SECURITIES AND EXCHANGE COMMISSION
Washington, D.C. 20549
Pursuant to Section 13 or 15(d)
of the Securities Exchange Act of 1934
Date of Report (Date of earliest event reported): November 8, 2019
(Exact name of registrant as specified in its charter)
(State or other jurisdiction
11099 N. Torrey Pines Road, Suite 190
La Jolla, California
|(Address of principal executive offices)||(Zip Code)|
Registrants telephone number, including area code: (858) 750-4789
Check the appropriate box below if the Form 8-K filing is intended to simultaneously satisfy the filing obligations of the registrant under any of the following provisions:
Written communications pursuant to Rule 425 under the Securities Act (17 CFR 230.425)
Soliciting material pursuant to Rule 14a-12 under the Exchange Act (17 CFR 240.14a-12)
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Securities registered pursuant to Section 12(b) of the Act:
Title of each class
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|Common Stock, par value $0.001 per share||THOR||The Nasdaq Global Select Market|
Indicate by check mark whether the registrant is an emerging growth company as defined in Rule 405 of the Securities Act of 1933 (§230.405 of this chapter) or Rule 12b-2 of the Securities Exchange Act of 1934 (§240.12b-2 of this chapter).
Emerging growth company ☒
If an emerging growth company, indicate by check mark if the registrant has elected not to use the extended transition period for complying with any new or revised financial accounting standards provided pursuant to Section 13(a) of the Exchange Act. ☒
|Item 8.01|| |
On November 9, 2019, certain members of the management team of Synthorx, Inc. (the Company) will be presenting two posters (the THOR-707 Poster and the IL-10 Poster) at the Society for Immunotherapy of Cancer 2019 Annual Meeting & Pre-Conference. A copy of the THOR-707 Poster is attached as Exhibit 99.1 to this Current Report on Form 8-K and is incorporated herein by reference. A copy of the IL-10 Poster is attached as Exhibit 99.2 to this Current Report on Form 8-K and is incorporated herein by reference.
On November 10, 2019, certain members of the management team of the Company will be presenting a poster (the THOR-809 Poster) at the American College of Rheumatologys 2019 Annual Meeting. A copy of the THOR-809 Poster is attached as Exhibit 99.3 to this Current Report on Form 8-K and is incorporated herein by reference.
|Item 9.01|| |
Financial Statements and Exhibits.
|99.1||THOR-707 Poster, dated November 9, 2019.|
|99.2||IL-10 Poster, dated November 9, 2019.|
|99.3||THOR-809 Poster, dated November 10, 2019|
Certain statements contained in this report are forward-looking statements that involve a number of risks and uncertainties. Words such as believe, may, will, estimate, promise, plan, continue, anticipate, intend, expect, potential and similar expressions (including the negative thereof) are intended to identify forward-looking statements, though not all forward-looking statements necessarily contain these identifying words. For such statements, the Company claims the protection of the Private Securities Litigation Reform Act of 1995. Actual events or results may differ materially from the Companys expectations. Factors that could cause actual results to differ materially from those stated or implied by the Companys forward-looking statements are disclosed in the Companys filings with the Securities and Exchange Commission, including in the section captioned Risk Factors in the Companys Quarterly Report on Form 10-Q for the quarterly period ended September 30, 2019. These forward-looking statements represent the Companys judgment as of the time of this report. The Company disclaims any intent or obligation to update these forward-looking statements, other than as may be required under applicable law.
Pursuant to the requirements of the Securities Exchange Act of 1934, the Registrant has duly caused this report to be signed on its behalf by the undersigned hereunto duly authorized.
