2. Conditional CD137 agonism is dependent on theprevalence of PD-L1-expressing cells

Matthew A Lakins, Sylwia Marshall, Alexander Koers, Raffaella Giambalvo, Robert Hughes, Emma Goodman, Mateusz Wydro, Cristian Gradinaru, Sarah Batey, Daniel Gliddon, Michelle Morrow, Neil Brewis

F-star, Babraham Research Campus, Cambridge, UK

Clustering CD137 via Cell-expressed PD-L1 Crosslinking Avoided Fc-mediated Agonism and Resulted in Safe and Potent Conditional Lymphocyte Activation

AACR Virtual Annual Meeting 2020 | 22 – 24 June | Poster Number: 4547 | Contact: matthew.lakins@f-star.com

1. FS222 simultaneously binds PD-L1 and CD137 with sub-nanomolar affinity andresults in potent T cell activation superior to the combination of component parts

BackgroundActivating T cells via clustering tumor necrosis factor receptorsuperfamily (TNFRSF) members has shown promise in cancertherapy. This has typically been achieved by crosslinking oftargeting antibodies via Fc-gamma receptor (FcgR). TNFRSFmember CD137 is expressed by activated lymphocytes, and itsclustering results in lymphocyte proliferation and agonism. Firstgeneration CD137 antibodies induced liver immune pathology,causing clinical toxicity and death. It remains a promising targetfor bispecific antibodies designed to limit unwanted toxicities.

T cell

PD-L1+ cell

CD137

PD-L1

LALA mutation to abrogate FcgR binding

CD137

PD-L1

FS222

MethodsFS222 was generated by introducing a CD137-binding specificityinto a human IgG1 targeting PD-L1 mAb with reduced FcgRbinding. Cell binding and in vitro activity was used to evaluateFS222 agonism conditional on PD-L1 crosslinking. An anti-mouseCD137/PD-L1 mAb2 was generated to assess anti-tumor activity,liver pharmacology and PK/PD in murine models. To predict theclinical toxicity and PK/PD profiles of FS222, studies wereperformed in non-human primates.

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FS222 Simultaenously Binding to CD137 and PD-L1

Time (s)

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)

100 nM hCD137-mFc

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hCD137 binding tocaptured FS222

FS222 binding toimmobilised hPD-L1

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FS222 Binding Primary CD8+ T Cells

Antibody Concentration (nM)

MFI

FS222

CD137/Ctrl(HelD1.3) mAb2

PD-L1(E12v2) mAb

CD137(MOR7480.1) mAb

Ctrl(4420) mAb

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FS222 Activity in Human Primary MLR

Antibody Concentration (nM)

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CD137/Ctrl(HelD1.3) mAb2

PD-L1(E12v2) mAb

CD137/Ctrl(HelD1.3) mAb2

+ PD-L1(E12v2) mAb

Ctrl(4420) mAb

✱✱✱

Figure 1.A Surface plasmon resonance showing FS222 simultaneous binding to both human PD-L1 and human CD137.B Cell binding of FS222 to activated human primary CD8+ T cells that express both PD-L1 and CD137.C FS222 activity in MLR (EC50 0.07 nM) against monospecific component entities that make up the complete mAb² either alone or in combination and monospecific monoclonal antibodies either alone or in combination.(* crosslinked with anti-hFc)

A C

B

Study Day 15 End of Treatment Free Period

Control FS222 Control FS222

AST (U/L) 31-74 28-64 34-41 24-55

ALT (U/L) 31-96 22-106 39-44 30-71

ALP (U/L) 260-948 289-1414 322-978 542-877

TRIG (mmol/L) 0.53-1.39 0.33-5.67 0.38-0.89 0.38-1.24

TBIL (umol/L) 1.7-6.2 1.2-14.2 1.2-5 1-2.2

Table 1. Clinical chemistry parameters at the end of main study (Day 15) and treatment free period (Day 43)

• Histopathology showed minimal-moderate inflammatory changes consistent with expected pharmacology in some tissues which resolved by the end of the treatment –free period.

• No acute cytokine release or changes in clinical haematology • Clinical chemistry measurements relating to liver function showed little change outside of normal parameters (control columns)

6. FS222 has a good PK profile and elicits immune cell activation without liver toxicityin a pivotal GLP toxicity study in cynomolgus monkey

3. Tetravalency results in full PDx blockade and optimalCD137 agonism in a human primary MLR

Conclusion

FS222 was observed to be a potent anti-human CD137/PD-L1 tetravalent bispecific antibody with a novel mode of action as a conditional agonist. The anti-mouse CD137/PD-L1 mAb2 (FS222 surrogate mAb2) had potent activity in vitro and potent anti-tumor activity in syngeneic mouse tumor models eradicating implanted MC38 tumors leading to 100% survival. FS222 surrogate mAb2 had self-limiting immunopharmacology outside the tumor microenvironment, suggesting a well-tolerated and effective mechanism of action with a broad therapeutic window.

