Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Reporter Gene Immunotherapy Bioassays Expand the Tool Box for Drug Development in Individual and Combination Immunotherapy Jey Cheng, Ph.D. Promega Corporation Promega Corporation ©2015 Promega Corporation. BEBPA Bioassay Conference September 30, 2016 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 Promega Corporation ©2015 Promega Corporation. 2 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, >P40 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 Resuspend and Plate Cells for Assay Measure Luminescence B io lu m in e s c e n c e (R L U ) Thaw Cells Monitor Cell Line Stability 4 .0 1 0 03 3 .0 1 0 03 T IG IT E ffe c to r c e ll lin e p a s s a g e # p16 p28 p40 2 .0 1 0 03 1 .0 1 0 03 p54 0 Time to complete: <24 hours Promega Corporation ©2015 Promega Corporation. -3 -2 -1 0 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 plate Effector Cells Add Test • Lab equipped luminescence readers Biologic 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 Induce (6-24 h) Plate Antigenexpressing Tumor Cells Add Bio-Glo™ Reagent Measure Luminescence Promega Corporation ©2015 Promega Corporation. 4 Considerations in developing Reporter Gene Immunotherapy Bioassays 3. Pre-qualified according to ICH guideline M e a s u r e d R e la t iv e P o t e n c y , % specificity precision accuracy linearity Range Robustness Dilutional Linearity/Range 250 Y=1.0883X-0.0838 R2=0.997 200 150 100 50 0 0 50 100 150 200 250 E x p e c t e d R e la t iv e P o t e n c y , % Promega Corporation ©2015 Promega Corporation. 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 384-well format by automation Promega Corporation ©2015 Promega Corporation. Pre- & Clinical Studies Antibody Manufacture Stability-Indicating Post-Launch Monitoring Human Serum Tolerance 6 Case 1: T Cell Activation Bioassays Platform assays for modulators of T cell activation Inhibition: • Orencia Promega Corporation ©2015 Promega Corporation. Redirected activation • CD3 bispecific Ab • CAR-T 7 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 . IL-2 promoter luciferase 2000000 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 ) NFAT-RE EC50= 40ng/ml Fold induction = 53 1500000 1000000 500000 0 -4 TCR/CD3 (IL-2) Effector Cell: • Jurkat cells engineered with an IL-2 promoter driving luciferase expression. • Responds to TCR/CD3 and CD28 stimulation. -3 -2 -1 L o g [a n ti- C D 3 ], g /m l Promega Corporation ©2015 Promega Corporation. 0 luciferase 12000 EC50= 28ng/ml Fold induction = 39 9000 6000 3000 0 -4 -3 -2 -1 0 L o g [a n ti- C D 3 ], g /m l 8 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. 80000 60000 40000 20000 TCR/CD3 (NFAT) 1500000 TCR/CD3 (IL-2) 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 100000 A reminder to choose the right assay system: a Fit-for-Purpose Bioassay 1000000 500000 0 0 -5 -4 -3 -2 -1 L o g [a b a ta c e p t] u M Promega Corporation ©2015 Promega Corporation. 0 1 -5 -4 -3 -2 -1 0 1 L o g [a b a ta c e p t] u M 9 T Cell Activation Bioassay for Anti-CD3 Bispecific Antibody Assay Design Antigen-expressing Target Cells antigen Bispecific Ab B io lu m in e s c e n c e ( R L U ) TCR/CD3 (NFAT) Adherent SK-BR3 as target cells 4 1 0 06 3 1 0 06 2 1 0 06 1 1 0 06 0 -3 -2 -1 0 1 2 Catumaxomab (Removab) CD3xEpCAM L o g [c a tu m a x o m a b ], n g /m l T Cell Receptor RE Luciferase B io lu m in e s c e n c e (R L U ) 1 .2 1 0 Suspension Raji as target cells 07 R a ji c e lls n o R a ji c e lls 8 .0 1 0 06 4 .0 1 0 06 Blinatumomab (Blincyto) CD3xCD19 Bispecific T Cell Engager (BiTE) 0 -3 -2 -1 0 1 2 3 L o g [b lin a tu m o m a b ], p M TCR/CD3 Effector Cells Promega Corporation ©2015 Promega Corporation. Similar results seen with TCR/CD3 (IL-2) Effector Cells 10 T Cell Activation Bioassay for CAR-T Cell Activity Assay Design Antigen-expressing Target Cells antigen aCD20-CAR T Cell Receptor CAR aCD19-CAR aCD19-CAR RE aCD20-CAR Luciferase TCR/CD3 Effector Cells Promega Corporation ©2015 Promega Corporation. Similar results seen with TCR/CD3 (IL-2) Effector Cells 11 Case 2: Immune Checkpoint Bioassays Co-inhibitory PD-1 TIGIT CTLA-4 LAG3 Promega Corporation ©2015 Promega Corporation. Co-stimulatory CD40 GITR 4-1BB OX40 HVEM 12 MOA of Immune Checkpoint Modulation Blocking Ab for immune inhibitory receptors (PD1, CTLA4) release the brakes. Agonist Ab for immune co-stimulatory receptors (GITR, OX40, CD27, CD137) push the gas. T cells are activated and kill the tumor cells. Promega Corporation ©2015 Promega Corporation. 