WHAT IS THE QUESTION?: Thoughts for PK/PD experts from a clinical immunotherapist

1. WHAT IS THE QUESTION?: Thoughts for PK/PD experts from a clinical immunotherapist Richard P Junghans, PhD, MD Associate Professor of Medicine Boston University School of Medicine Director, Biotherapeutics Development Lab Roger Williams Medical CenterProvidence, RI, USA No commercial relationships to disclose.

2. ABSTRACT: The classic two-compartment model for pharmacokinetics transformed the understanding of drug delivery more than 50 years ago. Since this time, the recognition of drug interactions with the host has led to new complexity in these models. The best modeling is based on mechanisms and not merely on model independent curve-fitting. Mechanism driven modeling allows the use of clinical data for hypothesis testing to probe those postulated mechanisms. In this presentation, I will pose the questions that we as clinical therapists would like to see addressed with the help of PK/PD modeling in the particular interest areas of antibodies, cytokines and adoptive cellular therapies.

3. Modeling • Linear models • Assume all drug movement is scale independent • Low dose and high dose behave the same • Tested by PK with different doses • Non-linear models • Allow for drug interactions

4. Utility • Dose scheduling • Predicting blood concentrations • BUT is this all there is? • Can we mine PK data for more? • Can we gain information on processes?

5. Pitfalls • Understand assay and units • How they may be flawed • Interfering substances • In vivo versus in vitro state of agent (e.g., IL15 vs IL15-IL15Ra) • What questions to ask • Understand the biological system • Examples to follow…

6. Example 1: Antibodies • Macromolecules: 150 kDa • Regulated catabolism • Restricted extravascular penetration • Key property: interacts with specific and non-specific ligands/receptors

7. What is the question? • What is the half life? • Two-compartment modeling • Alpha phase 1-2d • Beta phase 23d • Vd 6L (1:1 intravascular to extravascular) • Dosing once per 2-3w: maintains plasma levels • Is that the real question?.... NO. • What is effective concentration at the site of action?

8. What is the question? • What is effective concentration at the site of action? • What does it mean to have a Vd of 6 L? • 3L IV + 3L EV • Total ECF = 15L, 3L IV + 12L EV • Can you infer concentration in EV space? • PK models say peak EV conc ~0.5 of C0 • Yet 0.5 C0 > 3L/12L C0 • What is distribution in the tissues? • Measurements needed

9. What is question? • “What is effective concentration at the site of action?” • In vitro testing gives concentration values for effects (receptor blockade, apoptosis, ADCC) • In vivo infer effective systemic levels by doses in escalation • Correspondence? Not truly known: studies needed!

10. What is question? • “When is effective concentration NOT effective concentration?” • Soluble Antigen • May not affect Ab catabolism/blood levels • May render Ab inactive • Antigen Target Load • May reduce free Ab below levels sufficient for blockade • Less important for tracer distribution (radioAb) • May be different in different clinical settings

11. Soluble antigen generation

12. Soluble Ag binds and block Ab

13. Modeling of binding and activity Inputs: PK [Ab], [Ag], d[Ag]/dt, Ka binding affinity

14. Co-model complex interactions and their PK

15. Ab Ag Antibody may look okay, but not be okay…

16. What is question? • “When is effective concentration NOT effective concentration?” • Soluble Antigen • May not affect Ab catabolism/blood levels • May render Ab inactive • Antigen Target Load • May reduce free Ab below levels sufficient for blockade • Less important for tracer distribution (radioAb) • May be different in different clinical settings

17. Patient specific PK differences

18. Two leukemia patients, same 50 mg dose of 111In-anti-Tac

19. Hypothesis: Tumor load affects PK

21. Estimating Burden of Tumor Ag

22. Disappearance at 2 days correlated with tumor Ag burden Divergence of PK from model may reveal processes

23. Example 2: Cellular therapies • “Living drugs” • Complex interactions • Suppression • Activation • Growth factors • Effectiveness may increase with dose • Means needed to quantify • At site of action!

24. T cells

25. What is the question? • PK: “What is disappearance from blood” • Administration IV • Can be followed in blood (flow cytometry) • Rapid disappearance • Engraftment protocols • What is the REAL question? • “What is T cell concentration at site of action?” • “What is T cell activity status at site of action?” • Not measured routinely: biopsy, imaging • Studies needed!

26. TCR Gene-Modified TCR Anti-Cancer T Cell Gene Therapy

27. Pharmacokinetics“Drug disappearance in the body” Rapid Systemic Loss…

28. 1600 1400 1200 1000 800 600 400 200 0 CEA T Cells Increasing pain CEA (NG/ML) Pain resolved -28 -21 -14 -7 0 7 14 21 28 Day of Treatment Response: Proof-of-principle BUT! Time-Limited in Duration…

29. Phase I Trial in Prostate Cancer T Cell Harvest Ex vivo gene therapy Non-myeloablative (NMA) Conditioning CIR+ +IL2 low dose (outpatient) Anti-PSMA designer T cells Hematologic Recovery CIR+ Tumor Response Phase I dose escalation: 10^9 cells 10^10 cells 10^11 cells CD3+