A favorable safety profile and immunopharmacology was observed with FS222 in a primate GLP toxicity study making for a good potential therapeutic index for the treatment of human cancer. FS222 did not cause evident toxicity in cynomolgus monkey upon repeated dosing which we believe further encourages clinical development whereby we target tumours for which a significant unmet medical need exists in immunotherapy.

In many tumour settings at present, checkpoint inhibitors only provide moderate clinical benefit, and for many of those there is a strong mechanistic rationale for clinical outcomes to be improved with FS222 administration.

Figure 6. A Pharmacokinetic prolife of FS222 in cynomolgus monkey scaled to 10mg/kg. FS222 has dose-dependent linear PK in NHP, t1/2 = 210.5 ± 65.5 hours.B Maximum increases in frequency of proliferating NK cells, C CD8+ central memory cells and D activated CD8+ effector memory cells. PT – pre-treatments values

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Primary Mouse OT-I T Cell Assay

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ml

PD-L1(S70) mAb CD137(Lob12.3) + anti-hCH2 mAb

PD-L1(S70) mAb + CD137(Lob12.3) + anti-hCH2Surrogate FS222

mCD137/Ctrl(HelD1.3) mAb2

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MC38 Syngeneic Model, Survival (1 mg/kg)

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PD-L1(S70) + CD137(Lob12.3) mAb4/1233%

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Surrogate FS22212/12100%

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.B Survival data for mice treated with FS222 surrogate mAb² in MC38 syngeneic tumor model.C Individual plots for mice inoculated with MC38 tumor cell line and subsequently treated on day 7, 9, and 11 with 1 mg/kg FS222 surrogate mAb².Additionally, FS222 surrogate mAb2 shows significant tumor growth inhibition in B16-F10 and CT26 syngeneic mouse tumor models (data not shown).

100% survival

A B

C

4. FS222 surrogate mAb² has potent conditional in vitroactivity and significant in vivo survival benefit, superior to combination/monotherapy

5. FS222 surrogate mAb² induces dose-dependent serumcytokines and self-limiting immunopharmacology inthe liver of tumor-bearing mice

Figure 5.Serum cytokine levels from FS222 surrogate treated tumor-bearing mice as determined by MSD analysis for; AInterferon gamma, B TNF-alpha, C IL-6 and D IL-10E CD3+ T cells (as a percentage of total CD45+ immune cells) in the liver of FS222 treated treated tumor-bearing mice as determined by flow cytometryF Proliferating CD8+ T cells present in the liver of treated mice, using Ki67 expression as a proliferation marker, as determined by flow cytometry

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CD137(20H4.9)

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Proportion ofHEK.hPD-L1

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Figure 2.FS222 activity in a human primary CD8+ T-cell activation assay with varying ratios of HEK cells that are positive for PD-L1 to HEK cells that are negative for PD-L1.

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Tetravalent bispecific antibodies are the most efficient way to induce receptor clustering and activation

Human Primary MLR assayCD4+ T cells + iDCs

FS222 based molecules with a different valency to either target were produced and test in an MLR assay

EC50

(nM)Emax

(pg/mL)

0.03 2062

0.03 1428

1.13 2283

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PT 0 Low Mid.1Mid.2 High0

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Ki67 expression onNK cells

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central memory T cells

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CD69 expression on CD8+

effector memory T cells

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FS222 was well tolerated

HNSTD30mg/kg/dose

• GLP toxicity study was performed in cynomolgus monkeys

• FS222 or vehicle control were administered on days 1 and 8

• Study was terminated either on day 15 or day 43 (end of treatment free period)

• Study end-points included standard toxicological parameters and PD

• T cell proliferation and activation observed at all dose levels

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CD137 agonism via FS222 is dependent upon cross-linking via PD-L1 in a human primary T cell assay

Figure 3.FS222 activity in a human primary MLR assay against valency-variants of the same molecule. Blue; tetravalent (FS222) bivalent for both targets. Purple; bivalent for PD-L1, monovalent for CD137. Red; monovalent for PD-L1, bivalent for CD137. Yellow; monovalent for PD-L1 and monovalent for CD137.

Lakins et al. (2020). Clin Cancer Res April 28 2020 DOI: 10.1158/1078-0432.CCR-19-2958

t1/2 = 211 hrs

PD-L1

CD137

FS222