P779 Tetra-specific MATCH4 antibodies mediate concomitant ......e X. T ell ed t ell ling and CD8+...
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0.01 1 100 10000 -20 0 20 40 60 80 100 Her2 - /PD-L1 + CHO-PD-L1 target cell killing Co-cultured with hPBMCs Concentration [pM] Specific Killing [%] Her2/CD3/PD-L1 Low /hSA Her2/CD3 scDb Her2/CD3/dummy/hSA Trastuzumab + Nivolumab 0.01 1 100 10000 -20 0 20 40 60 80 100 Her2 + /PD-L1 + HCC827 target cell killing Co-cultured with hPBMCs Concentration [pM] Specific killing [%] Her2/CD3 scDb Her2/CD3/PD-L1 Low /hSA Her2/CD3/dummy/hSA Trastuzumab + Nivolumab 0.01 1 100 10000 -20 0 20 40 60 80 100 Her2 + /PD-L1 - MCF-7 target cell killing Co-cultured with hPBMCs Concentration [pM] Specific Killing [%] Her2/CD3/PD-L1 Low /hSA Her2/CD3 scDb Her2/CD3/dummy/hSA Trastuzumab + Nivolumab 0.01 1 100 10000 -20 0 20 40 60 80 100 Her2 +++ /PD-L1 + HCC1954 target cell killing Co-cultured with hPBMCs Concentration [pM] Specific Killing [%] Her2/CD3/PD-L1 Low /hSA Her2/CD3 scDb Her2/CD3/dummy/hSA Trastuzumab + Nivolumab Combination approach Cytolytic synapse-restricted PD-L1 blockade PD-L1 Her2 + /PD-L1 + Cell PD-1 T cell CD3 HSA Her2 Her2 mediated avidity binding PD-L1 T cell CD3 HSA Her2 + /PD-L1 - Cell PD-1 T cell CD3 HSA Her2 Her2 - /PD-L1 + Cell T cell activation level T cell activation level T cell activation level PD-L1 Her2 + /PD-L1 + Cell Her2 T cell CD3 PD-1 T cell activation level PD-L1 Her2 PD-L1 HER2 & PD-L1 expressing cancer cell line: HCC1954 Labeled PD-1 ECD PD-L1 expressing cancer cell line: HCC827 Labeled PD-1 ECD HER +++ /PD-L1 + HER + /PD-L1 + HER + /PD-L1 - HER - /PD-L1 + 0 20 40 60 80 100 T cell activation potency ratio Fold-change in potency MATCH4 to scDB 0.1 1 10 100 1000 10000 100000 0 200000 400000 600000 800000 1000000 Activation of NFAT/luc-reporter T cells Co-cultured with Her2 - /PD-L1 + CHO PD-L1 cells Concentration [pM] T cell activation (RLU) Her2/CD3/PD-L1 Low /hSA Her2/CD3/PD-L1 Low /hSA + Nivolumab dummy/CD3/PD-L1 Low /hSA dummy/CD3/PD-L1 Low /hSA + Nivolumab Her2/CD3/dummy/hSA Her2/CD3/dummy/hSA + Nivolumab Her2/CD3/dummy/hSA + PD-L1 Low scFv 1 100 10000 0 200000 400000 600000 800000 1000000 Activation of NFAT/luc-reporter T cells Co-cultured with Her2+++ HCC1954 Concentration [pM] T cell activation (RLU) Her2/CD3/dummy/hSA Her2/CD3/dummy/hSA + PD-L1 Low scFv Her2/CD3/PD-L1 Low /hSA dummy/CD3/PD-L1 Low /hSA Her2/CD3/dummy/hSA + Nivolumab Her2/CD3/PD-L1 Low /hSA + Nivolumab dummy/CD3/PD-L1 Low /hSA + Nivolumab A B T cell activation level Her2 High / PD-L1 + T cell activation level Her2 Low / PD-L1 + C T cell activation level Her2 Low / PD-L1 - T cell activation level Her2 - / PD-L1 + D Tetra-specific MATCH4 antibodies mediate concomitant blockade of PD-L1 and potent T cell-stimulation, locally restricted to Her2 + tumors Alexandre Simonin 1 , Tea Gunde 1 , Christian Hess 1 , Stefan Warmuth 1 , Matthias Brock 1 , Dan Snell 1 , Julia Tietz 1 , Eva Oswald 2 , Robin Heiz 1 , Naomi Flückiger 1 , Julia Zeberer 1 , Dania Diem 1 , Dana Mahler 1 , Diego Morenzoni 1 , Simone Muntwiler 1 , Benjamin Küttner 1 , Sebastian Meyer 1 , Timothy Egan 3 , David Urech 1 1) Numab Therapeutics AG, Einsiedlerstrasse 34, 8820 Wädenswil, Switzerland; 2) Charles River Discovery Service Germany GmbH, Am Flughafen 12-14, 79108 Freiburg, Germany; 3) Numab US LLC, P.O. Box 902 Northboro, 01532 Massachusetts, US Tumor-targeted T cell activation mechanisms have proved highly effective in the redirection of the immune system towards cancer cells. Of particular note is the use of CD3 T cell engagers and the approval of the molecule Blincyto for the treatment of patients suffering from B-ALL. One issue in identifying appropriate tumor associated antigens (TAAs) for CD3 T cell engagement is ensuring an appropriate expression level in tumor tissue vs normal tissue to preferentially drive T cell mediated cytotoxicity to the tumor. We sought to address this issue and open the therapeutic window of CD3 engagers by the generation of an affinity tailored MATCH4 molecule targeting PD-L1, Her2, CD3 and human serum albumin (hSA) to preferentially mediate T cell killing of tumor cells. We demonstrate that PD-L1/PD-1 antagonism is retained within this molecule which has increased potency compared to a conventional CD3 x Her2 engager, in a single-chain diabody format (scDb), on both Her2 high as well as Her2 low cells, in the presence of simultaneous PD-L1 expression. The activity is not enhanced on PDL1-negative cells and therefore we demonstrate the potential to open the therapeutic window towards higher activity on cancer cells in the context of PD-L1 