|Dated: November 8, 2019||By:|
|Laura Shawver, Ph.D.|
|President and Chief Executive Officer|
An Open-Label, Multicenter Phase 1/2 Dose Escalation and Expansion Study of THOR-707 as a Single Agent and in Combination with Pembrolizumab in Adult Subjects with Advanced or Metastatic Solid Tumors
David Luo;1 Raghad Abdul-Karim;2 Arun Azad;3 Joanna Bendell;4 Hui Gan;5 Filip Janku;6 Shiraj Sen;7 Tira Tan;8 Judy Wang;9 Lisa Schechet;1 Lauren Baker;1 Joseph Leveque;1 Tarek Meniawy10
1Synthorx, Inc., La Jolla, CA; 2NEXT Oncology, Texas Oncology, San Antonio, Tx; 3Peter MacCallum Cancer Centre, Melbourne, Victoria, Aus; 4Sarah Cannon Research Institute/Tennessee Oncology, Nashville, TN; 5Austin Health, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Aus; 6Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX; 7Sarah Cannon Research Institute at HealthONE, Denver, CO; 8Division of Medical Oncology, National Cancer Centre Singapore; 9Sarah Cannon Research Institute/Florida Cancer Specialists, Sarasota FL; 10Linear Clinical Research, Nedlands, Western Australia, Australia
BACKGROUND Synthorx Expanded Genetic Code Platform: A Synthetic DNA Base Pair Encodes Novel Amino Acids to Create Optimized Biologics X-Y Base Pair Creates New Codons That Specifically Encode Novel Amino Acid Chemistry Into Proteins Site-Specific Bioconjugation " Novel amino acid installation creates a dedicated, specific and stable chemical attachment site " Designed to bioconjugate moieties for improved properties: e.g. PEG Specificity " Improved target selectivity through altered receptor binding Polymer-Conjugates " Increased half-life " Epitope shielding through covalent and stable PEG attachment via bio-orthogonal chemistry Engineered Cells Install a Novel Amino Acid Utilizing X-Y to Produce Therapeutic Proteins with Optimized Properties Production System for Synthorins in E. coli Novel Amino Acid enters cells; used X and YTPs enter via by aminoacyl tRNA synthetase to transporter charge X-Y tRNAs X-Y containing plasmids encode product with X-Y codon and matching X-Y tRNA gene mRNA with X-Y codon matches with tRNA displaying anticodon Translation machinery decodes X-Y codons to introduce nAA into Synthorin proteins Dual Pharmacology of IL-2 is Mediated by 23 and 23 Receptor Sub-Types Receptor Immune Cells Immune Type Activated Response 2 3 Suppression High affinity Treg -11 CD4+ Treg up-regulation a (Kd ~ 10 M) Tcon Suppression Primarily Treg IL-2 2 3 Stimulation Intermediate affinity Tcon Proliferation of Tcon (K ~ 10-9M) reactive to Auto-antigens d Broadly expressed CD4 Th, CD8+ T, and NK cells
THOR-707 Strong Preclinical Rationale for Moving THOR-707 into Clinical Drives CD8+ Teff Cell Expansion and Tumor Infiltration at Levels Comparable to Tumor Microenvironment Re-Programmed Following Treatment Development Immune Checkpoint Inhibitors With THOR-707; Enhanced When Combined with Anti-PD-1 " THOR-707, as a single agent, elicits CD8 T cell activation and tumor infiltration, Select Immune Checkpoint Inhibitors THOR-707 including effector and memory subtypes, and improved survival. In combination with anti-PD-1, THOR-707 leads to durable anti-tumor responses and rejection * * * * * * upon re-challenge 80 Predose Predose TIL 60 " THOR-707 does not significantly expand Tregs, nor does it lead to increases in 3 mg/kg T of 60 eosinophils in NHPs % These results suggest that THOR-707 may CD3+ 40 " THOR-707 + anti-PD1 increases T Cell Receptor-mediated IFN-3 release which 40 in reprogram the tumor microenvironment to suggests that the combination strongly activates effector T cells; IFN-3 is a key Teff% express checkpoint inhibitory receptors (PD-1, in inducer of tumor antigen presentation CTLA-4) and ligands (PD-L1, PD-L2), 20 8+ 20 " On the other hand, anti-PD-1 did not increase IFN-3 release observed with THOR- CD8+ CD potentially reversing PD-1i/PD-L1i