Multiple Bispecific Checkpoint Combinations Promote T Cell Activation Matthew J

Home , BTLA, CD80, CD86

Multiple Bispecific Checkpoint Combinations Promote T cell activation Matthew J. Bernett, Gregory L. Moore, Michael Hedvat, Christine Bonzon, Seung Y. Chu, Rumana Rashid, Kendra N. Avery, Alex Nisthal, Umesh Muchhal, John R. Desjarlais

ItIntrod ucti on TILs co-express chkitheckpoint recept ors Bispec ific an tibodi es sel ecti vel y t arget

• Tumor infiltrating lymphocytes (TILs) co-express multiple checkpoint A A dual-checkpoint positive T cells NN NN receptitors, in con ttttrast to lymphtfdithihhocytes found in the periphery Receptor occupancy of PD-1 and LAG-3 on human CD3+ T cells stimulated with SEB (Matsuzaki et al PNAS 2010, Fourcade et al Cancer Res 2012, Gros

et al JCI 2014) PD-1 mR PD-1 mR Negative Control PD-1xLAG1 x LAG-3 Bispecific p < 0.001 for all tumor types p < 0.001 for all tumor types 15.6 %17.4 % 12.0 % 0.7 %

• TILs that co-express multiple checkpoint receptors may be resistant Log Log

to single-checkpoint blockade Log CTLA-4 mRNA Log LAG-3 mRNA • We produced PD-1 x CTLA-4, PD-1 x LAG-3, CTLA-4 x LAG-3, and PD-1 x BTLA bispecific antibodies and characterized their T cell mRNA -3 mRNA

activation activity in vitro and in vivo (huPBMC-engrafted NSG mice) 11

59.0 % 8.0 % 74.2 % 13.1 %

p < 0.001 for all tumor types p < 0.001 for all tumor types D-1 Log PD- Log LAG

Summary PP

• Bispecific antibodies selectively target T cells co-expressing multiple Log BTLA mRNA Log CTLA-4 mRNA LAG-3 checkpoint receptors The results shown are based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/ • All bispecific antibody pairs enhanced IL-2 production in an in vitro SEB stimulation assay relative to control, indicating functional checkpoint blockade Comppyonent antibody domains block check ppgpoint receptor / ligand interactions • Bispecific antibodies promote human T cell proliferation in mice PD-1 / PD-L1 PD-1 / PD-L2 LAG-3 / MHC II CTLA-4 / CD80 CTLA-4 / CD86 BTLA / HVEM 4000 0.4 nm) nm) nm) I) I) I) engrafted with human PBMCs (( (( (( 020.2 030.3 3000 • CTLA-4 x LAG-3 bispecific combines productively with anti-PD-1 to 0.2 2000 0.1 0.1 G-3 (MF

promote triple checkpoint blockade and strong T cell stimulation sponse sponse sponse D-L1 (MF D-L2 (MF ee ee ee 1000 AA 000.0 PP PP L R R 0.0 R 293T-PD-1 293T-PD-1 Daudi 200 400 600 0 100 200 300 0 -4 -3 -2 -1 0 1 2 Time (s) Time (s) Time (s)  PD-1 one-arm  PD-1 one-arm  LAG-3 one-arm No Antibody No Antibody No Antibody Dual-checkpoint bispecific antibody design (Log µg/ mL) (Log µg/ mL) (Log µg/ mL)  CTLA-4 one-arm  CTLA-4 one-arm  BTLA one-arm

 Checkpoint X  Check po int YFY scFv • Hig h y ie lding s ta ble ce ll lines Bispecific checkpoint blockade promotes T cell activation in vitro • Protein A + Ion exchange chromatography SEB-stimulated human lymphocytes (multiple healthy donors) Xtend® ** * ** * n.s. 10 1.6 X 100 1.8 X 3.3 X 100 1.5 X 4 )

• Modified Fc domain eliminates FcR interactions ee 3 • Modified Fc domain with Xtend technology to promote long half-life hang -2

cc 5 10 10 2

• Fc substitutions promote heterodimer formation and facilitate LL I purification by standard methods 1 1 6.0 X • Optimized checkpoint receptor antibodies were plugged into the (Fold- 0 platform without further reformatting ** 1 0  PD-1 PD-1  PD-1 CTLA-4 CTLA-4  PD-1 PD-1  PD-1 PD-1 Bivalent x Bivalent x x Bivalent x Bivalent x TIL acti vati on with bi specifi c antib odi es CTLA-4 LAG-3 LAG-3 LAG-3 BTLA p ≤ 0.01 + **  PD-1 Periphery Tumor * p ≤ 0050.05 Bivalent Microenvironment Checkpoint Checkpoint X Y Bispecific checkpoint blockade promotes human T cell proliferation in huPBMC huPBMC-NSG mice

Tumor ** n.s. * * * * 27 X 34X3.4 X 19X1.9 X 21X2.1 X 22X2.2 X 104

4 ) 104 10 ells s s

C 104 + 45 Count ( (

D 2.7 X

D 27X 103 C 3.3 X ** 103 103 ** 103 VhilVehicle  PD-1 PD-1  PD-1 Vehicle  PD-1 CTLA-4 CTLA-4 VhilVehicle  PD-1 PD-1 VhilVehicle  PD-1 PD-1 Bivalent x Bivalent Bivalent x x Bivalent x Bivalent x CTLA-4 + LAG-3 LAG-3 LAG-3 BTLA Peripheral T cells Multi-checkpoint positive TILs  CTLA-4 + Bivalent  PD-1 Weak bispecific-antibody interactions Avid bispecific antibody binding Bivalent No T cell activation TIL activation