expression. Conclusions: • A next-generation MATCH4 tetra-specific (Her2/CD3/PD- L1 low /hSA) molecule displays exquisite anti-tumoral efficacy in Her2 + cancer models • Increased therapeutic potential on Her2 + /PD-L1 + cells whilst not increasing activity on Her2 + /PD-L1 - cells (Fig 6A-C) • No T cell activity in the presence of Her2 - PD-L1 + cells due to the use of a low-affinity anti-PD-L1 domain (Fig. 6D) • PD-L1 / PD-1 antagonism is dependent on Her2 expression and delivers an additive effect to T cell activation at concentrations >100pM (Fig. 4) • The anti-tumoral activity is predominantly driven by an avidity-effect of engaging both Her2 and PD-L1 • Potential to open the therapeutic window of Her2/CD3 activity by increasing selectivity towards Her2 PD-L1 double-positive cells and therefore may present a safer Her2 targeting T cell engager Tumor targeted PD-L1 blockade PD-L1 binding domain substantially increases molecule potency A B Concept Figure 6. T cell mediated target cell killing and CD8+ cell activation in presence of A) Her2 +++ /PD-L1 + HCC1954 ductal breast carcinoma cells, B) Her2 + /PD-L1 + HCC827 NSCLC cells, C) Her2 + /PD-L1 - MCF-7 breast adenocarcinoma cells, D) Her2 - /PD-L1 + CHO PD-L1 cells. Freshly isolated human PBMCs were co-cultured for 16h with indicated target cells in presence of the different molecules. The MATCH4 Her2/CD3/PD-L1 low /hSA showed an increased potency in target cell killing and T cell activation compared to the Her2/CD3 scDb on Her2 +++ /PD-L1 + cells and Her2 + /PD-L1 + cells. Activity of the MATCH4 molecule is similar to that of the Her2/CD3 scDb in the presence of Her2 + /PD-L1 - cells. Importantly, no killing activity or T cell activation is observed in the presence of Her2 - /PDL1 + cells. A B Efficient tumor regression with the MATCH4 molecule αPD-L1 binding-domain substantially increases potency Figure 1. A) Conventional combination of a Her2/CD3 bispecific molecule with PD-L1 blockade results in potent T cell- activation and target cell lysis. B) αHer2-/αPD-L1-mediated avidity-binding elicits more potent T cell-activation and target cell lysis than the combination therapy in the presence of Her2 + PD-L1 + cancer cells, of both Her2-high and Her2-low phenotypes. Targeting of Her2 + PD-L1 − cells is similarly effective as combination therapy, whilst importantly Her2 − PD-L1 + cells are spared from cytolysis. Affinity-optimized anti-PD-L1 domain specifically targeting Her2/PD-L1 expressing cancer cells Figure 2. Neutralization of PD-1-binding to PD-L1 + cells. Effect of PD-L1-affinity on potency of blockade of PD-1-binding to A) Her2 +++ PD-L1 + HCC1954 ductal breast carcinoma cells and B) Her2 + PD-L1 + HCC827 NSCLC cancer cells. PD-L1-blockade is restricted to high Her2 expressing cancer cells. Careful selection of the affinity of the αPD-L1 domain (10’000-fold lower than Avelumab) allows considerable extension of the therapeutic window. Her2/CD3/PD-L1 low /hSA tetra-specific MATCH4 molecule A B C Figure 3. A) Schematic representation of Her2/CD3/PD-L1 low /hSA tetra-specific MATCH4 molecule: αCD3 (VL1/VH1 in green), αhSA (VL2/VH2 in orange), αHer2 (VL3/VH3 in purple), αPD-L1 (VL4/VH4 in blue), inter-chain disulfide bridge indicated in yellow. Domains are connected by Gly-Ser peptide linkers, as indicated by red lines. B) SDS-PAGE analysis of the MATCH4, NR: non-reducing, R: reducing, M: molecular weight marker C) Representative SE-HPLC chromatogram of the MATCH4. Figure 4. Blockade of PD-L1-signaling in the immunological synapse. CD3-engagement and blockade of PD-1-signaling by MATCH4 molecules was assessed in a transgenic NFAT-Luciferase reporter Jurkat cell-line. PD-1-signaling is induced by co- cultivation of PD-1 + reporter Jurkat cells with A) Her2 +++ PD-L1 + HCC1954 cancer cells or B) Her2−PD-L1 + CHO cells. CD3 activation results in reporter gene activation and PD-L1-inhibition enhances maximum T cell-activation. CD3 activation requires the presence of Her2 + cells, and immunological synapse-restricted PD-L1-blockade elevates the efficacy of T cell-activation similarly to Nivolumab. When co-administered as an scFv, the low-affinity αPD-L1 domain has no effect. Figure 5. T cell-mediated target cell-killing. Freshly isolated human PBMCs were co-cultured for 40h with Her2 +++ PD-L1 + HCC1954 cells in the presence of the different molecules and target cell-killing was assessed by annexin V staining. MATCH4 molecules were tested in the presence or absence of a saturating concentration of Nivolumab (1 μg/ml). MATCH4 harboring the low-affinity αPD-L1 domain is more potent than the MATCH 4 molecule lacking the αPD-L1 