30. Peripheral Blood Recovery • Chemo d-8 to d-2 • T cells d0 • IL2 start d0 x 28d • ANC=0 for 5-8 d • Recovery by d8 to d10

31. CFSE stained total T cells

32. Rhodamine staining of IgTCR+ T cells

33. Totaland IgTCR-modified T cells

34. PK for JN Modified CD8V5+

35. 1.1% 2.5% CD3 Engraftment Blood sample Day +14 CIR+ 7.3% CIR+ 61% Dose CD3+

36. What is the question? • PK: what are dTc levels? • Measured in blood… • Calculations: • Estimate 10^12 T cells in body • Never measured!!! Need this. • 10% x 10^12 T cells = 10^11 cells • 10^9 dose =>100x expansion • But are dTc everywhere? Or just where we measure? Bone marrow, LN, spleen, etc. • Important to calculation of ?? • What is concentration at site of action??? (Tumor) • Data needed (biopsy, imaging)

37. Response

38. Dose-Response • Response in low dose, not high dose • Numbers small (2/3 PR low dose; 0/2 PR high dose) • Against expectation • With 50-70% PSA reduction in low dose, one could hope for 100% PSA reduction in high dose • Unclear difference between responders and non-responders • Age, disease stage, PS, renal function, prior therapies… • During data analysis, became apparent IL2 levels differed

39. Example 3: Cytokine • Mainly small proteins (e.g., IL2, 15 kDa) • Growth factors for T cells • Renal filtration dTc need IL2

40. What is the question? • Administration: Bolus versus continuous infusion • T1/2 1-3 hr • Vd 8L • What is the REAL question? • “What is concentration at site of action?” • “What is persistence of activity at site of action?”

41. IL2 via Continuous Infusion 20 IU/ml 75,000 IU/kg/d ~ 3 MIU/m2/d

42. IL2 levels pts 1-5 Significance: 1 Cetus unit IL2 = ½ max stimulation of aTc 6 IU ~ 1 Cetus unit [know your units and their meaning?] 30 IU/ml = 5x ½ max stim 3-6 IU/ml =< ½ max stim (and lower in tumor…) [what is level in tumor??] Therefore, the range plausible for difference in therapeutic activity

43. Response versus IL2 RESPONSE NO YES LOW 3 0 IL2 HIGH 0 2 P = 0.1 by Fischer exact test (ns) [underpowered = too few samples]

44. Causes for Low IL2 • Artifact? • Repeat assays together instead of sequential • Mixing assays to rule out ELISA inhibitor • Delivery problems? • Pharmacy verified pumps function normal • Cassette volumes appropriately depleted • Bioactivity problem? • Novartis confirmed bioactivity nominal for all lots • Same lot used for patients 2 (hi IL2) and 3 (lo IL2) • Catabolic rates differ between individuals? • Would need 10-fold difference in rates; no precedent • CONCLUDE: IL2 levels genuinely different • WHY THEN? • Only dose sizes are variables between patients… • Maybe the T cells?

45. Hypothesis: T Cells Depleting IL2? • Hypothesis: Are high levels of engrafted activated T cells (aTc) binding and reducing IL2 to too low a level? • Calculation: • Total body 10^12 T cells [NEVER MEASURED!!] • 10% engraftment = 10^11 T cells • 100-fold expansion from 10^9 T cells [DISTRIBUTION UNPROVEN!] • 1000 IL2R per T cell => 10^14 R (~2 nmole) [NOT MEASURED!!] • bind 3 ug IL2 vs total 16 ug at steady state (10 ug/h) , 19% reduction • 50% engraftment => 95% reduction • Plausible, in range: depending on several factors: actual IL2R, internalization/cycling rates, number of T cells, etc. • SO MANY UNCERTAINTIES! (“plausible is not data”)

46. Estimating Cell Load for IL2 Binding • Infused T cells are all activated T cells (aTc), with elevated IL2R • Modified = dTc, CAR+ • Unmodified = CAR- • Recovering T cells = endogenous (non-activated) + infused (activated) • Recovery complete by 2 weeks • To estimate relative cell load, use fractional engraftment of aTc • Calculate total aTc from dTc using the original modified dose fraction 61%CAR+ CAR+ Dose 7.3% CAR+ Blood CD3+

47. Deriving Fraction of aTc in Engraftment

48. IL2 versus aTc Engraftment

49. Summary of Data • Higher dTc doses NOT higher response • Against expectation • High engraftments of aTc => non-response • Low IL2 correlates with high engraftment • Non-response correlates with low IL2 • Plausible: • MDI sufficient for low engraftment • MDI insufficient for high engraftment • Results affect plan: • Benefit of higher doses of dTc will be realized with adequate IL2 (use high dose IL2) [BUT I WISH I KNEW TISSUE LEVELS…] • Instead of 50-70% PSA reductions, may obtain 100% PSA reductions

50. Summary:What are the questions? • What is concentration of drug at site of action? • What are mechanisms of action in vivo? • What is activity state of drug at the site? • What are interactions that can impact the drug activity?