Development of an ICOSL and BAFF bispecific inhibitor AMG 570 for systemic lupus erythematosus treatment M. Zhang1, F. Lee1, A. Knize1,2, F. Jacobsen1, S. Yu1,3, K. Ishida1, K. Miner1, K. Gaida1, J. Whoriskey1, C. Chen1, K. Gunasekaran1,4, H. Hsu1 1Amgen Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA; 2Abide Therapeutics, San Diego, CA; 3Shire Pharmaceuticals, Lexington, MA; 4Denali Therapeutics, South San Francisco, CA, USA. Objective Systemic lupus erythematous (SLE) is a heterogeneous disease lacking highly effective treatment options. Here we tested if targeting both BAFF and ICOSL has superior efficacy than single target inhibition in the mouse arthritis and lupus models. We also generated AMG 570, an ICOSL and BAFF bispecific inhibitory molecule, for potential treatment of autoimmune diseases such as SLE. Methods Murine BAFF/ICOSL bispecific, combination of BAFF and ICOSL inhibitors or single inhibitor was evaluated in the sheep red blood cell (SRBC) challenge model, mouse collagen induced arthritis (CIA) model, or NZB/NZW lupus models. AMG 570 was tested for human and cyno BAFF and ICOSL binding affinities by Kinexa A. AMG 570 dual target blocking activities was evaluated in human and cyno BAFF and ICOSL mediated B cell and T cell assay, respectively. Pharmacodynamics effect of AMG 570 was evaluated in cynomolgus monkey. Results Treatment with murine ICOSL/BAFF bispecific or combination therapy was more efficacious than single ICOSL or BAFF inhibitor in mouse NZB/NZW lupus model. Dual ICOSL and BAFF inhibition was also more effective in the mouse collagen induced arthritis (CIA) model. AMG 570 was developed as the clinical bispecific lead. AMG 570 inhibits human and cynomolgus monkey ICOSL and BAFF. B cell reduction was observed after AMG 570 treatment in cynomolgus monkeys, consistent with the pharmacological effect of BAFF inhibition. Conclusion By targeting both ICOSL and BAFF, AMG 570 has the potential to achieve superior efficacy in treatment of autoimmune diseases such as SLE and rheumatoid arthritis. Key words systemic lupus erythematosus, ICOSL, BAFF, T cell, B cell Clinical and Experimental Rheumatology 2019; 37:Clinical 906-914. and Experimental Rheumatology 2019 Development of ICOSL/BAFF bispecific for SLE treatment / M. Zhang et al. Ming Zhang Introduction and dendritic cell. It is released by pro- Francis Lee Systemic lupus erythematosus (SLE) teolytic cleavage and functions in the Anna Knize, remains an indication with high unmet periphery (14). BAFF inhibition results Frederick Jacobsen medical needs. Benlysta® (belimumab), in a significant reduction of B cell num- Steven Yu Katsu Ishida an anti-BAFF antibody with modest ef- ber in human, cynomolgus monkey, and Kent Miner ficacy, provides clinical validation for rodents, a hallmark of pharmacody- Kevin Gaida the BAFF pathway yet also indicates namics (PD) for BAFF inhibition (15- John Whoriskey the need for additional therapeutics 17). BAFF blocking antibody Benlysta Ching Chen with better efficacy (1). SLE is a het- is approved for SLE treatment, albeit Kannan Gunasekaran erogeneous disease. Thus, desirable ef- with modest efficacy. Hailing Hsu ficacy is likely achievable by targeting Given both aberrant B cell and T cell Please address correspondence to: multiple pathways. Among many cell are involved in SLE pathogenesis, we Dr Hailing Hsu, types and pathways involved in SLE hypothesise that addition of a BAFF Amgen Inc., One Amgen Center Drive, pathogenesis, aberrant B cells and T inhibitor to an anti-ICOSL antibody, is Thousand Oaks, CA 91320, USA. cells are critical drivers in autoantibody likely to achieve superior efficacy than E-mail: [email protected] production, inflammatory cytokine pro- BAFF and ICOSL inhibitor alone in Received on October 9, 2018; accepted in duction, and tissue damage. SLE treatment. We demonstrated that revised form on December 17, 2018. ICOSL is critical for TFH cell develop- dual ICOSL and BAFF inhibition, ei- © Copyright CLINICAL AND ment and T memory cell homeostasis ther by bispecific or combination treat- EXPERIMENTAL RHEUMATOLOGY 2019. (2). ICOSL is mainly expressed on ment, was more efficacious than single B cells and antigen presenting cells, inhibitor treatment in lupus and arthritis whereas its receptor ICOS is expressed models in mice. AMG 570 was selected on activated T cells. ICOSL knockout as the clinical lead. By targeting both (KO) mice or mice administered with BAFF and ICOSL, AMG 570 has the anti-ICOSL antibody had an impaired potential to achieve superior efficacy in T-dependent antibody response (3-5). treatment of autoimmune diseases such Elevation of ICOS expression on T as SLE and rheumatoid arthritis. cells was reported in patients with SLE, RA and Sjögren’s syndrome (6-8). Evi- Material and methods dence of the role of ICOS/ICOSL in hu- Reagents man immune function comes from both Recombinant human, cynomolgus patients deficient in ICOS function as monkey and murine BAFF or ICOSL- well as in lupus patients