domain. Absence of the αHer2 domain results in a 10000-fold lower potency. Nivolumab treatment, in combination with the Her2/CD3/dummy/hSA tetra-specific MATCH4 molecule, did not change potency of the molecule, providing evidence that the improved potency the Her2/CD3/PD-L1 LOW /hSA MATCH4 is exclusively due to high avidity conferred by concomitant binding to both PD-L1 and Her2. Figure 8. Human PBMC-substituted NOG mice were engrafted with HCC1954 ductal breast carcinoma cells (n=8 per group). Mice were treated on day 0, 5, 10, 15, 20, 25 and 30 (dotted vertical lines). Tumor growth and body weight was recorded twice weekly. The Her2/CD3/PD-L1 low /hSA tetra-specific MATCH4 molecule elicited considerable tumor regression in all treated mice. The MATCH4 molecule demonstrated greater anti-tumoral efficacy than the Nivolumab + Trastuzumab combination. Nivolumab shows limited anti- tumoral activity in this model and the control molecule Her2/PD-L1 low /hSA shows a similar anti- tumoral activity despite a 5-fold lower potency in vitro (data not shown), confirming effective tumor-targeted PD-L1 blockade. No body weight loss was observed (not shown). cell lines Potency for T cell activation EC 50 (pM) MATCH4 scDb HCC1954 0.39 35.91 HCC827 23.72 314.6 MCF-7 25.33 54.29 CHO-PD-L1 NC NC Fold potency difference Her2 high / PDL1 + vs Her2 low / PDL1 - 65x 1.5x Figure 7. T cell activation potencies of MATCH4 and scDb on cells expressing different Her2 and PD-L1 levels. A) EC50 values for T cell activation mediated by MATCH4 and scDb molecules. B) Calculated potency ratios between MATCH4 and scDb. C) Her2 antibody binding sites on different cancer cells. The MATCH4 Her2/CD3/PD-L1 low /hSA showed an increased potency on PD-L1 positive Her2 expressing cancer cells in comparison to the scDb regardless of Her2 expression levels. Data suggest an improved therapeutic window for the MATCH4 over the scDb in the context of PD-L1 expression. Importantly a greater difference in potency can be demonstrated for the MATCH4 molecule between Her2 high / PD-L1 + cells and Her2 low / PD-L1 - cells compared to a scDb. NC = not calculable A B C cell lines Her2 expression Antibody binding sites HCC1954 61937 MCF-7 12670 HCC827 8908 A B Her2 + /PD-L1 + cancer cell PD-1 NFAT-Luciferase reporter Jurkat cell CD3 HSA Her2 Luciferase N VL1 VH2 VH2 VH1 VL4 VH4 Peptide linker C N C VL2 VL3 VH3 Inter-chain disulfide bridge 5 6 7 8 9 10 0 50 100 Retention time [min] Normalized Absorbance 280 nm [%] Her2 + /PD-L1 + cancer cell PD-1 T cell CD3 HSA Her2 Target cell lysis 50 kDa 100 kDa 75 kDa 37 kDa 25 kDa NR R M 0.01 1 100 10000 -20 0 20 40 60 80 100 Her2 +++ /PD-L1 + HCC1954 target cell killing Co-cultured with hPBMCs Concentration [pM] Specific Killing [%] Her2/CD3/PD-L1 Low /hSA + Nivolumab Her2/CD3/dummy/hSA Her2/CD3/dummy/hSA + Nivolumab Her2/CD3/dummy/hSA + PD-L1 Low scFv dummy/CD3/PD-L1 Low /hSA dummy/CD3/PD-L1 Low /hSA + Nivolumab Her2/CD3/PD-L1 Low /hSA 0 2 6 9 13 16 20 23 27 30 33 0 500 1000 1500 HCC1954 xeno in humanized NOG Tumor localized CD3 activation/PD-L1 blockade days Relative Tumor Volume Her2/PD-L1 low /hSA (5 mg/kg) Palivizumab (5 mg/kg) Nivolumab (5 mg/kg) Her2/CD3/PD-L1 low /hSA (2.5 mg/kg) Trastuzumab (5 mg/kg) + Nivolumab (5 mg/kg) Her2/PD-L1 low /hSA (25 mg/kg) 34th Annual Meeting of the Society for Immunotherapy of Cancer, 2019. National Harbor, Maryland, USA. Poster P779 P779 10 100 1000 10000 100000 0 5000 10000 15000 20000 25000 Inhibition of PD-1 binding to Her2 + HCC827 cells Concentration [ng/ml] PD-1 binding (MFI) Avelumab Her2/CD3/PD-L1 med Her2/CD3/dummy Her2/CD3/PD-L1 low dummy/CD3/PD-L1 low 197’532 ng/ml 1’204 ng/ml 56 ng/ml 150 fold 10 100 1000 10000 100000 0 5000 10000 15000 20000 25000 30000 Inhibition of PD-1 binding to Her2 +++ HCC1954 cells Concentration [ng/ml] PD-1 binding (MFI) Avelumab Her2/CD3/PD-L1 med Her2/CD3/dummy Her2/CD3/PD-L1 low dummy/CD3/PD-L1 low 352 ng/ml 205 ng/ml 135 ng/ml 0.01 1 100 10000 0 20 40 60 80 100 CD8 + cell activation in presence of Her2 + /PD-L1 - MCF-7 cells Concentration [pM] CD69 pos. CD8 + cells [%] 0.01 1 100 10000 0 20 40 60 80 100 CD8 + cell activation in presence of Her2 + /PD-L1 + HCC827 cells Concentration [pM] CD69 pos. CD8 + cells [%] 0.01 1 100 10000 0 20 40 60 80 100 CD8 + cell activation in presence of Her2 +++ /PD-L1 + HCC1954 cells Concentration [pM] CD69 pos. CD8 + cells [%] 0.01 1 100 10000 0 20 40 60 80 100 CD8 + cell activation in presence of Her2 - /PD-L1 + CHO-PD-L1 cells Concentration [pM] CD69 pos. CD8+ cells [%]
Transcript of P779 Tetra-specific MATCH4 antibodies mediate concomitant ......e X. T ell ed t ell ling and CD8+...