resistance 707 ex vivo, suggesting a low risk to exacerbate auto-immune effects of anti-PD-1s These results suggest that an IFN-3 mediated 0 0 " THOR-707 +/- anti-PD-1 does not induce release of cytokines associated with VLS Untreated Fvax CTLA-4 PD-1 PD-L1 PD-1 + Day 0 Day 1 Day 2 Day 3 Day 5 Day 7 Day 10 effect from the combination of THOR-707 and a CTLA-4 PD-1i may potentiate antigen-driven TCR or cytokine release syndrome (CRS) Following 3 Doses IV of CPIs Following Single Dose of THOR-707 responses in T effector cells and neoantigen " Similar exposure levels observed vs re-dosing in non-human primates: no B16F10 Model1 B16F10 model presentation via MHC expression to overcome indication of anti-drug antibodies PD-1i resistance in some refractory phenotypes
STUDY DESIGN Dose Escalation (3+3 Design) Dose Expansion Part 1 Part 3 THOR-707 Single Agent Single Agent and Combination Basket Studies in Select Populations All Comers Population Additional Optimal Single THOR-707 Q3W RP2D Dose Levels Agent and Dose Level N Cohort D Cohort A up to the Combination Dose 16 ug/kg MTD and Frequency - THOR-707 Single Agent RP2D THOR-707 8 ug/kg Recommended Q2W or Q3W Q3W Additional Phase 2 Dose THOR-707 Q2W RP2D Dose Levels Dose Level N (RP2D) up to the Cohort B MTD Dose Level 2 THOR-707 8 ug/kg Q2W Part 2 Cohort E THOR-707 + Anti-PD-1 Combo Combo RP2D Anti-PD-1 Sensitive Populations Combo RP2D Additional Q3W Dose Levels Q3W Dose Level N up to Cohort C Dose Level 2 the MTD Combo Q3W Dose Level 1 Clinical Sites Key Biomarkers CD8+ T effector cell counts in the periphery Ki67 expression levels CD4+ T regulatory cell counts NK cell counts in the and in the tumor microenvironment in peripheral blood immune cells in the periphery and in the periphery and in the tumor (Expansion to 1.5x above baseline the threshold for considering (Expression level between 40% and 60% the threshold for tumor microenvironment microenvironment a combination with a PD-1i) considering a combination with a PD-1i)
STUDY OBJECTIVES Primary Objectives: Secondary Objectives: " Evaluate the safety and tolerability of THOR-707 as a " Evaluate preliminary anti-tumor activity of THOR-707 as a single agent and in combination with a checkpoint inhibitor single agent and in combination with a checkpoint inhibitor (identify DLTs, AEs/serious adverse event profile) by determination of the ORR defined according to RECIST " Define the MTD and/or the RP2D of THOR-707 as a single Version 1.1 agent and in combination with a checkpoint inhibitor " Determine TTR, DOR,PFS, OS, and DCR of THOR-707 as a single agent and in combination with a checkpoint inhibitor STUDY ENROLLMENT " Each cohort will enroll approximately 3-6 subjects as per protocol. Up to 3 cohorts (dose schedules) may be open simultaneously " Once a dose level is declared safe and adequate PD activity is demonstrated at that dose level, up to 10 additional subjects may be enrolled into a Safety Expansion Cohort at the discretion of the Safety Review Committee (SRC) " After reaching the MTD and/or defining the RP2D, up to 15 additional subjects may be enrolled at the RP2D of the respective dosing schedule to further evaluate safety, pharmacodynamic effects, and anti-tumor activity Key Inclusion Criteria: Key Exclusion Criteria: " Males or females aged e18 years at " Inadequate washout from prior systemic anti-cancer therapy, surgery, and/or radiation screening " Primary central nervous system or leptomeningeal disease " ECOG performance status of 0 or 1 " Active autoimmune disease requiring systemic treatment within the past 3 months or " Life expectancy greater than or equal documented history of clinically severe autoimmune disease that requires systemic to 12 weeks as determined by the steroids or immunosuppressive agents Investigator " Abnormal pulmonary function " Histologically or cytologically confirmed " Active second