Pre-clinical Case Studies of Biologic Therapeutics

Transcription

1 Pre-clinical Case Studies of Biologic Therapeutics A Multi-Faceted Strategy of Testing Immunotoxic Potential and Pharmacodynamic Properties of Immunomodulatory Monoclonal Antibodies Jennifer Wheeler Bristol-Myers Squibb Drug Safety Evaluation, Department of Immunotoxicology MASOT Spring Webinar May 17 th, 2016

2 Biologics Represent a Diverse Class Proteins Peptides Nucleic Acids Vaccines Blood-derived Proteins Cell Therapies

3 Biologics Represent a Diverse Class Proteins Monoclonal antibodies (mabs) Antibody variations Bispecifics Antibody-Drug/Toxin conjugates Cytokines Plasma Factors Growth Factors Enzymes

4 Recruiting the immune system in the fight against cancer Challenge to fighting cancer: Cancer cells are experts at evading detection by the immune system! Express inhibitory receptors that essentially turn off immune response/detection Solution I: Non-specific cytotoxic therapies Chemotherapy and radiation kill tumor cells, but also healthy tissue Solution II: Targeted therapies Small molecules that target cancer cell-specific proteins Monoclonal antibodies (mabs) that target immune receptors to activate immune response and/or prevent inhibition of response Immuno-oncology (I-O) mabs

5 Small Molecule vs I-O mab Attribute Small Molecule I-O mab Targets Preclinical Tox Studies Intracellular Extracellular Ligand receptor Non-host (anti-viral) Traditional design/strategy Special Concerns - Typically extracellular (Costimulatory or checkpoint inhibitor) Less traditional case-by-case basis Lack of relevant species Immunogenicity Species Selection Based on metabolic/exposure considerations Pharmacologic activity secondary Generally one rodent and one non-rodent Based on shared pharmacology Limited species specificity Identification of non-rodent species difficult Adapted from J. Cavagnaro and C. Horvath in Preclinical Safety Evaluation of BioPharmaceuticals, John Wiley & Sons. 2008

6 Considerations for Immune Assessments on I-O mab Studies Agonist vs antagonist (co-stimulatory vs checkpoint inhibitor) Blocking or non-blocking endogenous ligand Target-cell expression Constitutive vs activation induced Expression at tumor microenvironment vs non-tumor site (blood, LN, spleen) Pharmacology, pharmacodynamics (PD)? Cell type(s) specific assays Anticipated toxicities from literature and early exploratory studies? Pardoll (2012) Nat Rev Cancer

7 Assessing Immunotoxicity and Pharmacology Toxicities are not routinely observed on I-O mab studies Majority of FIH-enabling studies use monkey Human I-O mabs often are pharmacologically active in monkeys Findings often limited to anti-drug antibody (ADA)- mediated reactions and expected pharmacology Immune system enhancement expected pharmacology Pharmacodynamics can be monitored through: Receptor occupancy (RO) analysis Specialized immunophenotyping Functional assays

8 Utilizing TDAR to Assess PD of I-O mabs TDAR routinely used to assess overall immune response function Keyhole limpet hemocyanin (KLH) most commonly used Predictable, robust response across multiple species Historical data for comparison Other antigens available, but experience is limited (HBsAg, tetanus toxoid, etc.) How much KLH for I-O studies? 10 mg/monkey to assess immunosuppressive drugs Concern for maxing out antibody response, enhancement due to PD may be missed/abrogated

9 Case Study I: Assessing Immunotox and PD mab Y target expressed on several lymphocyte populations 1 month (1 dose/week) with 10 week recovery, immunized with KLH D1 (5 mg) 4 dose groups (including vehicle control; 5/group/sex) Dosing Dosing Dosing Dosing Dosing Recovery (2/group/sex) KLH D1 D8 D15 D22 D29 D99 ADA ADA ADA ADA ADA ADA ADA ADA ADA ADA TBNK/memory TBNK/memory (blood) (blood) (Spleen, memory only) TDAR TDAR TDAR TDAR TDAR Ex vivo recall Ex vivo recall ANA ANA = Anti-nuclear antibody ADA = Anti-drug antibody TBNK = T cells, B cells, NK cells TBNK/memory (blood) ANA