ND022: superiority over scDb for targeting cells PD-L1 positive with very low Her2 expression
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Figure X. T cell mediated target cell killing and CD8+ cells activation in presence of A) Her2+/-/PD-L1+ HCC827 NSCLC cells B) Her2-/PD-L1+ CHO PD-L1 cells. In thisassay, freshly isolated human PBMCs were co-cultured for 16h with indicated target cells in presence of the different molecules tested. The MATCH4 Her2/CD3/PD-L1low/hSA showed a 20-fold better potency than the Her2/CD3 scDb on Her2+/-/PD-L1+ as compared to PD-L1 negative cells were only a slightly different potency isobserved. In presence of Her2 negative PD-L1 positive cells only a minor target cell killing and CD8+ cells activation was observed at high concentrations of the
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Figure X. T cell mediated target cell killing and CD8+ cells activation in presence of A) Her2+/-/PD-L1+ HCC827 NSCLC cells B) Her2-/PD-L1+ CHO PD-L1 cells. In thisassay, freshly isolated human PBMCs were co-cultured for 16h with indicated target cells in presence of the different molecules tested. The MATCH4 Her2/CD3/PD-L1low/hSA showed a 20-fold better potency than the Her2/CD3 scDb on Her2+/-/PD-L1+ as compared to PD-L1 negative cells were only a slightly different potency isobserved. In presence of Her2 negative PD-L1 positive cells only a minor target cell killing and CD8+ cells activation was observed at high concentrations of the
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Her2+/PD-L1- MCF-7 target cell killingCo-cultured with hPBMCs
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Figure X. T cell mediated target cell killing and CD8+ cells activation in presence of A) Her2+++/PD-L1+ HCC1954 ductal carcinoma cells, B) Her2+/PD-L1- MCF-7 breastadenocarcinoma cells In this assay, freshly isolated human PBMCs were co-cultured for 16h with indicated target cells in presence of the different molecules tested.The MATCH4 Her2/CD3/PD-L1low/hSA showed a 50 to 100-fold better potency than the Her2/CD3 scDb on Her2+++/PD-L1+ as compared to PD-L1 negative cellswere only a slightly different potency is observed.
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Figure X. T cell mediated target cell killing and CD8+ cells activation in presence of A) Her2+++/PD-L1+ HCC1954 ductal carcinoma cells, B) Her2+/PD-L1- MCF-7 breastadenocarcinoma cells In this assay, freshly isolated human PBMCs were co-cultured for 16h with indicated target cells in presence of the different molecules tested.The MATCH4 Her2/CD3/PD-L1low/hSA showed a 50 to 100-fold better potency than the Her2/CD3 scDb on Her2+++/PD-L1+ as compared to PD-L1 negative cellswere only a slightly different potency is observed.
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Combination approach Cytolytic synapse-restricted PD-L1 blockade
PD-L1
Her2+/PD-L1+ Cell
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HSA
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T cell activation levelT cell activation level T cell activation level
PD-L1
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CD3PD-1
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PD-L1Her2
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HER2 & PD-L1 expressing
cancer cell line: HCC1954
LabeledPD-1ECD
PD-L1 expressingcancer cell line:
HCC827
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ND022: Potency in cell killing and T cell activation
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cell linesPotency for T cell activation
IC50 (pM)
MATCH4 scDb
HCC1954 0.3861 35.91
HCC827 23.72 314.6
MCF-7 25.33 54.29
CHO-PD-L1 0.00 0.00
HER+++ /P
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Activation of NFAT/luc-reporter T cells
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Activation of NFAT/luc-reporter T cellsCo-cultured with Her2+++ HCC1954
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Her2/CD3/dummy/hSA + Nivolumab
Her2/CD3/PD-L1Low/hSA + Nivolumab
dummy/CD3/PD-L1Low/hSA + Nivolumab
0.1 1 10 100 1000 10000 1000000
200000
400000
600000
800000
1000000
Activation of NFAT/luc-reporter T cells
Co-cultured with Her2-/PD-L1+ CHO PD-L1 cells
Concentration [pM]
T c
ell a
ctiv
atio
n (R
LU) Her2/CD3/PD-L1Low/hSA
Her2/CD3/PD-L1Low/hSA + Nivolumab
dummy/CD3/PD-L1Low/hSA
dummy/CD3/PD-L1Low/hSA + Nivolumab
Her2/CD3/dummy/hSAHer2/CD3/dummy/hSA + Nivolumab
Her2/CD3/dummy/hSA + PD-L1Low scFv
A
B
T cell activation level
Her2 High / PD-L1+
T cell activation level
Her2 Low / PD-L1+
C
T cell activation level
Her2 Low / PD-L1-
T cell activation level
Her2- / PD-L1+
D
Tetra-specific MATCH4 antibodies mediate concomitant blockade of PD-L1 and potent T cell-stimulation, locally restricted to Her2+ tumors