malignancy, or history of previous malignancy that would impact the diagnosis of advanced and/or metastatic assessment of any study endpoints solid tumors with at least one tumor History " of allogenic or solid organ transplant lesion with location accessible to safely " Uncontrolled diabetes mellitus or other uncontrolled immune-related endocrinopathies biopsy per clinical judgment of the " Known HIV or active infection with hepatitis B or C Investigator " Clinically significant bleeding d2 weeks prior to initial THOR-707 dose " Measurable disease per RECIST v1.1 " Refusal of SOC, no reasonable SOC " Deep vein thrombosis or pulmonary embolism d3 months available, or standard therapy intolerable, " Severe or unstable cardiac condition d6 months prior to study treatment not effective, not accessible " Grade 3 or higher immune-related toxicity from prior immuno-oncology therapy " Prior anti-cancer therapy is allowed, " Is pregnant or breastfeeding, or expecting to conceive or father children within the including prior immunotherapy treatment, projected duration of the trial as long as any treatment related toxicity " History of non-pharmacologically induced prolonged corrected QT interval is resolved to an appropriate level. " Any serious medical condition (including pre-existing autoimmune disease or " Adequate cardiovascular, hematological, inflammatory disorder), laboratory abnormality, psychiatric condition, or any other liver, and renal function significant or unstable concurrent medical illness that in the opinion of the Investigator " Adequate laboratory parameters would preclude protocol therapy or would make the subject inappropriate for the study SAFETY SUMMARY " AEs (type, incidence, severity, HAMMER is a three-part global Phase 1/2 dose escalation and expansion timing, seriousness, and study evaluating the safety and anti-tumor activity of THOR-707, our Synthorin relatedness) not-alpha IL-2, in patients with advanced or metastatic solid tumors: " Vital sign measurements (blood " Part 1 of the study will determine the RP2D of THOR-707 as a single agent pressure, pulse, respiratory rate, " Part 2 of the study will determine the RP2D of THOR-707 in combination body temperature, pulse oximetry) with a PD-1 inhibitor " Physical examinations " Part 3 of the study will continue to evaluate safety along with the efficacy of " Clinical laboratory tests THOR-707 alone or in combination with a PD-1 or PD-L1 inhibitor as well as " Electrocardiograms in combination or sequenced with other established and emerging " Concomitant medications and immunooncology therapies via dose expansion procedures, including all supportive Safety and tolerability data at the 8 ug/kg Q3W starting dose were reviewed care provided by the Safety Review Committee and the study is currently enrolling subjects at 16 ug/kg Q3W and 8 ug/kg Q2W; the threshold for advancing Part 2 of the study in combination with a PD-1i was exceeded in the first cohort.
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
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
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
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
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
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
THOR-809: An IL-2 Engineered From an Expanded Genetic Alphabet for the Potential Treatment of Autoimmune Disorders
Marcos E. Milla; Carolina E. Caffaro; Lina Ma; Ingrid B. Joseph; David B. Chen; Taylor Ismaili; Kristine M. San Jose; Yelena Pavlova; Namit Singh; Lilia K. Koriazova; Hans R. Aerni; Michael J. Pena; Jerod L. Ptacin
