PowerPoint Presentation · Background FS222 surrogate mAb2 Median survival (days) p-values Log-rank...
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Conclusions FS222, a tetravalent CD137/PD-L1 mAb² was developed which binds PD-L1 and, in a PD-L1- dependent manner, stimulates CD137 agonism to elicit potent T cell activation in vitro with greater potency than a combination approach. FS222 has comparable in vitro potency in cynomolgus monkeys and so a preliminary toxicity study was undertaken in this species which showed PD responses related to dose and no evidence of liver toxicity (as seen clinically with CD137 agonists). A surrogate mouse CD137/PD-L1 mAb 2 with mechanism matched to FS222 significantly reduced tumour growth in two models with greater survival benefit than a combination approach in the MC38 model. In a CT26 model, a dose-dependent significant survival benefit was seen at doses between 0.1 and 0.3 mg/kg and above. This was coincident with intra-tumoural and peripheral T cell expansion. Within these two compartments FS222 surrogate mAb 2 showed rapid distribution to T cells via PD-L1 receptor occupancy and drug binding assays. This data supports the further development of FS222, a best-in-class CD137/PD-L1 tetravalent bispecific mAb 2 with IgG format for the treatment of patients with advanced malignancies. Matthew A. Lakins, Alexander Koers, Jose Munoz-Olaya, Raffaella Giambalvo, Robert Hughes, Daniel Jones, Sarka Pechouckova, Emma Goodman, Sylwia Marshall, Mateusz Wydro, Cristian Gradinaru, Francisca Wollerton, Sarah Batey, Daniel Gliddon, Michael Davies, Michelle Morrow , Mihriban Tuna, Neil Brewis F-star, Cambridge, UK FS222 mAb², a Bispecific Conditional Agonist Antibody Targeting CD137 and PD-L1, Induces Potent Lymphocyte Activation and has a Favourable Safety Profile 5. FS222 surrogate mAb² causes dose-dependent survival benefit concomitant with peripheral and tumour expansion of T cells B Background FS222 surrogate mAb 2 Median survival (days) p-values Log-rank (group wise comparison with lower dosed group) 10mg/kg 39 0.2 1mg/kg 29 0.02 0.3mg/kg 24 0.007 0.1mg/kg 21 0.6 IgG ctrl 21 0 20 40 60 0 50 100 Days post inoculation Percent survival 10 mg/kg 1 mg/kg 0.3 mg/kg 0.1 mg/kg IgG ctrl ns * ** ns A E Compound IgG ctrl PD-L1 mAb CD137 mAb PD-L1 mAb + CD137 mAb FS222 surrogate mAb 2 Tumour-free animals Number 0/12 2/12 2/12 4/12 12/12 Percentage 0 % 16 % 16 % 33 % 100 % Figure 4. A FS222 surrogate mAb² activity in a CD8 + OT-1 mouse T cell activation assay with cell-based crosslinking provided by B16-F10 tumour cells pulsed with ovalbumin peptide and that express mouse PD-L1. Survival data for mice treated with surrogate FS222 mAb² in B MC38, C CT26, and D individual plots for mice inoculated with MC38 tumour cell line and subsequently treated on day 7, 9, and 11 with 1mg/kg FS222 surrogate mAb² - E Summary table of tumour-free animals by end of study for MC38 experiment described above. Additionally, FS222 surrogate mAb 2 shows significant tumour growth inhibition in B16-F10 syngeneic mouse tumour model (data not shown). 10 20 30 40 50 0 500 1000 1500 Days post inoculation Tumour Volume mm 3 IgG ctrl 10 20 30 40 50 0 500 1000 1500 Days post inoculation PD-L1 mAb 10 20 30 40 50 0 500 1000 1500 Days post inoculation CD137 mAb 10 20 30 40 50 0 500 1000 1500 Days post inoculation PD-L1 mAb + CD137 mAb 10 20 30 40 50 0 500 1000 1500 Days post inoculation FS222 surrogate mAb² B A C D 0 20 40 60 0 50 100 Days Percent survival FS222 surrogate mAb 2 PD-L1 mAb PD-L1 mAb + CD137 mAb CD137 mAb IgG ctrl MC38 (1mg/kg) 0 20 40 60 80 0 50 100 Days Percent survival IgG ctrl FS222 surrogate mAb 2 CT26 (10mg/kg) PD-L1 mAb CD137 mAb AACR Annual Meeting 2019 | 29 Mar-03 Apr | Atlanta | Poster Number: 1540 Strictly for personal use - DO NOT POST ONLINE T cell PD-L1 + cell CD137 PD-L1 Fcab™ Fc TARGET B TARGET B TARGET A TARGET A mAb²™ Directed amino acid substitutions to create new binding sites in Fc Rapid conversion into tetravalent IgG i.e. bivalent for both targets Plug-and-play into any mAb 1. FS222 simultaneously binds to both PD-L1 and CD137 with sub-nanomolar affinity Figure 1. A Representation of FS222, a tetravalent bispecific CD137/PD-L1 antibody on a human IgG1 backbone with FcgR-binding significantly reduced by the L234A and L235A (LALA) mutation highlighted in green. The anti-PD-L1 IgG1 with a distinct CD137 binding capability in the Fc region consists of two homodimers, each comprised of a heavy and light chain. The complementarity- determining regions (CDRs) of the heavy and light chains are highlighted in orange. The CH3 domain AB and EF binding loops are highlighted in cyan. B Surface plasmon resonance showing FS222 simultaneous binding to both human PD-L1 and human CD137. C –E Cell binding of FS222 to: C DO11.10 T cells expressing human CD137, D HEK cells expressing human PD-L1, or E activated human primary CD4 + and CD8 + T cells. C 0 20 40 60 80 100 Time (h) Post-dose % Ki67 + 2 6 24 48 72 96 120 192 0 20 40 60 80 100 Time (h) Post-dose % Ki67 + 2 6 24 48 72 96 120 192 0 20 40 60 80 100 Time (h) Post-dose % Ki67 + 2 6 24 48 72 96 120 192 0 20 40 60 80 100 Time (h) Post-dose % Ki67 + 2 6 24 48 72 96 120 192 IgG ctrl (10 mg/kg) FS222 surrogate mAb² (10 mg/kg) FS222 surrogate mAb² (1 mg/kg) Figure 6. A PD-L1 receptor occupancy. Free PD-L1 was determined using a competing anti-mouse PD-L1 antibody. The data presented shows the positive population as a percentage of PD-L1 receptor occupancy compared to a 100% mAb 2 saturated sample of CD8 + or CD4 + T cells from the tumour or blood. PD-L1 receptor occupancy is maintained longer in the tumour microenvironment than the blood. B FS222 binding data to T cells ex vivo. Anti-human Fc secondary antibody detected the human Fc region of FS222 mAb² bound to cells and the positive population is presented as a percentage of all CD8 + or all CD4 + T cells from the tumour or blood. FS222 mAb² rapidly binds both CD8 + and CD4 + T cells in both tumour and blood and the percentage positive T cell population is positively correlated with dose level. 6. FS222 surrogate mAb² shows dose-dependent PD-L1 receptor occupancy and T cell binding with rapid intratumoral and peripheral distribution E A -600 -400 -200 0 200 0 100 200 300 400 500 600 700 Time FS222 binding to immobilised human PD-L1-Avi-His-Biot Response (RU) B Human CD137-mFc binding to captured FS222 CD137 binding sites PD-L1 binding sites LALA mutation C D 0.01 1 100 0 5000 10000 15000 Antibody concentration (nM) MFI AF647 FS222 mAb² CD137/mock mAb² CD137 mAb IgG ctrl 0.01 1 100 0 50000 100000 150000 Antibody concentration (nM) MFI AF647 FS222 mAb² CD137/mock mAb² PD-L1 mAb IgG ctrl Cell binding on DO11.10 overexpressing human CD137 Cell binding on HEK overexpressing human PD-L1 0.001 0.01 0.1 1 10 100 1000 2000 3000 4000 5000 Antibody concentration (nM) MFI AF647 FS222 mAb² CD137/mock mAb² PD-L1 mAb CD137 mAb IgG ctrl 0.001 0.01 0.1 1 10 100 1000 2000 3000 4000 5000 6000 Antibody concentration (nM) MFI AF647 FS222 mAb² CD137/mock mAb² PD-L1 mAb CD137mAb IgG ctrl CD4 + T cells CD8 + T cells 0 20 40 60 80 100 Time (h) Post-dose % positive for FS222 surrogate mAb² or IgG ctrl 2 6 24 48 72 96 120 192 0 20 40 60 80 100 Time (h) Post-dose 2 6 24 48 72 96 120 192 0 20 40 60 80 100 Time (h) Post-dose % positive for FS222 surrogate mAb² or IgG ctrl 2 6 24 48 72 96 120 192 0 20 40 60 80 100 Time (h) Post-dose 2 6 24 48 72 96 120 192 FS222 surrogate mAb² (10mg/kg) FS222 surrogate mAb² (1mg/kg) IgG ctrl (10mg/kg) Blood Tumour 0 20 40 60 80 100 Time (h) post-dose PD-L1 