treated with an- Fc protein were generated at Amgen. ti-ICOSL antibody. ICOS null patients Anti-mouse ICOSL antibody 1B7 are immunodeficient showing impaired was described previously (Hu et al., T effector memory cell homoeostasis 2009). Anti-mouse BAFF peptibody and humoral immune responses (9, 10). was described previously (Hsu et al., AMG 557 is a fully human IgG2 an- 2012). Murine ICOSL/BAFF bispecific tagonist antibody against ICOSL and is molecule was generated by fusion of currently being investigated in a Phase two tandem copies of BAFF-binding 1 clinical trial in lupus arthritis patients peptides (Hsu et al., 2012) to the C- and primary Sjögren’s syndrome (pSS). terminus of anti-mouse ICOSL block- Single and multiple doses of AMG 557 ing antibody 1B7 as described (Hu et demonstrated ICOSL occupancy on B al., 2009). AMG 570 was generated by cells in the peripheral blood of SLE fusion of two tandem copies of BAFF- subjects in a dose-related and reversible binding peptides to the C-terminus of manner. A significant reduction in ant- anti-ICOSL antibody AMG 557 (Cheng KLH (Keyhole Limpet Hemocyanin) et al., 2018). PE labelled anti-B220, IgG response was observed after AMG FITC labelled anti-CD3 mAb, PE la- 557 treatment (11). AMG 557 showed belled anti-CD69 mAb, PE labelled an- safety and potential efficacy in patients ti-CD62L, FITC anti-CD44 were from with active lupus arthritis (12). BD Biosciences. Complete Freund’s BAFF (TNFSF13B) supports B cell Adjuvant (CFA) and Incomplete Fre- Competing interests: All the authors survival through binding to BAFF re- und’s Adjuvant (IFA) were from Difco. were Amgen employees and stock holders ceptor (BAFFR) expressed on B cells Bovine type II collagen were from Dr. at the time when this research work was (13). BAFF is a type II transmembrane Marie Griffiths University of Utah. Cy- conducted. protein mainly expressed by monocyte nomolgus monkey spleen was provided 907 Development of ICOSL/BAFF bispecific for SLE treatment / M. Zhang et al. by Shin Nippon Biomedical Laborato- dine in the last 18 hours of pulse. For Animal Research Facility at Amgen ries. Human PBMC for T cell and B cell cynomolgus monkey BAFF assay, cyn- Thousand Oaks. Mice (female, 6-8wk purification was from Amgen donors. omolgus monkey splenocytes were cul- old) were immunised with a single tured in RPMI media plus 10% heat in- intraperitoneal injection of 2X108 Binding affinities activated FBS in duplicates in 96- well SRBC (Lampire Biological Laborato- The binding affinities of AMG 570 to flat-bottom plates in the presence of 100 ries, Pipeville, PA) in 0.2 ml of PBS human and cynomolgus monkey BAFF ng/ml cynomolgus monkey BAFF pro- at day 1 and were boosted with 2X108 were measured by KinExA™. In brief, tein, 10 ug/ml Goat F(ab’ )2 anti-human SRBC on day 28. Mice were treated AMG 570 was incubated with various IgM from Jackson ImmunoResearch, with anti-ICOSL, anti-BAFF pepti- concentrations of human or cynomol- (Cat.# 109-006-129, West Grove, PA) body, combination of anti-ICOSL and gus monkey BAFF before running and a serially diluted AMG 570. For anti-BAFF inhibitors, murine ICOSL/ through the human or cynomolgus mouse BAFF assay, mouse spleen B BAFF bispecific, mouse IgG1 isotype monkey BAFF-coated Reacti-Gel 6x cells were stimulated with 200 ng/mL control twice per week until day 35. On beads. The amount of the bead-bound of murine BAFF and 0.1 μg/mL Goat day 35, spleen cells were collected for AMG 570 was quantified by fluores- F(ab’)2 anti-IgM. B cell proliferation FACS analysis. In brief, splenocytes cent (Cy5) labelled goat anti-human-Fc was measured by 3H thymidine incorpo- were pre-incubated with unlabelled antibody. The binding signal is propor- ration as described above. anti-CD16/32 to block nonspecific tional to the concentration of free anti- For ICOSL mediated T cell assay, 96- binding of the staining mAbs to FcγR. body at equilibrium with a given BAFF well tissue culture plates were first Cells were incubated on ice with 1:100 concentration. Dissociation equilib- coated with 2 μg/mL anti-CD3 and 10 diluted FITC anti-B220 for 30 min- rium constant (Kd) was obtained from μg/mL anti-human Fc overnight at 4°C. utes for B cell staining; 1:100 diluted non-linear regression of the competi- The plates were washed with 200 μl of FITC anti-CD44 and 1:200 diluted PE tion curves using a curve one-site ho- PBS, and coated with 3 μg/mL human anti-CD62L for 30 minutes for effector mogeneous binding model provided in ICOSL-Fc for 4 hours at 37°C. Approx- memory T cell staining. B cell were de- KinExA™ software. imately 105 purified T cells from PBMC fined as B220+ cells, effector memory T + + The binding affinities of AMG 570 to using Pan T cell isolation kits (Miltenyi cells (TEM) were defined as CD3 CD4 human and cynomolgus monkey ICOSL Biotec, Auburn, CA) were added to an- CD44hiCD62Llo cells and activated T were measured using BIAcore based ti-CD3/ICOSL-Fc coated plates in the cells were defined as CD3+CD69+ cells.