INTRODUCTION " CD4+ regulatory T cells (Tregs) play a crucial role in maintaining immune homeostasis. Treg dysfunction is associated with multi-organ autoimmune (AI) and inflammatory-related diseases1. At high doses, recombinant interleukin-2 is approved to treat melanoma and renal cell carcinoma. At low doses, IL-2 selectively induces proliferation of Tregs through activation of the high-affinity alpha-beta-gamma IL-2 receptor (IL-2R23), resulting in immune suppression, without activating the medium-affinity beta-gamma IL-2 receptor (IL-2R23) expressed on CD8+ T effector cells (Teffs) and natural killer (NK) cells. Therapeutic benefit of low dose IL-2 has been demonstrated in chronic graft-versus-host disease and HCV-induced vasculitis2,3. Treg-mediated downmodulation by IL-2 of the immune effector function that prevents antigen recall may reset immune tolerance in select AI disorders. " We applied our expanded genetic alphabet platform using a novel, fully-synthetic DNA base pair, to create optimized biologics with enhanced pharmacological properties. Using this platform, we developed a site-specific, covalently bound mono-pegylated IL-2 that selectively expands peripheral Tregs in mice and non-human primates (NHP). " THOR-809 was identified as an IL-2 variant that maximized proliferation of peripheral Tregs, with an optimal PK/PD profile and a strong preference for Treg proliferation relative to Teffs (no expansion) and NK cells (minimal expansion). These properties correlated with extended half-life and sustained exposure. In NHPs, subcutaneous dosing of THOR-809 demonstrated dose-dependent proliferation of peripheral Tregs with no detectable proliferation of Teffs or NK cells up to 0.2 mg/kg. " The size of the PEG bioconjugate on THOR-809 modestly affects potency yet significantly modulates exposure and the level of Treg expansion in cynomolgus monkey. " Our results support continued investigation of THOR-809 as a therapeutic for AI disorders. Synthorx Expanded Genetic Code Platform: A Synthetic DNA Base Pair Encodes Novel Amino Acids to Create Optimized Biologics X-Y Base Pair Creates New Codons That Specifically Encode Novel Amino Acid Chemistry Into Proteins Site-Specific Bioconjugation " Novel amino acid installation creates a dedicated, specific and stable chemical attachment site " Designed to bioconjugate moieties for improved properties: e.g. PEG Specificity " Improved target selectivity through altered receptor binding Polymer-Conjugates " Increased half-life " Epitope shielding through covalent and stable PEG attachment via bio-orthogonal chemistry Engineered Cells Install a Novel Amino Acid Utilizing X-Y to Produce Therapeutic Proteins with Optimized Properties Production System for Synthorins in E. coli Novel Amino Acid enters cells; used X and YTPs enter via by aminoacyl tRNA synthetase to transporter charge X-Y tRNAs X-Y containing plasmids encode product with X-Y codon and matching X-Y tRNA gene mRNA with X-Y codon matches with tRNA displaying anticodon Translation machinery decodes X-Y codons to introduce UAA into Synthorin proteins
INTRODUCTION Dual Pharmacology of IL-2 is Mediated by 23 and 23 Receptor Sub-Types Receptor Immune Cells Immune Type Activated Response 2 3 Suppression Treg Treg up-regulation a High affinity -11 CD4+ (Kd ~ 10 M) Tcon Suppression Primarily Treg IL-2 2 3 Stimulation Intermediate affinity Tcon Proliferation of Tcon (K ~ 10-9M) reactive to Auto-antigens d Broadly expressed CD4 Th, CD8+ T, and NK cells THOR-809 is Designed to Activate Tregs Without Stimulating Conventional T cells (Tcons) THOR-809s Key Differentiation 1. IL-2 receptor 23 complex bias Site-specific PEG tunes IL-2 pharmacology for preferential Treg activation and expansion over Tcon 2. Ease of use pegylation allows for Q2W dosing or less frequent 3. Low risk for ADAs Sites targeted for pegylation have low risk of MHC-II binding and presentation. Covalent, stable PEG shields new amino acid Properties of PEG-IL-2 Synthorin for AI Stable PEG covalently attached to the novel amino acid installed where affinity at the 23 chain of the IL-2 receptor is reduced- potency at the IL-2 receptor requires the chain IL-2 R IL-2 R THOR-809 IL-2 PEG IL-2 R2 IL-2 R2 IL-2 R3 IL-2 R3