receptor occupancy % 2 6 24 48 72 96 120 192 0 20 40 60 80 100 Time (h) post-dose 2 6 24 48 72 96 120 192 0 20 40 60 80 100 Time (h) post-dose PD-L1 receptor occupancy % 2 6 24 48 72 96 120 192 0 20 40 60 80 100 Time (h) post-dose 2 6 24 48 72 96 120 192 FS222 surrogate mAb² (10mg/kg) FS222 surrogate mAb² (1mg/kg) IgG ctrl (10mg/kg) + spike IgG ctrl (10mg/kg) 0.0001 0.001 0.01 0.1 1 10 100 0 10000 20000 30000 Antibody concentration (nM) mIFNg pg/ml PD-L1 mAb CD137 mAb (crosslinked) CD137 mAb + PD-L1 mAb FS222 surrogate mAb² CD137/mock mAb² 4. FS222 surrogate mAb² in vitro activity replicates that of FS222 mAb² and leads to significant in vivo survival benefit, superior to combination/monotherapy 0.001 0.01 0.1 1 10 100 0 2500 5000 7500 10000 Antibody concentration (nM) hIL2 (pg/ml) FS222 (100% HEK.hPD-L1) FS222 (50% HEK.hPD-L1) FS222 (25% HEK.hPD-L1) FS222 (12.5% HEK.hPD-L1) FS222 (6.5% HEK.hPD-L1) FS222 (0% HEK.hPD-L1) CD137 mAb (0% HEK.hPD-L1) IgG ctrl (0% HEK.hPD-L1) 0.001 0.01 0.1 1 10 100 5000 10000 15000 20000 Antibody concentration (nM) hIFNg (pg/ml) FS222 mAb² CD137/mock mAb² PD-L1 mAb CD137/mock mAb² + PD-L1 mAb IgG ctrl Figure 2. A FS222 activity in a CD8 + T cell activation assay with varying mixed populations of HEKs that are positive or negative for PD-L1. B FS222 activity in MLR (EC 50 0.07 nM) against monospecific component entities that make up the complete mAb² either alone or in combination. A B 2. FS222 is superior to combinations in human T cell activation assays, with CD137-activity dependent on binding to PD-L1 Figure 3. A Pharmacokinetic prolife of FS222 in cynomolgus monkey has a half-life of ~6 days. (SD = single dose) B Changes in clinical chemistry parameters relating to liver function of cynomolgus monkey in FS222 repeat dose phase. No change outside of normal parameters (lower and upper limit columns) was observed. C-F Kinetic changes after 4x weekly repeated dose of FS222 in cynomolgus monkey C frequency of peripheral CD4 + central memory cells expressing Ki67 and D NK cells expressing Ki67. E Frequency of peripheral CD8 + central memory cells expressing Ki67 and F profile of serum soluble PD-L1. PD-1/L1 axis blockade shows durable responses and extended overall survival across cancer types in a subset of patients. Tumour Necrosis Factor Receptor (TNFR) superfamily activation is also being tested clinically to improve patient responses. Current interventions using therapeutic CD137 (4-1BB) agonists to activate T cells are restricted by severe dose limiting toxicities and poor efficacy as monotherapies. The generation of a bispecific agonist of CD137 dependent on PD-L1 crosslinking creates a greater therapeutic window with improved safety and efficacy. Here, we show how a novel tetravalent bispecific antibody (mAb²™) binds to both CD137 and PD-L1, induces potent in vitro T cell activity in a PD-L1-dependent manner and results in significant tumour control across two syngeneic tumour models without toxicity. Intra-tumoural and peripheral pharmacodynamic changes implicate an increase in the proliferative CD8 + T cell response as a mechanism of action. CD8 + T cell activation assay Mixed Lymphocyte Reaction A B 0 100 200 300 400 0.001 0.01 0.1 1 10 100 1000 10000 Time post-dose (h) FS222 mAb² concentration (μg/ml) Male 30mg/kg Female 30mg/kg Male 10mg/kg Female 10mg/kg Male 3mg/kg (SD) Female 3mg/kg (SD) Male 1mg/kg Female 1mg/kg Male 0.1mg/kg Female 0.1mg/kg A B 3. FS222 mAb² has a dose proportional PK profile and elicits immune activation with no liver toxicity in a preliminary toxicity study in cynomolgus monkey 0 20 40 60 80 Time (d) Relative (%) -5 1 4 8 11 15 18 22 25 0 200 400 600 0 1000 2000 3000 4000 Time (h) Serum PD-L1 (pg/mL) Male 0.1mg/kg Female 0.1mg/kg Male 1mg/kg Female 1mg/kg Male 10mg/kg