Reporter Gene Immunotherapy Bioassays

Reporter Gene Immunotherapy Bioassays Expand the Tool Box for Drug Development in Individual and Combination Immunotherapy Jey Cheng, Ph.D. 215. BEBPA Bioassay Conference September 3, 216

Presentation Outline Considerations in developing reporter gene immunotherapy Bioassays Case studies of assay designs for MOA-based bioassays T Cell Activation (Anti-CD3 bispecific antibody, CAR) Immune checkpoint modulation Combination therapy Summary 215. 2

B io lu m in e s c e n c e (R L U ) Considerations in developing Reporter Gene Immunotherapy Bioassays 1. Control cells as critical reagent Use engineered cell lines to replace primary cells Thaw-and-Use cells: no need of cell culture, convenient and timesaving, day to day consistency Cell line stability monitored and established, >P4 Multi-tiers of cell banks and controlled cell manufacture (CellSTACK, spinner, G-Rex, Triple-layer) QC tests: Cell ID, STR, mycoplasma, viability, function assay Thaw-and-Use Format Monitor Cell Line Stability 4. 1 3 3. 1 3 2. 1 3 T IG IT E ffe c to r c e ll lin e p a s s a g e # p 1 6 p 2 8 p 4 p 5 4 Thaw Cells 215. Resuspend and Plate Cells for Assay Time to complete: <24 hours Measure Luminescence 1. 1 3-3 - 2-1 1 2 L o g [a n ti-t IG IT ], g /m l 3

Considerations in developing Reporter Gene Immunotherapy Bioassays 2. Optimized and robust protocol Simple and streamlined assay procedure, no washing and spinning steps: Add-Mix-Read format Short assay time: one day; hands on time: a couple of hours Standard assay reagents and instrument: Medium, serum from multiple vendors Lab equipped luminescence readers Identify sources of assay variations (single factor and DOE): cell number, E:T ratio, incubation time, assay buffer Standard 96-well format, compatible for 384-format by automation Add Test Biologic Plate Antigenexpressing Tumor Cells plate Effector Cells Induce (6-24 h) Add Bio-Glo Reagent Measure Luminescence 215. 4

M e a s u r e d R e la tiv e P o te n c y, % Considerations in developing Reporter Gene Immunotherapy Bioassays 3. Pre-qualified according to ICH guideline specificity precision accuracy linearity Range Robustness 2 5 2 Dilutional Linearity/Range Y=1.883X-.838 R 2 =.997 1 5 1 5 5 1 1 5 2 2 5 E x p e c te d R e la tiv e P o te n c y, % 215. Assay qualification for PD-1/PD-L1 Blockade Bioassay 5

Considerations in developing Reporter Gene Immunotherapy Bioassays 4. Test suitability for drug development life cycle Early research and candidate screening Potency determination Stability study Neutralizing antibody monitoring Target Discovery Screen and Optimization Pre- & Clinical Studies Antibody Manufacture Post-Launch Monitoring 384-well format by automation Stability-Indicating Human Serum Tolerance 215. 6

Case 1: T Cell Activation Bioassays Platform assays for modulators of T cell activation Inhibition: Orencia Redirected activation CD3 bispecific Ab CAR-T 215. 7

B io lu m in e s c e n c e (R L U ) B io lu m in e s c e n c e (R L U ) T Cell Activation Assay: Assay Design Two TCR/CD3 Effector Cells Respond Similarly to TCR/CD3 signaling TCR/CD3 (NFAT) Effector Cell: Jurkat cells engineered with an NFAT-RE driving luciferase expression. Responds to TCR/CD3 activation, and responds minimally to CD28 co-stimulation. TCR/CD3 (IL-2) Effector Cell: Jurkat cells engineered with an IL-2 promoter driving luciferase expression. Responds to TCR/CD3 and CD28 stimulation. NFAT-RE luciferase IL-2 promoter luciferase 2 1 5 EC 5 = 4ng/ml Fold induction = 53 1 2 9 EC 5 = 28ng/ml Fold induction = 39 1 6 5 3-4 - 3-2 - 1 L o g [a n ti-c D 3 ], g /m l - 4-3 - 2-1 L o g [a n ti-c D 3 ], g /m l 215. 8