RESULTS Screening in Mice Identified Multiple IL-2 Synthorins With Differentiated Ex Vivo Pharmacology " Analysis of pSTAT5 activation in freshly isolated human PBMCs identified Synthorins with diverse potency profiles " Multiple Synthorins covering a broad Treg potency range and CD8/Treg ratios were selected for mouse PK/PD profiling 1000 THOR-designed N88R novel constructs 50 800 EC V91K THOR-designed 600 constructs based on THOR-809 IL-2 sites targeted on 400 select benchmark CD8/Treg compounds 200 IL-2 N88R/D109 0 N88 10 100 1000 10000 100000 1000000 EC50 at Tregs (pg/mL) Key Findings The pSTAT5 signal reports on receptor engagement and activation. We observed loose coupling between pSTAT5 and readouts of cell proliferation, including Ki67 and Treg counts. As a result, compound profiling for Treg expansion and activation in vivo (mouse) was required to select candidates with the best pharmacological properties. Functional Screen For Treg Expansion in Mice Identified THOR-809 Maximum Fold Increase in Treg % in Total Blood Cells A single subcutaneous dose (~0.9 mg/kg) of the indicated Synthorin variant was administered to C57/Bl6 mice. Flow cytometry was used to quantitate Treg (CD4+ CD25+ FoxP3+) expansion in peripheral blood. % 30 Treg of Singlets 20 Change in 10 mutein 809 Fold - Gylated V91K Fc -mutein THOR Max PEN88R 0 Vehicle A B C D E F G H I J K THOR-809 Synthorin Construct THOR-809 Robustly Expands Tregs in Mice Comparison of THOR-809 and a PEGylated IL-2 N88R mutein construct for Treg expansion in peripheral blood using flow cytometry. Vehicle THOR-809 0.9 mg/kg PEG N88R mutein 0.3 mg/kg Treg % in Total Blood Cells CD8+ T Cell % in Total Blood Cells 7 7 6 6 5 Singlets 5 in Singlets 4 4 in % % 3 Cell 3 2 T 2 Treg 1 CD8+ 1 0 0 0 1 2 3 4 5 6 7 8 Pre- 1 2 3 4 5 6 7 8 (2 h) Day dose Day
RESULTS THOR-809 Induces Treg Activation and Biomarkers of Suppressive Function in Mice Comparison of THOR-809 and a PEGylated IL-2 N88R mutein construct for Treg induction of biomarkers that correlate with suppressive function. Vehicle THOR-809 0.9 mg/kg PEG N88R mutein 0.3 mg/kg CD25 FoxP3 30,000 60,000 20,000 40,000 (MFI) Intensity 10,000 20,000 0 0 Pre- 0 1 2 3 4 5 6 7 8 Pre- 0 1 2 3 4 5 6 7 8 dose (2 h) Day dose(2 h) Day Helios ICOS Fluorescence 20,000 15,000 Mean 15,000 10,000 Treg 10,000 5,000 5,000 0 0 Pre- 0 1 2 3 4 5 6 7 8 Pre- 0 1 2 3 4 5 6 7 8 dose (2 h) Day dose (2 h) Day Pharmacokinetics of THOR-809 in NHP THOR-809 Pharmacokinetic Parameters in Non-human Primates Single subcutaneous dose of THOR-809 administered at t=0 THOR-809 0.12 mg/kg THOR-809 0.67 mg/kg 10000 Test Article Mean (ng/mL) Dose Unit Parameter value 1000 h t 17.2 1/2 THOR-809 809 h*ng/mL AUC last 18,349 - 0.12 mg/kg 100 ng/mL Cmax 361 THOR h t1/2 18.0 10 THOR-809 h*ng/mL AUC last 71,940 0.67 mg/kg ng/mL Cmax 1,358 Plasma 0 0 24 48 72 96 120 144 168 192 Hours Post-dose THOR-809 Induces Treg Expansion in NHP THOR-809 induced large-scale expansion of peripheral Tregs in blood Treg Percentage in Total Blood Cells Pre-dose Vehicle THOR-809 0.67 mg/kg 25 THOR-809 0.2 mg/kg THOR-809 0.07 mg/kg BUV395 Singlets 20 15 A::CD25 7 days post-dose in - % 387 - 10 371 Treg - CD4 5 Comp 0 Como-561-582-A :: FOXP3 PE D-1 Pre 24 36 72 120 168 192 240 312 360 528 Flow cytometry plot showing Treg population (CD25+ Hours Post-dose FoxP3+) in CD4+ cells pre-and post-dose