Alexandre Simonin1, Tea Gunde1, Christian Hess1, Stefan Warmuth1, Matthias Brock1, Dan Snell1, Julia Tietz1, Eva Oswald2, Robin Heiz1, Naomi Flückiger1, Julia Zeberer1, Dania Diem1, Dana Mahler1, Diego Morenzoni1, Simone Muntwiler1, Benjamin Küttner1, Sebastian Meyer1, Timothy Egan3, David Urech1
1) Numab Therapeutics AG, Einsiedlerstrasse 34, 8820 Wädenswil, Switzerland; 2) Charles River Discovery Service Germany GmbH, Am Flughafen 12-14, 79108 Freiburg, Germany; 3) Numab US LLC, P.O. Box 902 Northboro, 01532 Massachusetts, US
Tumor-targeted T cell activation mechanisms have proved highlyeffective in the redirection of the immune system towards cancer cells.Of particular note is the use of CD3 T cell engagers and the approval ofthe molecule Blincyto for the treatment of patients suffering from B-ALL.One issue in identifying appropriate tumor associated antigens (TAAs) forCD3 T cell engagement is ensuring an appropriate expression level intumor tissue vs normal tissue to preferentially drive T cell mediatedcytotoxicity to the tumor.We sought to address this issue and open the therapeutic window of CD3engagers by the generation of an affinity tailored MATCH4 moleculetargeting PD-L1, Her2, CD3 and human serum albumin (hSA) topreferentially mediate T cell killing of tumor cells.We demonstrate that PD-L1/PD-1 antagonism is retained within thismolecule which has increased potency compared to a conventional CD3 xHer2 engager, in a single-chain diabody format (scDb), on both Her2 highas well as Her2 low cells, in the presence of simultaneous PD-L1expression. The activity is not enhanced on PDL1-negative cells andtherefore we demonstrate the potential to open the therapeutic windowtowards higher activity on cancer cells in the context of PD-L1expression.
Conclusions:
• A next-generation MATCH4 tetra-specific (Her2/CD3/PD-L1low/hSA) molecule displays exquisite anti-tumoralefficacy in Her2+ cancer models
• Increased therapeutic potential on Her2+/PD-L1+ cellswhilst not increasing activity on Her2+/PD-L1- cells (Fig 6A-C)
• No T cell activity in the presence of Her2- PD-L1+ cells dueto the use of a low-affinity anti-PD-L1 domain (Fig. 6D)
• PD-L1 / PD-1 antagonism is dependent on Her2 expressionand delivers an additive effect to T cell activation atconcentrations >100pM (Fig. 4)
• The anti-tumoral activity is predominantly driven by anavidity-effect of engaging both Her2 and PD-L1
• Potential to open the therapeutic window of Her2/CD3activity by increasing selectivity towards Her2 PD-L1double-positive cells and therefore may present a saferHer2 targeting T cell engager
Tumor targeted PD-L1 blockade
PD-L1 binding domain substantially increases molecule potency
A
B
Concept
Figure 6. T cell mediated target cell killing and CD8+ cell activation in presence of A) Her2+++/PD-L1+ HCC1954 ductal breast carcinoma cells, B) Her2+/PD-L1+ HCC827 NSCLCcells, C) Her2+/PD-L1- MCF-7 breast adenocarcinoma cells, D) Her2-/PD-L1+ CHO PD-L1 cells. Freshly isolated human PBMCs were co-cultured for 16h with indicated target cells inpresence of the different molecules. The MATCH4 Her2/CD3/PD-L1low/hSA showed an increased potency in target cell killing and T cell activation compared to the Her2/CD3scDb on Her2+++/PD-L1+ cells and Her2+/PD-L1+ cells. Activity of the MATCH4 molecule is similar to that of the Her2/CD3 scDb in the presence of Her2+/PD-L1- cells.Importantly, no killing activity or T cell activation is observed in the presence of Her2-/PDL1+ cells.
A B
Efficient tumor regression with the MATCH4 molecule
αPD-L1 binding-domain substantially increases potency
Figure 1. A) Conventional combination of a Her2/CD3 bispecific molecule with PD-L1 blockade results in potent T cell-activation and target cell lysis. B) αHer2-/αPD-L1-mediated avidity-binding elicits more potent T cell-activation and targetcell lysis than the combination therapy in the presence of Her2+PD-L1+ cancer cells, of both Her2-high and Her2-lowphenotypes. Targeting of Her2+PD-L1− cells is similarly effective as combination therapy, whilst importantly Her2−PD-L1+
cells are spared from cytolysis.
Affinity-optimized anti-PD-L1 domain specifically targeting Her2/PD-L1 expressing cancer cells
A
Figure 2. Neutralization of PD-1-binding to PD-L1+ cells. Effect of PD-L1-affinity on potency of blockade of PD-1-binding toA) Her2+++PD-L1+ HCC1954 ductal breast carcinoma cells and B) Her2+PD-L1+ HCC827 NSCLC cancer cells. PD-L1-blockadeis restricted to high Her2 expressing cancer cells. Careful selection of the affinity of the αPD-L1 domain (10’000-foldlower than Avelumab) allows considerable extension of the therapeutic window.