Female 10mg/kg Male 30mg/kg Female 30mg/kg 100% survival Lower limit of normal range FS222 Upper limit of normal range AST (U/L) 20 23-69 94 ALT (U/L) 21 19-111 112 ALP (U/L) 140 485-1310 1350 TBIL (mg/dL) 0.06 0.07-0.38 0.43 0 10 20 30 40 50 Time (d) Relative (%) -5 1 4 8 11 1518 2225 0 10 20 30 40 50 60 Time (d) Relative (%) -5 1 4 8 11 1518 2225 Ki67 expression on CD4 + central memory T cells Ki67 expression on NK cells No change in clinical chemistry parameters relating to liver function C Serum levels of soluble PD-L1 E F Blood Tumour Ki67 Expression on CD8 + central memory T cells D Figure 5. A Kaplan-Meier of dose-range finding study in CT26 of FS222 surrogate mAb² in the range 0.1 to 10 mg/kg. B Increased dose of FS222 surrogate mAb² is correlated with increased survival. C Pharmacodynamic changes observed in the tumour and the periphery for CT26 tumour-bearing mice upon treatment with a single dose (10mg/kg or 1mg/kg) of FS222 surrogate mAb² . CD4 + T cells CD8 + T cells Cell binding on human primary T cells: FS222 PD-L1 receptor occupancy on CD8 + T cells PD-L1 receptor occupancy on CD4 + T cells FS222 surrogate mAb² bound on CD8 + T cells FS222 surrogate mAb² bound on CD4 + T cells
Transcript of PowerPoint Presentation · Background FS222 surrogate mAb2 Median survival (days) p-values Log-rank...
Conclusions
FS222, a tetravalent CD137/PD-L1 mAb² was developed which binds PD-L1 and, in a PD-L1-dependent manner, stimulates CD137 agonism to elicit potent T cell activation in vitro withgreater potency than a combination approach. FS222 has comparable in vitro potency incynomolgus monkeys and so a preliminary toxicity study was undertaken in this species whichshowed PD responses related to dose and no evidence of liver toxicity (as seen clinically withCD137 agonists). A surrogate mouse CD137/PD-L1 mAb2 with mechanism matched to FS222significantly reduced tumour growth in two models with greater survival benefit than acombination approach in the MC38 model. In a CT26 model, a dose-dependent significantsurvival benefit was seen at doses between 0.1 and 0.3 mg/kg and above. This was coincidentwith intra-tumoural and peripheral T cell expansion. Within these two compartments FS222surrogate mAb2 showed rapid distribution to T cells via PD-L1 receptor occupancy and drugbinding assays.This data supports the further development of FS222, a best-in-class CD137/PD-L1 tetravalentbispecific mAb2 with IgG format for the treatment of patients with advanced malignancies.
Matthew A. Lakins, Alexander Koers, Jose Munoz-Olaya, Raffaella Giambalvo, Robert Hughes, Daniel Jones, Sarka Pechouckova, Emma Goodman, Sylwia Marshall, Mateusz Wydro, Cristian Gradinaru, Francisca Wollerton, Sarah Batey, Daniel Gliddon, Michael Davies, Michelle Morrow, Mihriban Tuna, Neil Brewis
F-star, Cambridge, UK
FS222 mAb², a Bispecific Conditional Agonist Antibody Targeting CD137 and PD-L1, Induces Potent Lymphocyte Activation and has a Favourable Safety Profile
5. FS222 surrogate mAb² causes dose-dependent survival benefit concomitant with peripheral and tumour expansion of T cells
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Figure 4. A FS222 surrogate mAb² activity in a CD8+ OT-1mouse T cell activation assay with cell-based crosslinkingprovided by B16-F10 tumour cells pulsed with ovalbuminpeptide and that express mouse PD-L1. Survival data formice treated with surrogate FS222 mAb² in B MC38, CCT26, and D individual plots for mice inoculated withMC38 tumour cell line and subsequently treated on day 7,9, and 11 with 1mg/kg FS222 surrogate mAb² - E Summarytable of tumour-free animals by end of study for MC38experiment described above.Additionally, FS222 surrogate mAb2 shows significanttumour growth inhibition in B16-F10 syngeneic mousetumour model (data not shown).