10 Case Study I: Assessing Immunotox and PD No adverse events or toxicities throughout the study No anti-nuclear antibodies (ANAs) detected at any dose No changes in lymphocytes counts, or in number or percentage of specific subsets both in spleen and peripheral blood Treatment-emergent ADA s detected in 8 animals No ADA-mediated toxicities observed

11 Endopoint Titer Case Study I: Assessing Immunotox and PD TDAR to KLH mg/kg 10 mg/kg 40 mg/kg 150 mg/kg KLH-specific IgG KLH-specific IgG Pretest Day 8 Day 15 Day 22 Day 29 Study Day Dose-independent increases in IgM and IgG

12 Case Study I: Assessing Immunotox and PD Ex Vivo Recall Response Pretest Study Day 22 Flow cytometric analysis CD69, IFNγ, TNFα CD4 and/or CD8 T cells Blood Isolate peripheral blood mononuclear cells (PBMCs) Brefeldin A after 4 hrs Incubate an additional 20 hrs Lympholyte M 1. Antigen (KLH), αcd28, αcd49d 2. αcd28α, CD49d (negative control) 3. αcd3 (positive control)

13 %IFN+ CD4 T cells %TNF+ CD4 T cells Case Study I: Assessing Immunotox and PD Ex Vivo Recall Response IFN+ CD4 T cells 0 mg/kg 10 mg/kg 40 mg/kg 150 mg/kg TNF+ CD4 T cells Males Females 0.0 Males Females Dose-independent increase in ex vivo recall to KLH at Day 22 Taken together, TDAR and ex vivo recall to KLH demonstrate PD of mab Y

14 Case Study I: Assessing Immunotox and PD Cytokine Release Assay (Human) Cytokine Release Assay (CRA) designed to identify super-agonists and risk for cytokine storm-like reactions mab Y αdt-igg1f αcd28 (TeGenero mimetic) Air-dry 3-5 hrs Incubate human PBMCs 18 & 66 hrs Multiplex Cytokine Analysis IL-1β, IL-2, IL-5, IL-6, IL-8, IL-10, IFNγ, TNFα Flow Cytometry Activated T cells, B cells, NK cells (CD25+ and/or CD69+) No evidence of mab Y as a super agonist or causing cytokine storm-like reactions in any tested donors

15 MFI Case Study I: Assessing Immunotox and PD PD on Human Cytokine Release Assay 1500 MFI CD25 on NK cells (66 Hrs) MFI CD25 on NK Cells (66 Hrs) Donor Dose-dependent increase in NK cell activation for most tested donors Pharmacology: target of mab Y expressed constitutively on NK cells BMS mab Y (µg/well) (g/well) anti-dt-igg1f (µg/well) (g/well) DPBS TGN 5.11A1 (µg/well) (g/well)

16 Case Study I: Assessing Immunotox and PD Summary Consider the dose of antigen when using TDAR as a PD marker on I-O mab studies Enhancement of TDAR (up to 4X compared to vehicle) Could a further enhancement have been observed at a lower dose of KLH? How low should we go for KLH? In addition to TDAR, existing assays can be used to assess PD ex vivo recall to KLH in monkeys, CRA in humans We should modify our approach to what is included on studies on a case-bycase basis, not one size fits all. No changes seen in immunophenotyping or ANA Should we assess ANA on every I-O study? What is the value added? Use WOE approach for including assessments (early tox studies, literature)

17 Case Study II: Going for PD! An Alternative Antigen for Assessing PD Recombinant, replication incompentent adenovirus (Ad-5) as an alternative antigen to KLH Express viral proteins Hypothesis: more relevant antigen for demonstrating PD for CD8-specific targets KLH response predominantly CD4 mediated Ability to look at antigen-specific responses (aside from TDAR) using specific viral peptide tetramer analysis. Image from

18 % CD8+CD3+ lymphocytes Case Study II: Going for PD! An Alternative Antigen for Assessing PD Recombinant, replication incompentent adenovirus (Ad-5) as an alternative antigen to KLH Express viral proteins Hypothesis: more relevant antigen for demonstrating PD for CD8-specific targets Antigen-specific CD8 T cells KLH response predominantly CD4 mediated 2 0 Vehicle mab X mab Y Ability to look at antigen-specific responses (aside from TDAR) using specific viral peptide tetramer analysis.

