bioconjugation Specificity Polymer-Conjugates " Novel amino acid installation " Improved target selectivity " Increased half-life creates a dedicated, specific when altered receptor binding " Epitope shielding through and stable chemical attachment is required for intended covalent and stable PEG site pharmacodynamic response attachment via bio-orthogonal " Designed to bioconjugate and/or safety chemistry moieties for improved properties: e.g. PEG IL-10 Pegylation is Required to Optimize its Pharmacological Effects IL-10 Background " IL-10 induces antigen-specific immunity: CD8+ T cell expansion and increased cytotoxicity " Antigen-specific recognition by TCR induces upregulation of IL-10R1 and PD-1 " Reduces inflammatory cytokine responses that contribute to a suppressive tumor microenvironment " IL-10 knock-out mice show increased tumor incidence and reduced immune surveillance " Clinical evidence suggests a benefit across multiple tumor types (NSCLC, PDAC and RCC) IL-10 Attributes " Highly potent cytokine with a very short half life " 160 amino acid polypeptide (~18 kDa), composed of a non-covalent homodimer " Contains 13 lysine residues susceptible to pegylation if conditions are not well-controlled Adapted from Martin Oft. Cancer Immunol Res. 2014 Synthorxs Platform Allows for Pegylation of IL-10 Away from the Receptor Interface IL-10 Synthorin " Pegylated at a specific site away from the receptor interface to maintain comparable potency to native PEG PEG IL-10 Covalent, irreversible click chemistry for high yield


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RESULTS IL-10 Synthorins are Produced as Stable Homogeneous Dipegylated Dimers WB CS Before After Before After " Homogeneous pegylation: Western Blot (WB) and Coomassie stain (CS) SDS- 75 kDa PAGE analysis under reducing 50 kDa Pegylated IL-10 conditions shows homogeneous 37 kDa pegylation of IL-10 Synthorin monomers 25 kDa 20 kDa Unpegylated IL-10 15 kDa " Homogeneous dimeric state: m/U molar mass determination by 2000 Native IL-10 size exclusion chromatography multiangle light scattering 1500 IL-10 Dipegylated (SEC-MALS) analysis is 1000 Synthorin consistent with IL-10 Synthorin 500 being a dipegylated dimer (1:1 protein:PEG ratio) 0 2 4 6 8 10 12 14 min m/U m/U 500 " Stable dimeric state: SEC 400 analysis of IL-10 Synthorin 400 300 200 dilutions shows no subunit 300 100 dissociation over the range of 0 -100 concentrations tested. No shift 200 in peak retention time or 2 3 4 5 6 7 8 9 min 100 monomer/dimer peak relative abundance (insert) 0 2.5 5 7.5 10 12.5 15 17.5 min IL-10 Synthorin Screening Algorithm for Identification of Pegylation Sites with Minimal Interference in Potency Screening Algorithm Assay Purpose MC/9 Proliferation Assay Concentration-Response Curves MC/9 proliferation Initial activity screen THOR-1003 (EC50=38,301 pg/mL) Native IL-10 (EC50=528 pg/mL) Ex vivo pSTAT3 in Receptor engagement in 0.8 mouse splenocytes human & mouse primary and human LRS ) cells 450 0.6 (OD DiscoverX 0.4 PathHunter(R) Cytokine Confirmation of In vitro Receptor assay receptor engagement Proliferation 0.2 TCR-dependent IFN3 0 Functional assay secretion 100 102 104 106 108 Concentration pg/mL In vivo PK/PD and THOR-1009 (EC =9,693 pg/mL) 50 efficacy studies Native IL-10 (EC =305 pg/mL) 0.3 50 THOR-1003 and THOR-1009 were Identified as ) 450 IL-10 Hits in the MC/9 Screening Assay (OD 0.2 Compound Relative potency (EC Synthorin/IL-10) 50 -1002 ~800 0.1 -1003 ~70 Proliferation -1004 ~100 -1015 ~400 0 -1009 ~30 100 102 104 106 Concentration pg/mL


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RESULTS IL-10 Synthorins Induce STAT3 Phosphorylation in Mouse Splenocytes Native IL-15 THOR-1003 THOR-1009 Balb/c Splenocytes C57BL/6 Splenocytes CD8+ T Cells CD8+ T Cells 1000 1000 800 800 pSTAT3 600pSTAT3 600 ofof MFI 400 MFI 400 0 0 100 101 102 103 104 105 106 107 108 100 101 102 103 104 105 106 107 108 pg/mL pg/mL NK Cells NK Cells 4000 4000 3000 3000 pSTAT3 2000pSTAT3 2000 ofof MFI 1000 MFI 1000 0 0 100 101 102 103 104 105 106 107 108 100 101 102 103 104 105 106 107 108 pg/mL pg/mL B Cells B Cells 4000 4000 3000 3000 pSTAT3 2000pSTAT3 2000 ofof MFI 1000 MFI 1000 0 0 100 101 102 103 104 105 106 107 108 100 101 102 103 104 105 106 107 108 pg/mL pg/mL IL-10 Synthorins Potently Induce STAT3 Phosphorylation in Mouse Splenocytes " No differences observed in IL-10R activation between mouse strains C57Bl/6 and Balb/c " THOR-1009 induced STAT3 phosphorylation in CD8+ T, NK and B cells with only ~10-fold reduction in potency relative to native IL-10 in both mouse strains " THOR-1003 activates the IL-10R with ~4-fold less potency than THOR-1009 EC50 (ng/mL) IL-10 THOR-1003 THOR-1009 Balb/c Splenocytes CD8+ T 4.07 165 37.5 NK 1.08 47.8 12.3 B cells 1.30 46.1 12.5 C57BL/6 Splenocytes CD8+ T 3.65 244 38.2 NK 1.06 60.9 17.3 B cells 0.83 36.9 9.18