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AACR Annual Meeting 2019 | 29 Mar-03 Apr | Atlanta | Poster Number: 1540 Strictly for personal use - DO NOT POST ONLINE
T cell
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Directed amino acid substitutions to
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Plug-and-play into any mAb
1. FS222 simultaneously binds to both PD-L1 and CD137 with sub-nanomolar affinity
Figure 1. A Representation of FS222, a tetravalent bispecific CD137/PD-L1 antibody on a human IgG1 backbone with FcgR-bindingsignificantly reduced by the L234A and L235A (LALA) mutation highlighted in green. The anti-PD-L1 IgG1 with a distinct CD137binding capability in the Fc region consists of two homodimers, each comprised of a heavy and light chain. The complementarity-determining regions (CDRs) of the heavy and light chains are highlighted in orange. The CH3 domain AB and EF binding loops arehighlighted in cyan. B Surface plasmon resonance showing FS222 simultaneous binding to both human PD-L1 and human CD137. C– E Cell binding of FS222 to: C DO11.10 T cells expressing human CD137, D HEK cells expressing human PD-L1, or E activatedhuman primary CD4+ and CD8+ T cells.
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Figure 6. A PD-L1 receptor occupancy. Free PD-L1 was determined using a competing anti-mouse PD-L1 antibody. The datapresented shows the positive population as a percentage of PD-L1 receptor occupancy compared to a 100% mAb2 saturatedsample of CD8+ or CD4+ T cells from the tumour or blood. PD-L1 receptor occupancy is maintained longer in the tumourmicroenvironment than the blood. B FS222 binding data to T cells ex vivo. Anti-human Fc secondary antibody detected thehuman Fc region of FS222 mAb² bound to cells and the positive population is presented as a percentage of all CD8+ or all CD4+ Tcells from the tumour or blood. FS222 mAb² rapidly binds both CD8+ and CD4+ T cells in both tumour and blood and thepercentage positive T cell population is positively correlated with dose level.
6. FS222 surrogate mAb² shows dose-dependent PD-L1 receptor occupancy and T cell binding with rapid intratumoral and peripheral distribution
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Figure 2. A FS222 activity in a CD8+ T cell activation assay with varying mixed populations of HEKs that are positive or negative forPD-L1. B FS222 activity in MLR (EC50 0.07 nM) against monospecific component entities that make up the complete mAb² eitheralone or in combination.
A B
2. FS222 is superior to combinations in human T cell activation assays, with CD137-activity dependent on binding to PD-L1
Figure 3. A Pharmacokinetic prolife of FS222 incynomolgus monkey has a half-life of ~6 days. (SD= single dose) B Changes in clinical chemistryparameters relating to liver function ofcynomolgus monkey in FS222 repeat dose phase.No change outside of normal parameters (lowerand upper limit columns) was observed. C - FKinetic changes after 4x weekly repeated dose ofFS222 in cynomolgus monkey C frequency ofperipheral CD4+ central memory cells expressingKi67 and D NK cells expressing Ki67. E Frequencyof peripheral CD8+ central memory cellsexpressing Ki67 and F profile of serum solublePD-L1.
PD-1/L1 axis blockade shows durable responses and extended overall survival across cancer types in asubset of patients. Tumour Necrosis Factor Receptor (TNFR) superfamily activation is also being testedclinically to improve patient responses. Current interventions using therapeutic CD137 (4-1BB)agonists to activate T cells are restricted by severe dose limiting toxicities and poor efficacy asmonotherapies. The generation of a bispecific agonist of CD137 dependent on PD-L1 crosslinkingcreates a greater therapeutic window with improved safety and efficacy.
Here, we show how a novel tetravalent bispecific antibody (mAb²™) binds to both CD137 and PD-L1,induces potent in vitro T cell activity in a PD-L1-dependent manner and results in significant tumourcontrol across two syngeneic tumour models without toxicity. Intra-tumoural and peripheralpharmacodynamic changes implicate an increase in the proliferative CD8+ T cell response as amechanism of action.
CD8+ T cell activation assay Mixed Lymphocyte Reaction
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3. FS222 mAb² has a dose proportional PK profile and elicits immune activation with no liver toxicity in a preliminary toxicity study in cynomolgus monkey
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Figure 5. A Kaplan-Meier of dose-range findingstudy in CT26 of FS222 surrogate mAb² in therange 0.1 to 10 mg/kg. B Increased dose of FS222surrogate mAb² is correlated with increasedsurvival. C Pharmacodynamic changes observedin the tumour and the periphery for CT26tumour-bearing mice upon treatment with asingle dose (10mg/kg or 1mg/kg) of FS222surrogate mAb² .
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FS222
PD-L1 receptor occupancy on CD8+ T cells
PD-L1 receptor occupancy on CD4+ T cells
FS222 surrogate mAb² bound on CD8+ T cells
FS222 surrogate mAb² bound on CD4+ T cells