19 Case Study II: Going for PD! An Alternative Antigen for Assessing PD Study #1: Early Exploratory Antagonistic anti-human mab X Early exploratory single dose IV TK, tolerability and PD study 4 dose groups (including vehicle control; 2-3 monkeys/group/sex) Monkeys immunized with KLH and Ad-5 vectors expressing viral proteins Dosing KLH Ad5 D1 D8 D15 D22 D29 D31 ADA ADA ADA ADA ADA D43 ADA BIO BIO BIO BIO RO RO RO RO RO RO RO RO TSA Ex vivo recall TDAR TDAR TDAR Ki67 Ki67 Ki67 Ki67 Mem Mem Mem Mem TBNK TSA Ex vivo recall TDAR TBNK BIO = Biomarker Assay TSA = T cell specific antigen Ki67 = T cell activation Mem = Memory T cell phenotyping

20 Case Study II: Going for PD! An Alternative Antigen for Assessing PD Study #1: Early Exploratory Antagonistic anti-human mab X Early exploratory single dose IV TK, tolerability and PD study 4 dose groups (including vehicle control; 2-3 monkeys/group/sex) Monkeys immunized with KLH and Ad-5 vectors expressing viral proteins Dosing KLH Ad5 D1 D8 D15 D22 D29 D31 ADA ADA ADA ADA ADA D43 ADA BIO BIO BIO BIO RO RO RO RO RO RO RO RO TSA Ex vivo recall TDAR TDAR TDAR Ki67 Ki67 Ki67 Ki67 Mem Mem Mem Mem TBNK TSA Ex vivo recall TDAR No changes in biomarker assay, ex vivo recall, TDAR, T cell and B cell numbers TBNK

21 Case Study II: Going for PD! An Alternative Antigen for Assessing PD Study #2: FIH-enabling study Data from the exploratory study allowed for narrowing/refining of assays included on the FIH enabling study Ex vivo recall, TDAR, standard immunophenotyping, biomarker assay excluded. NK cell activation (NKA) added 1 month (1 dose/week) with 10 week recovery, immunized with Ad-5 only 4 dose groups (including vehicle control; 5 monkey/group) Dosing Dosing Dosing Dosing Dosing Ad5 D1 Recovery (2/group/sex) D8 D15 D22 D29 D99 ADA ADA ADA ADA ADA ADA ADA ADA ADA RO RO RO RO RO RO RO RO RO RO TSA NKA NKA NKA NKA NKA NKA Ki67 Ki67 Ki67 Ki67 Mem Mem Mem Ki67 Mem

22 Percent of NK Cells % CD3+CD8+ lymphocytes % CD3+CD8+ lymphocytes Case Study II: Going for PD! Study #2: FIH-enabling study Ki67+ CD8 T cells Antigen-specific CD8 T cells mg/kg mab X 10 mg/kg mab X 30 mg/kg mab X 100mg/kg mab X Pre-test Day 8 Day 15 Day Study Day Dose mab X (mg/kg) 50 CD107a+ NK Cells Collectively demonstrates mab PD 10 0 pretest Day 3 Day 8 Day 15 Day 29 Study Day

23 Case Study II: Going for PD! Investigating Mechanisms Underlying Clinical Signs Immediately after 4 th dose (Day 22), one low-dose (10 mg/kg) female monkey developed clinical signs indicative of hypersensitivity Red discoloration of face (resolved within minutes) Erythema on limbs (resolved within 24 hours) Likely ADA-mediated (RO ~0% at Day 22) 4 other low-dose monkeys (2M/2F) also at ~0% RO at Day 22, but no clinical signs To investigate potential mechanisms underlying clinical signs, serum and plasma isolated from all vehicle and low-dose female monkeys at Day 29 Predose, 15 min, 4 hr, 24 hr Complement activation and serum cytokine analysis