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RESULTS IL-10 Synthorins Show Comparable Potency to Native IL-10 for Induction of pSTAT3 in Human Primary Immune Cells " THOR-1009 potency for induction of STAT3 phosphorylation is within 3-fold of native IL-10 " THOR-1003 is ~4-fold less potent than THOR-1009, as observed with mouse splenocytes Human LRS Native IL-15 THOR-1003 THOR-1009 2500 CD8+ T Cells 800 NK Cells 2000 600 pSTAT3 1500 pSTAT3 400 of 1000 of MFI MFI 500 200 0 0 10-1 100 101 102 103 104 105 106 107 108 10-1 100 101 102 103 104 105 106 107 108 pg/mL pg/mL 300 B Cells pSTAT3 250 200 of MFI 150 100 10-1 100 101 102 103 104 105 106 107 108 pg/mL EC50 (ng/mL) IL-10 THOR-1003 THOR-1009 Human LRS CD8+ T 28.0 401 89.7 NK 30.3 371 93.2 B cells 23.7 145 49.9 Orthogonal In Vitro Cell Assay Confirms Potency of THOR-1009 Variant IL-10 IL-10 drives R1/2 dimer formation " Pegylated variants tested via PathHunter" eXpress IL-10 R1/R2 dimerization assay IL-10R1 IL-10R2 (Eurofins/DiscoverX) " THOR-1009 shows ~15-fold decrease in EC50 relative to IL-10 standard https://www.discoverx.com/products-applications/cell-based-assays-for-biologics/interleukin-bioassays IL-10 Standard THOR-1009 100 100 (%) 75 75 (%) Activity 50 Activity 50 25 25 0 0 0.0001 0.001 0.01 0.10 1 0.001 0.01 0.10 1 10 uM uM Compound EC50 (uM) IL-10 0.0139 THOR-1009 0.2123


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RESULTS THOR-1009 Enhances CMV-specific CD8+ Memory T Cell Responses " Based on initial screens, THOR-1009 was CMV Peptide Presentation Assay selected for advance ex vivo profiling Isolate CD8+ T cells from PBMCs " CMV-specific recognition by TCR induces upregulation of PD-1 and IFN3 release CMV-peptide pulse non-CD8 T cell fraction " Human CD8+ T cells were sorted and expanded by co-culturing with antigen Co-culture CD8+ T cells with +/- CMV peptide presenting cells from the same donor, in the pulsed non-CD8s, +/- THOR-1009 or IL-10 presence or absence of CMV peptide, at different concentrations of THOR-1009 or IL-10 control Measure CMV Tetramer positive T cell response by flow cytometry THOR-1009 Increases IFN3 Release Upon Antigen-specific TCR Activation IL-10/peptide THOR-1009/peptide 10 10 8 8 (%) (%) 6 6 Gamma 4 Gamma 4 IFN IFN 2 2 0 0 IL-10/no peptide THOR-1009/no peptide 0 10 100 1000 0 10 100 1000 0 1000 0 1000 ng/mL ng/mL THOR-1009 and IL-10 Increase PD-1 Expression Independent of TCR Activation IL-10/peptide THOR-1009/peptide IL-10/no peptide THOR-1009/no peptide 100 100 80 80 (%)(%) 60 60 Positive 40Positive 40 PD1 PD1 20 20 0 0 0 10 100 1000 0 10 100 1000 0 1000 0 1000 ng/mL ng/mL CONCLUSIONS " IL-10 is known to induce antigen-specific immunity: CD8+ T cell expansion and increased cytotoxicity. It also reduces inflammatory cytokine responses that contribute to a suppressive tumor microenvironment " We have employed our Expanded Genetic Alphabet platform to pegylate IL-10 at a specific site away from the receptor interface. By employing orthogonal covalent click chemistry for highly efficient bioconjugation, we enabled production of stable, homogeneous dipegylated IL-10 Synthorin homodimers " Pegylated IL-10 Synthorin variants were designed for half-life extension with minimal interference of receptor engagement. Initial hits identified employing an MC/9 cell proliferation screening assay were confirmed employing additional cell-based orthogonal readouts for IL-10R1/2 assembly and induction of pSTAT3 in mouse and human primary cells. Our screening results underscore the importance of site-specific pegylation for cytokine activity. THOR-1009 stood out as the most active hit from those screened " THOR-1009 enhanced IFN3 expression in an ex vivo CMV-specific CD8+ T memory cell assay more efficaciously than IL-10 control protein " THOR-1009 will next be evaluated in vivo, in PK/PD and efficacy studies