Her2/CD3/PD-L1low/hSA tetra-specific MATCH4 moleculeA B C
Figure 3. A) Schematic representation of Her2/CD3/PD-L1low/hSA tetra-specific MATCH4 molecule: αCD3 (VL1/VH1 in green), αhSA (VL2/VH2 in orange), αHer2 (VL3/VH3 in purple), αPD-L1 (VL4/VH4 in blue), inter-chain disulfide bridge indicated in yellow. Domains are connected by Gly-Ser peptide linkers, as indicated by red lines. B) SDS-PAGE analysis of the MATCH4, NR: non-reducing, R: reducing, M: molecular weight marker C) Representative SE-HPLC chromatogram of the MATCH4.
Figure 4. Blockade of PD-L1-signaling in the immunological synapse. CD3-engagement and blockade of PD-1-signaling byMATCH4 molecules was assessed in a transgenic NFAT-Luciferase reporter Jurkat cell-line. PD-1-signaling is induced by co-cultivation of PD-1+ reporter Jurkat cells with A) Her2+++PD-L1+ HCC1954 cancer cells or B) Her2−PD-L1+ CHO cells. CD3activation results in reporter gene activation and PD-L1-inhibition enhances maximum T cell-activation. CD3 activation requiresthe presence of Her2+ cells, and immunological synapse-restricted PD-L1-blockade elevates the efficacy of T cell-activationsimilarly to Nivolumab. When co-administered as an scFv, the low-affinity αPD-L1 domain has no effect.
Figure 5. T cell-mediated target cell-killing. Freshly isolated human PBMCs were co-cultured for 40h with Her2+++PD-L1+
HCC1954 cells in the presence of the different molecules and target cell-killing was assessed by annexin V staining.MATCH4 molecules were tested in the presence or absence of a saturating concentration of Nivolumab (1 μg/ml).MATCH4 harboring the low-affinity αPD-L1 domain is more potent than the MATCH 4 molecule lacking the αPD-L1domain. Absence of the αHer2 domain results in a 10000-fold lower potency. Nivolumab treatment, in combination withthe Her2/CD3/dummy/hSA tetra-specific MATCH4 molecule, did not change potency of the molecule, providing evidencethat the improved potency the Her2/CD3/PD-L1LOW/hSA MATCH4 is exclusively due to high avidity conferred byconcomitant binding to both PD-L1 and Her2.
Figure 8. Human PBMC-substituted NOG mice wereengrafted with HCC1954 ductal breast carcinomacells (n=8 per group). Mice were treated on day 0,5, 10, 15, 20, 25 and 30 (dotted vertical lines).Tumor growth and body weight was recorded twiceweekly. The Her2/CD3/PD-L1low/hSA tetra-specificMATCH4 molecule elicited considerable tumorregression in all treated mice. The MATCH4molecule demonstrated greater anti-tumoralefficacy than the Nivolumab + Trastuzumabcombination. Nivolumab shows limited anti-tumoral activity in this model and the controlmolecule Her2/PD-L1low/hSA shows a similar anti-tumoral activity despite a 5-fold lower potency invitro (data not shown), confirming effectivetumor-targeted PD-L1 blockade. No body weightloss was observed (not shown).
cell lines
Potency for T cell activationEC50 (pM)
MATCH4 scDb
HCC1954 0.39 35.91
HCC827 23.72 314.6
MCF-7 25.33 54.29
CHO-PD-L1 NC NCFold potency difference
Her2 high / PDL1+ vs Her2 low / PDL1-
65x 1.5x
Figure 7. T cell activation potencies of MATCH4 and scDb on cellsexpressing different Her2 and PD-L1 levels. A) EC50 values for T cellactivation mediated by MATCH4 and scDb molecules. B) Calculatedpotency ratios between MATCH4 and scDb. C) Her2 antibody bindingsites on different cancer cells. The MATCH4 Her2/CD3/PD-L1low/hSAshowed an increased potency on PD-L1 positive Her2 expressingcancer cells in comparison to the scDb regardless of Her2 expressionlevels. Data suggest an improved therapeutic window for theMATCH4 over the scDb in the context of PD-L1 expression.Importantly a greater difference in potency can be demonstrated forthe MATCH4 molecule between Her2 high / PD-L1+ cells and Her2 low/ PD-L1- cells compared to a scDb. NC = not calculable
A
B
Ccell lines
Her2 expressionAntibody binding sites
HCC1954 61937MCF-7 12670
HCC827 8908
A
B
Her2+/PD-L1+ cancer cell
PD-1
NFAT-Luciferasereporter Jurkat cell
CD3
HSAHer2
Luciferase
N
VL1
VH2 VH2
VH1
VL4
VH4
Peptide linker
CN
C
VL2
VL3
VH3
Inter-chaindisulfidebridge
5 6 7 8 9 100
50
100
Retention time [min]No
rma
lize
d A
bs
orb
an
ce
28
0 n
m [
%]
Her2+/PD-L1+ cancer cell
PD-1
T cell