13 PD-1/PD-L1 Blockade Bioassay Assay Design Reflecting MOA Anti-PD-1 PD-1 RE Luciferase B io lu m in e s c e n c e (R L U ) Assay specificity T Cell Receptor 2 .5 1 0 06 2 .0 1 0 06 1 .5 1 0 06 1 .0 1 0 06 5 .0 1 0 05 P D -1 A b P D -L 1 A b P D -L 2 A b 0 -9 PD-1 Effector Cells Promega Corporation ©2015 Promega Corporation. + α-CTLA4 Anti-PD-L1 + α-PD-L1 TCR Activator PD-L1 - cells PD-L1 ❸ PD-L1 + cells ❶ ❷ + α-PD-1 PD-L1 aAPC Cells -8 -7 -6 -5 -4 L o g [te s t a n tib o d y ], g /m l 14 TIGIT/CD155 Blockade Bioassay Assay Design CD155 aAPC Cells TCR Activator Anti-TIGIT T Cell Receptor TIGIT CD226 B io lu m in e s c e n c e (R L U ) CD155 6 .0 1 0 A n ti- T IG IT 03 A n ti- P D -1 4 .0 1 0 03 2 .0 1 0 03 0 RE Luciferase TIGIT Effector Cells Promega Corporation ©2015 Promega Corporation. -2 -1 0 1 2 L o g [te s t a n tib o d y ], g /m l 15 CTLA-4 Blockade Bioassay Assay Design aAPC/Raji Cells a n ti-C T L A -4 ip ilim u m a b CD80/86 T Cell Receptor Anti-CTLA-4 CD28 CTLA-4 B io lu m in e s c e n c e (R L U ) TCR Activator 16000 a n ti-H E R 2 tra s tu z u m a b a n ti-P D -L 1 A b 12000 8000 4000 0 RE Luciferase -3 -2 -1 0 1 2 3 L o g [te s t a n tib o d y ], g /m l CTLA-4 Effector Cells Promega Corporation ©2015 Promega Corporation. 16 Case 3: Combination Therapy Bioassays Promega Corporation ©2015 Promega Corporation. PD-1 + CTLA-4 PD-1 + TIGIT PD-1 + CD3 bispecific Ab PD-1 + CAR 17 PD-1+CTLA4 Combination Bioassay Assay Design PD-L1+ CD80 aAPC Cells PD-L1 CD80 n iv o lu m a b CD28 CTLA-4 PD-1 RE Luciferase B io lu m in e s c e n c e (R L U ) 8 .0 1 0 03 Ip ilim u m a b n iv o lu m a b + ip ilim u m a b 6 .0 1 0 03 4 .0 1 0 03 2 .0 1 0 03 0 -9 PD-1+ CTLA-4 Effector Cells Promega Corporation ©2015 Promega Corporation. -8 -7 -6 -5 -4 L o g [te s t a n tib o d y ], g /m l 18 PD-1+TIGIT Combination Bioassay Assay Design PD-L1+CD155 aAPC Cells PD-L1 CD155 CD226 TIGIT PD-1 B io lu m in e s c e n c e (R L U ) 5 .0 1 0 Is o ty p e C o n tr o l 04 A n ti-P D -1 A b 4 .0 1 0 04 3 .0 1 0 04 2 .0 1 0 04 1 .0 1 0 04 A n ti-T IG IT A b A n ti-P D - 1 + A n ti-T IG IT 0 RE Luciferase -3 -2 -1 0 1 2 L o g [ t e s t a n t ib o d y ] , g /m l PD-1+TIGIT Effector Cells Promega Corporation ©2015 Promega Corporation. 19 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 r k a t/P D - 1 + H E K 2 9 3 /C D 1 9 /P D - L 1 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 CAR PD-1 RE Luciferase PD-1 Effector Cells Promega Corporation ©2015 Promega Corporation. B io lu m in e s c e n c e (R L U ) T Cell Receptor 2 .0 1 0 05 1 .5 1 0 05 1 .0 1 0 05 5 .0 1 0 04 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 0 -9 -8 -7 -6 -5 -4 L o g [n iv o lu m a b ], g /m l 20 Combination Bioassay for PD-1 Blockade and CD3 Bispecific Ab Assay Design PD-L1+ antigen-expressing Target Cells CD19 PD-L1 T a r g e t c e ll / C D 3 b is p e c if ic A b C D 1 9 + t a r g e r c e lls / N o b is p e c if ic 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 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 T Cell Receptor PD-1 RE Luciferase PD-1 Effector Cells Promega Corporation ©2015 Promega Corporation. B io lu m in e s c e n c e (R L U ) BiTE 1 .0 1 0 06 5 .0 1 0 05 0 -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. Promega Corporation ©2015 Promega Corporation. 22 Acknowledgement Pete Stecha Jamison Grailer Jun Wang Michael Beck Julia Gilden Jim Hartnett Mei Cong Frank Fan Promega Corporation ©2015 Promega Corporation. 23