L u m in e s c e n c e R L U L u m in e s c e n c e R L U TCR/CD3 (IL-2) or (NFAT) Effector Cells Respond Differentially to CD28 Activity Abatacept is a CTLA-4/IgG fusion protein used to prevent overactive immune system. 1 TCR/CD3 (IL-2) 8 6 4 2-5 - 4-3 - 2-1 1 L o g [a b a ta c e p t] u M 1 5 TCR/CD3 (NFAT) 1 5-5 - 4-3 - 2-1 1 L o g [a b a ta c e p t] u M A reminder to choose the right assay system: a Fit-for-Purpose Bioassay 215. 9

B io lu m in e s c e n c e (R L U ) B io lu m in e s c e n c e (R L U ) T Cell Activation Bioassay for Anti-CD3 Bispecific Antibody Assay Design TCR/CD3 (NFAT) Antigen-expressing Target Cells 4 1 6 3 1 6 Adherent SK-BR3 as target cells antigen 2 1 6 T Cell Receptor Bispecific Ab 1 1 6 1.2 1 7-3 - 2-1 1 2 L o g [c a tu m a x o m a b ], n g /m l Suspension Raji as target cells R a ji c e lls Catumaxomab (Removab) CD3xEpCAM n o R a ji c e lls 8. 1 6 RE Luciferase TCR/CD3 Effector Cells 4. 1 6-3 - 2-1 1 2 3 L o g [b lin a tu m o m a b ], p M Blinatumomab (Blincyto) CD3xCD19 Bispecific T Cell Engager (BiTE) 215. Similar results seen with TCR/CD3 (IL-2) Effector Cells 1

T Cell Activation Bioassay for CAR-T Cell Activity Assay Design Antigen-expressing Target Cells antigen T Cell Receptor CAR acd2-car acd19-car RE 215. Luciferase TCR/CD3 Effector Cells acd19-car Similar results seen with TCR/CD3 (IL-2) Effector Cells acd2-car 11

Case 2: Immune Checkpoint Bioassays Co-inhibitory PD-1 TIGIT CTLA-4 LAG3 Co-stimulatory CD4 GITR 4-1BB OX4 HVEM 215. 12

MOA of Immune Checkpoint Modulation Blocking Ab for immune inhibitory receptors (PD- 1, CTLA4) release the brakes. Agonist Ab for immune co-stimulatory receptors (GITR, OX4, CD27, CD137) push the gas. T cells are activated and kill the tumor cells. 215. 13

B io lu m in e s c e n c e (R L U ) PD-L1 - cells PD-L1 + cells + α-pd-l1 + α-pd-1 + α-ctla4 PD-1/PD-L1 Blockade Bioassay Assay Design PD-L1 aapc Cells Reflecting MOA ❶ ❷ ❸ PD-L1 TCR Activator Anti-PD-L1 T Cell Receptor Anti-PD-1 2.5 1 6 Assay specificity P D -1 A b 2. 1 6 P D -L 1 A b PD-1 1.5 1 6 P D -L 2 A b 1. 1 6 RE 215. Luciferase PD-1 Effector Cells 5. 1 5-9 - 8-7 - 6-5 - 4 L o g [te s t a n tib o d y ], g /m l 14

B io lu m in e s c e n c e (R L U ) TIGIT/CD155 Blockade Bioassay Assay Design CD155 aapc Cells CD155 TCR Activator 6. 1 3 A n ti- T IG IT A n ti- P D -1 T Cell Receptor CD226 Anti-TIGIT TIGIT 4. 1 3 2. 1 3 RE Luciferase TIGIT Effector Cells - 2-1 1 2 L o g [te s t a n tib o d y ], g /m l 215. 15