24 Percent Case Study II: Going for PD! Investigating Mechanisms Underlying Clinical Signs Equivalent units Receptor Occupancy 10 mg/kg mab Z Females CH50 in serum pretest Study Day Time relative to dose (Hrs) Three females that had ~0% RO at Day 22 exhibited mild clinical signs that quickly resolved following dosing on Day 29 Serum from these animals defective in ability to activate complement Suggests depletion of complement cascade proteins due to immune complex fixation of complement

25 Case Study II: Going for PD! Summary Let your early exploratory studies be your guide! Immunization with Ad5 instead of KLH Relevant antigen for assessing immune response Tools to assess effect of I-O mabs Elimination of several endpoints (TDAR, biomarker assessment, etc) Modification of NK phenotyping to include activation marker (CD107a) Timing of assessments (Ki67, NK cell activation) Multiple PD assessments to get a more global view of pharmacology Toxicities rarely happen for I-O mab studies, but should always be prepared to identify and collect samples for future analysis Monitoring RO throughout studies to ID animals with risk for ADA-mediated hypersensitivity Collecting samples following unscheduled euthanasia Tissue for IHC, serum, plasma, etc.

26 Case Study III: You Don t Know What You Don t Know Agonistic anti-human mab Z 1 month (1/week) with 10 week recovery, immunized with KLH D1 (5 mg) 0, 30, 60, 120 mg/kg/monkey (5/group/sex) Dosing Dosing Dosing Dosing Dosing KLH D1 Recovery (2/group/sex) D8 D15 D22 D29 D99 ADA ADA ADA ADA ADA ADA ADA ADA ADA ADA ADA TDAR Ex vivo recall TBNK Memory Ki67 Ki67 Ki67 Ki67 TDAR TDAR TDAR TDAR Ex vivo recall ANA TBNK Memory No changes in T cell activation, immunophenotyping, or ANA ANA TBNK Memory

27 Endopoint Titer Endopoint Titer Case Study III: You Don t Know What You Don t Know KLH-specific IgM 0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg Dose-independent suppression of TDAR to KLH IgM up to 75% IgG up to 88% No treatment-emergent ADA 0 Pretest Day 8 Day 15 Expected with suppressed antibody responses KLH-specific IgG 0 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg Suppressed ex vivo recall response to KLH Low dose significantly above projected human efficacious dose Pretest Day 8 Day 15 Day 22 Day 29 Expected enhancement, how to explain suppression?

28 Case Study III: You Don t Know What You Don t Know A Possible Hypothesis FcR mab Z Z ligand Z receptor Fc Receptor T-cell APC ACTIVATION

29 Case Study III: You Don t Know What You Don t Know A Possible Hypothesis FcR mab Z Z ligand Z receptor Fc Receptor T-cell APC ACTIVATION APC APC T-cell T-cell Excess mab Z: 1) Prevents Fc x-linking 2) Blocks the endogenous receptor/ligand at 2 interfaces (APC and B-cell) Suppressed TDAR

30 Case Study III: You Don t Know What You Don t Know Summary You can t always make the assumption that without tox, you can increase doses without impacting efficacy! Bell-shaped curve dose response Blocking antibodies can disrupt mab target signaling pathways and interactions with endogenous ligands Do we need to take more consideration of PD when selecting doses for FIH-enabling studies? Strategy: Keep low-dose low enough to monitor PD, but high enough for reasonable exposure multiples Not always feasible in FIH-enabling studies Assess PD in early exploratory studies at lower doses

31 So What Have We Learned? That We are Still Learning! Consider the dose (and type) of antigen when using TDAR as a PD marker on IO mab studies While existing assays can be used as PD assessments, assays should be evaluated on a case-by-case basis, not one size fits all Let early exploratory studies guide and refine assessments that will go on FIH-enabling tox study Although drug-mediated toxicities rarely happen for IO mab studies, be prepared to identify and collect samples for future analysis While higher doses are necessary for safety margins, PD may affected at higher doses.