CD3
HSAHer2Target cell
lysis
50 kDa
100 kDa75 kDa
37 kDa25 kDa
NR R M
0.01 1 100 10000-20
0
20
40
60
80
100
Her2+++/PD-L1+ HCC1954 target cell killingCo-cultured with hPBMCs
Concentration [pM]
Sp
ec
ific
Killi
ng
[%
]
Her2/CD3/PD-L1Low/hSA + Nivolumab
Her2/CD3/dummy/hSA
Her2/CD3/dummy/hSA + Nivolumab
Her2/CD3/dummy/hSA + PD-L1Low scFv
dummy/CD3/PD-L1Low/hSA
dummy/CD3/PD-L1Low/hSA + Nivolumab
Her2/CD3/PD-L1Low/hSA
0 2 6 9 13 16 20 23 27 30 330
500
1000
1500
HCC1954 xeno in humanized NOGTumor localized CD3 activation/PD-L1 blockade
days
Rel
ativ
e T
umor
Vol
ume
Her2/PD-L1low/hSA (5 mg/kg)
Palivizumab (5 mg/kg)
Nivolumab (5 mg/kg) Her2/CD3/PD-L1low/hSA (2.5 mg/kg)
Trastuzumab (5 mg/kg) + Nivolumab (5 mg/kg)
Her2/PD-L1low/hSA (25 mg/kg)
34th Annual Meeting of the Society for Immunotherapy of Cancer, 2019. National Harbor, Maryland, USA. Poster P779
P779
Optimal low affinity PD-L1 domain to maximize therapeutic window
«CONFIDENTIAL» 4
ND022-0070
10 100 1000 10000 1000000
5000
10000
15000
20000
25000
30000
Inhibition of PD-1 binding to Her2+++ HCC1954 cells
Concentration [ng/ml]
PD
-1 b
indi
ng
(M
FI)
Avelumab
Her2/CD3/PD-L1med
Her2/CD3/dummy
Her2/CD3/PD-L1low
dummy/CD3/PD-L1low
352 ng/ml
205 ng/ml
135 ng/ml
Neutralization of PD-L1/PD-1 interaction assessed by FC
10 100 1000 10000 1000000
5000
10000
15000
20000
25000
Inhibition of PD-1 binding to Her2+ HCC827 cells
Concentration [ng/ml]
PD
-1 b
ind
ing
(M
FI)
Avelumab
Her2/CD3/PD-L1med
Her2/CD3/dummy
Her2/CD3/PD-L1low
dummy/CD3/PD-L1low
197’532 ng/ml
1’204 ng/ml
56 ng/ml
150 fold
Figure X. Neutralization potency of PD-1 binding to PD-L1 expressing cells. A) Effect of PD-L1-affinity on potency to block PD-1 binding toHer2+++/PD-L1+ HCC1954 breast ductal carcinoma cells. B) Effect of PD-L1-affinity on potency to block PD-1 binding to Her2+/PD-L1+ HCC827NSCLC cancer cells. PD-L1 blockade is restricted to Her2 expressing cancer cells. Careful selection of the anti-PD-L1 domain affinity allowsconsiderable extension of the therapeutic window.
Optimal low affinity PD-L1 domain to maximize therapeutic window
«CONFIDENTIAL» 4
ND022-0070
10 100 1000 10000 1000000
5000
10000
15000
20000
25000
30000
Inhibition of PD-1 binding to Her2+++ HCC1954 cells
Concentration [ng/ml]
PD
-1 b
ind
ing
(M
FI)
Avelumab
Her2/CD3/PD-L1med
Her2/CD3/dummy
Her2/CD3/PD-L1low
dummy/CD3/PD-L1low
352 ng/ml
205 ng/ml
135 ng/ml
Neutralization of PD-L1/PD-1 interaction assessed by FC
10 100 1000 10000 1000000
5000
10000
15000
20000
25000
Inhibition of PD-1 binding to Her2+ HCC827 cells
Concentration [ng/ml]
PD
-1 b
ind
ing
(M
FI)
Avelumab
Her2/CD3/PD-L1med
Her2/CD3/dummy
Her2/CD3/PD-L1low
dummy/CD3/PD-L1low
197’532 ng/ml
1’204 ng/ml
56 ng/ml
150 fold
Figure X. Neutralization potency of PD-1 binding to PD-L1 expressing cells. A) Effect of PD-L1-affinity on potency to block PD-1 binding toHer2+++/PD-L1+ HCC1954 breast ductal carcinoma cells. B) Effect of PD-L1-affinity on potency to block PD-1 binding to Her2+/PD-L1+ HCC827NSCLC cancer cells. PD-L1 blockade is restricted to Her2 expressing cancer cells. Careful selection of the anti-PD-L1 domain affinity allowsconsiderable extension of the therapeutic window.
ND022: More efficient targeting of cancer cell lines expressing Her2 and PD-L1
11
0.01 1 100 10000-20
0
20
40
60
80
100
Her2+++/PD-L1+ HCC1954 target cell killingCo-cultured with hPBMCs
Concentration [pM]
Spe
cifi
c K
illin
g [%
] Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
0.01 1 100 10000-20
0
20
40
60
80
100
Her2+/PD-L1- MCF-7 target cell killingCo-cultured with hPBMCs
Concentration [pM]
Spe
cifi
c K
illin
g [%
] Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
Figure X. T cell mediated target cell killing and CD8+ cells activation in presence of A) Her2+++/PD-L1+ HCC1954 ductal carcinoma cells, B) Her2+/PD-L1- MCF-7 breastadenocarcinoma cells In this assay, freshly isolated human PBMCs were co-cultured for 16h with indicated target cells in presence of the different molecules tested.The MATCH4 Her2/CD3/PD-L1low/hSA showed a 50 to 100-fold better potency than the Her2/CD3 scDb on Her2+++/PD-L1+ as compared to PD-L1 negative cellswere only a slightly different potency is observed.