B io lu m in e s c e n c e (R L U ) CTLA-4 Blockade Bioassay Assay Design aapc/raji Cells TCR Activator CD8/86 Anti-CTLA-4 1 6 1 2 a n ti-c T L A -4 ip ilim u m a b a n ti-h E R 2 tra s tu z u m a b a n ti-p D -L 1 A b T Cell Receptor CD28 CTLA-4 8 4 215. RE Luciferase CTLA-4 Effector Cells - 3-2 - 1 1 2 3 L o g [te s t a n tib o d y ], g /m l 16

Case 3: Combination Therapy Bioassays PD-1 + CTLA-4 PD-1 + TIGIT PD-1 + CD3 bispecific Ab PD-1 + CAR 215. 17

B io lu m in e s c e n c e (R L U ) PD-1+CTLA4 Combination Bioassay Assay Design PD-L1+ CD8 aapc Cells PD-L1 CD8 8. 1 3 n iv o lu m a b Ip ilim u m a b n iv o lu m a b + ip ilim u m a b CD28 CTLA-4 6. 1 3 4. 1 3 PD-1 RE Luciferase PD-1+ CTLA-4 Effector Cells 2. 1 3-9 - 8-7 - 6-5 - 4 L o g [te s t a n tib o d y ], g /m l 215. 18

B io lu m in e s c e n c e (R L U ) PD-1+TIGIT Combination Bioassay Assay Design PD-L1+CD155 aapc Cells PD-L1 CD155 CD226 5. 1 4 4. 1 4 3. 1 4 Is o ty p e C o n tr o l A n ti-p D -1 A b A n ti-t IG IT A b A n ti-p D -1 + A n ti-t IG IT PD-1 TIGIT 2. 1 4 1. 1 4 PD-1+TIGIT Effector Cells 215. RE Luciferase - 3-2 - 1 1 2 L o g [te s t a n tib o d y ], g /m l 19

B io lu m in e s c e n c e (R L U ) Combination Bioassay for PD-1 Blockade and CAR Assay Design PD-L1+ antigen-expressing Target Cells PD-L1 antigen Effector Cells + antigen+ target cells J u rk a t/p D -1 + H E K 2 9 3 /C D 1 9 J u rk a t/p D -1 + H E K 2 9 3 /C D 1 9 /P D -L 1 T Cell Receptor 2. 1 5 J u rk a t/p D -1 /a -C D 1 9 -C A R + H E K 2 9 3 /C D 1 9 J u rk a t/p D -1 /a -C D 1 9 -C A R + H E K 2 9 3 /C D 1 9 /P D -L 1 CAR 1.5 1 5 PD-1 1. 1 5 215. RE PD-1 Effector Cells Luciferase 5. 1 4-9 - 8-7 - 6-5 - 4 L o g [n iv o lu m a b ], g /m l 2

B io lu m in e s c e n c e (R L U ) Combination Bioassay for PD-1 Blockade and CD3 Bispecific Ab Assay Design PD-L1+ antigen-expressing Target Cells PD-L1 CD19 T a rg e t c e ll / C D 3 b is p e c ific A b C D 1 9 + ta rg e r c e lls / N o b is p e c ific A b C D 1 9 + ta r g e t c e ll / A d d B lin a tu m o m a b BiTE C D 1 9 /P D -L 1 + ta r g e t c e ll / A d d B lin a tu m o m a b 1. 1 6 PD-1 T Cell Receptor 5. 1 5 PD-1 Effector Cells 215. RE Luciferase - 9-8 - 7-6 - 5-4 L o g [n iv o lu m a b ], g /m l 21

Summary Reporter-based immunotherapy bioassays are able to quantitatively measure the desired biological activities for targeted drug candidates. Combination bioassays can be used to monitor individual and synergetic effects from multiple immunotherapy strategies. 215. 22

Acknowledgement Pete Stecha Jamison Grailer Jun Wang Michael Beck Julia Gilden Jim Hartnett Mei Cong Frank Fan 215. 23