RESULTS THOR-809 Does Not Expand CD8+ T or NK Cells in NHP Minimal changes in peripheral CD8+ T cells and NK cells in blood post-dose Vehicle THOR-809 0.12 mg/kg THOR-809 0.67 mg/kg Fold Change in CD8+ T Cell % Fold Change NK Cell % in 10 in Total Blood Cells 10 Total Blood Cells Change 8 Change 8 Fold 6 Fold 6 Singlets 4 4 Singlets in 2 2 in CD8+% 0 NK% 0 D-1 Pre 24 36 72 120 168 192 240 312 360 528 D-1 Pre 24 36 72 120 168 192 240 312 360 528 Hours Post-dose Hours Post-dose Plots normalized to pre-dose percentage of each cell type. THOR-809 Induces Treg Biomarkers of Activation and Proliferation in NHP Quantitation of induction of biomarkers in peripheral blood Tregs using flow cytometry (pSTAT5, Ki-67, CD25) Vehicle THOR-809 0.12 mg/kg THOR-809 0.67 mg/kg pSTAT5 Ki-67 100 100 80 80 - 67+ 60 60 PSTAT5+ Ki 40 Treg 40 Cells % 2 % 2 0D-1 Pre 2 8 24 36 72 120 168 192 240 312 360 528 0D-1 Pre 2 8 24 36 72 120168192240312360528 Hours Post-dose Hours Post-dose 5000 CD25 4000 MFI CD25 3000 2000 Treg 1000 0 D-1 Pre 2 8 24 36 72 120168192240 528312360 Hours Post-dose THOR-809 Induces Treg Biomarkers of Differentiation and Suppressive Function in NHP Quantitation of induction of biomarkers that correlate with Treg suppressive function in peripheral blood Tregs using flow cytometry. FoxP3 and Helios are Treg transcription factors that control gene programs for Treg differentiation and suppressive activity. Vehicle THOR-809 0.12 mg/kg THOR-809 0.67 mg/kg FoxP3 Helios 10000 2500 8000 2000 MFI 6000MFI FoxP3 Helios 1500 4000 Treg 2000 Treg 1000 500 0 D-1Pre 2 8 24 36 72 120168192240 528312360 D-1Pre 2 8 24 36 72 120168192240 528312360 Hours Post-dose Hours Post-dose
RESULTS PEG Length Modulates THOR-809 Exposure and Pharmacodynamics in NHP THOR-809 30 kDa 0.12 mg/kg EC 50 THOR-809 30 kDa THOR-809 50 kDa THOR-809 50 kDa 0.2 mg/kg EC50 pSTAT5 Activation in Tregs PK: Plasma Levels vs Time 8000 (Ex Vivo) (ng/mL) 10000 MFI 6000 1000 T5 4000 Plasma 100 pSTA in 800000 3.4X 600000 2000 809 10 400000 Treg - AUC/dose 200000 1 (h*kg*ng/mL/mg) 0 0 THOR 104 105 106 107 108 0 25 50 75 100 125 150 175 Dose (pg/mL) Time (h) Peripheral Treg Expansion at 25 EC Dose 50 20 Vehicle THOR-809 30 kDa Singlets 15 THOR-809 50 kDa in % 10 Treg 5 0 D-1 Pre 24 36 72 120 168 192 240 312 360 528 Time (h) Larger THOR-809 PEG Bioconjugate Increases Peripheral Treg Expansion at Equivalent Predicted Receptor Occupancy " THOR-809 precursor protein was conjugated to 30 kDa and 50 kDa linear mPEGs " THOR-809 with a 50 kDa PEG had a similar potency (~2-fold reduction in EC50) relative to THOR-809 with a 30 kDa PEG for pSTAT5 activation in Treg cells (top left panel) " A single subcutaneous dose of each compound was administered to cynomolgus monkeys, dose calculated for half-maximal IL-2 receptor occupancy (EC50) in Tregs at Cmax Analysis of compound levels vs time demonstrated that THOR-809 with a 50 kDa PEG had a much larger exposure relative to the 30 kDa PEG bioconjugate (3.4-fold dose-normalized increase in AUC; top right panel) THOR-809 with a 50 kDa PEG induced a larger and more persistent expansion of peripheral Treg cells relative to the 30 kDa PEG bioconjugate (bottom panel) " We conclude that peripheral Treg expansion in response to THOR-809 is exposure (AUC) driven CONCLUSIONS " Ex vivo screening for PEGylated identified IL-2 R biased IL-2 compounds Screening identified multiple Synthorin IL-2 constructs biased for IL-2R Identified compounds- broad range of Treg potency and Tcon:Treg ratios " Screening in mice identified THOR-809 as a specific/effective Treg activator " In mice, a single subcutaneous dose of THOR-809 induced: Sustained pSTAT5 signaling in Treg cells Specific expansion of Tregs, not Tcons Increase in markers of Treg differentiation and suppressive function Upregulation of FoxP3, CD25, Helios, and ICOS " In cynomolgus monkey, a single subcutaneous dose of THOR-809 induced: Dose-dependent expansion and activation of Tregs, but not Tcons Increased Treg biomarkers for differentiation and suppressive function " The size of the PEG bioconjugate modulates both the exposure and level of Treg expansion REFERENCES 1. Sakaguchi S, et al. Cell. 2008;133:77587. 2. Saadoun D, et al. N Engl J Med. 2011;365:206777. 3. Rosenzwajg M, et al. J Autoimmun. 2015;58,4858.