0.01 1 100 100000
20
40
60
80
100
CD8+ cell activation
in presence of Her2+++/PD-L1+ HCC1954 cells
Concentration [pM]
CD
69 p
os. C
D8+ c
ells
[%]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
0.01 1 100 100000
20
40
60
80
100
CD8+ cell activation
in presence of Her2+/PD-L1- MCF-7 cells
Concentration [pM]
CD
69
pos
. C
D8
+ cel
ls [%
]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
ND022: superiority over scDb for targeting cells PD-L1 positive with very low Her2 expression
12
0.01 1 100 10000-20
0
20
40
60
80
100
Her2-/PD-L1+ CHO-PD-L1 target cell killingCo-cultured with hPBMCs
Concentration [pM]
Spe
cifi
c K
illin
g [%
] Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
0.01 1 100 100000
20
40
60
80
100
CD8+ cell activation
in presence of Her2+/PD-L1+ HCC827 cells
Concentration [pM]
CD
69
pos
. C
D8
+ cel
ls [%
]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
0.01 1 100 100000
20
40
60
80
100
CD8+ cell activation
in presence of Her2-/PD-L1+ CHO-PD-L1 cells
Concentration [pM]
CD
69
pos
. C
D8
+ ce
lls [%
]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
Figure X. T cell mediated target cell killing and CD8+ cells activation in presence of A) Her2+/-/PD-L1+ HCC827 NSCLC cells B) Her2-/PD-L1+ CHO PD-L1 cells. In thisassay, freshly isolated human PBMCs were co-cultured for 16h with indicated target cells in presence of the different molecules tested. The MATCH4 Her2/CD3/PD-L1low/hSA showed a 20-fold better potency than the Her2/CD3 scDb on Her2+/-/PD-L1+ as compared to PD-L1 negative cells were only a slightly different potency isobserved. In presence of Her2 negative PD-L1 positive cells only a minor target cell killing and CD8+ cells activation was observed at high concentrations of the
0.01 1 100 10000-20
0
20
40
60
80
100
Her2+/PD-L1+ HCC827 target cell killingCo-cultured with hPBMCs
Concentration [pM]
Spe
cifi
c ki
lling
[%]
Her2/CD3 scDb
Her2/CD3/PD-L1Low/hSA
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
ND022: More efficient targeting of cancer cell lines expressing Her2 and PD-L1
11
0.01 1 100 10000-20
0
20
40
60
80
100
Her2+++/PD-L1+ HCC1954 target cell killingCo-cultured with hPBMCs
Concentration [pM]
Spe
cifi
c K
illi
ng
[%
]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
0.01 1 100 10000-20
0
20
40
60
80
100
Her2+/PD-L1- MCF-7 target cell killingCo-cultured with hPBMCs
Concentration [pM]
Spe
cifi
c K
illi
ng
[%
]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
Figure X. T cell mediated target cell killing and CD8+ cells activation in presence of A) Her2+++/PD-L1+ HCC1954 ductal carcinoma cells, B) Her2+/PD-L1- MCF-7 breastadenocarcinoma cells In this assay, freshly isolated human PBMCs were co-cultured for 16h with indicated target cells in presence of the different molecules tested.The MATCH4 Her2/CD3/PD-L1low/hSA showed a 50 to 100-fold better potency than the Her2/CD3 scDb on Her2+++/PD-L1+ as compared to PD-L1 negative cellswere only a slightly different potency is observed.
0.01 1 100 100000
20
40
60
80
100
CD8+ cell activation
in presence of Her2+++/PD-L1+ HCC1954 cells
Concentration [pM]
CD
69
pos
. C
D8
+ cel
ls [%
]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
0.01 1 100 100000
20
40
60
80
100
CD8+ cell activation
in presence of Her2+/PD-L1- MCF-7 cells
Concentration [pM]
CD
69
po
s. C
D8
+ cel
ls [
%]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
ND022: superiority over scDb for targeting cells PD-L1 positive with very low Her2 expression
12
0.01 1 100 10000-20
0
20
40
60
80
100
Her2-/PD-L1+ CHO-PD-L1 target cell killingCo-cultured with hPBMCs
Concentration [pM]
Spe
cifi
c K
illin
g [%
] Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
0.01 1 100 100000
20
40
60
80
100
CD8+ cell activation
in presence of Her2+/PD-L1+ HCC827 cells
Concentration [pM]
CD
69
po
s. C
D8
+ cel
ls [%
]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
0.01 1 100 100000
20
40
60
80
100
CD8+ cell activation
in presence of Her2-/PD-L1+ CHO-PD-L1 cells
Concentration [pM]
CD
69
pos
. C
D8
+ ce
lls
[%]
Her2/CD3/PD-L1Low/hSA
Her2/CD3 scDb
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
Figure X. T cell mediated target cell killing and CD8+ cells activation in presence of A) Her2+/-/PD-L1+ HCC827 NSCLC cells B) Her2-/PD-L1+ CHO PD-L1 cells. In thisassay, freshly isolated human PBMCs were co-cultured for 16h with indicated target cells in presence of the different molecules tested. The MATCH4 Her2/CD3/PD-L1low/hSA showed a 20-fold better potency than the Her2/CD3 scDb on Her2+/-/PD-L1+ as compared to PD-L1 negative cells were only a slightly different potency isobserved. In presence of Her2 negative PD-L1 positive cells only a minor target cell killing and CD8+ cells activation was observed at high concentrations of the
0.01 1 100 10000-20
0
20
40
60
80
100
Her2+/PD-L1+ HCC827 target cell killingCo-cultured with hPBMCs
Concentration [pM]
Spe
cifi
c ki
llin
g [%
]
Her2/CD3 scDb
Her2/CD3/PD-L1Low/hSA
Her2/CD3/dummy/hSA
Trastuzumab + Nivolumab
