Challenges in Bioassay Development for ADCs and Their Utility for Measuring In-vitro Activity of Conjugate Variants

Transcription

1 Challenges in Bioassay Development for ADCs and Their Utility for Measuring In-vitro Activity of Conjugate Variants Sonia Connaughton, Ph.D. Senior Scientist, Bioanalytical Science Bioassays 214: Scientific Approaches & Regulatory Strategies March 24th, 214

2 Talk Outline Challenges developing Cell based Potency assays for Lot Release and Stability Testing Utility of Bioassays in Conjugate Variant Activity Relationship (CVAR) studies 2

3 Antibody-Maytansinoid Conjugates (AMC) ImmunoGen Approach Tumor-targeting antibody With or without intrinsic functions Potent cell-killing agent (DM1, DM4) Derivatives of maytansine, potent antimicrotubule agent Linker (disulfide, thioether) Non-cleavable and cleavable (inside cell) designs Keeps conjugate intact in blood Releases fully active payload inside cancer cell Conjugation approach (through lysines) retains native Ab functions Design goal: add potent tumor cell killing mechanism (payload) while retaining favorable properties of native antibodies 3

4 Bioassays for AMCs Bioassays are needed to provide a link between the structural integrity of the AMC (physicochemical assays) and biological activity of the AMC Used for monitoring manufacturing release, stability & lot to lot consistency Used for characterization of new process & formulations (comparability studies, ICH Q5E) Not all AMCs have the same set of bioactivities All AMCs have antibody-directed binding and maytansinoid-directed cytotoxicity Some AMCs have additional antibody-directed functions Bioassays should reflect the activities of the AMC Antibody component is tested using same bioassays where appropriate 4

5 Bioassay Qualification Path: Applying ICH Q2 (R1) Accuracy Expected vs observed potency should be 1% ± 2% Linearity and range Observed vs expected potency should follow a straight line with a slope of and R 2 >.95 within the specification range Repeatability (intra-assay) Compare results of sample repeated within an experiment Intermediate precision (inter-assay) Compare results across assays: days, analysts, reagents, equipment Parallelism Show that curves for different samples have the same shape and are parallel (same upper and lower asymptotes, and slope) Only difference between curves is the EC 5 Specificity Show that related Abs or conjugates cannot pass assay acceptance 5

6 Response Ideal Assay Curve for Cell Based Potency Assessment 15% Sample 5% Sample Max/ Min response, well defined upper, lower asymptotes Slope of the dose-response curve Linearity of potency response across intended specification range Pass Parallelism test for similarity to reference [Conjugate], Log M 6

7 CPS Importance of Cell Number for Optimal S/N 5 day assay, Cell Titer-Glo: 12 minutes PL1 5 cells PL2 5 cells PL3 1 cells PL4 1 cells PL5 2 cells PL6 2 cells Cell Number S/N Ratio Mean S/N Ratio 5 cells/well PL cells/well PL2 3 1 cells/well PL cells/well PL cells/well PL cells/well PL [Antibody-Conjugate], M Higher Signal/Noise (S/N) results in a more robust response Less plate to plate variability in upper asymptote response 7

8 RLU Specific Cytotoxicity: Plate Uniformity RLU RLU Cell Seeding Uniformity 24 hrs post seeding Growth Uniformity 94 hrs post seeding Cell Killing Uniformity 94 hrs post seeding S7 S1 S3S S1 S3S5S S1 S3S5S7 Interassay PL 1,2 &3 PL1 Intra-assay PL2 PL3 Mean %CV Interassay PL 1,2 &3 PL1 Intra-assay PL2 PL3 Mean %CV Interassay PL 1,2 &3 PL1 Intra-assay PL2 PL3 Mean %CV Uniform cell seeding and signal across plate is the first critical step in controlling assay variability 8

9 Typical Specific Cytotoxicity Assay Setup A media media media media media media media media media media media media B media Ref no AMC C media TA1 no AMC D media TA2 no AMC E media Ref no AMC F media TA1 no AMC G media TA2 no AMC H media media media media media media media media media media media media Non-random, non-clustered plate layout 1 point dilution curve with 2 pseudo-replicates per sample 3 independent plates / assay Each plate has a True Replicate of Test Article and Reference Standard Each plate has 2 Pseudo-replicates of the True Replicate Results are combined across all three plates (6 values per point) 9

10 Observed Relative Potency Challenges in Balancing Slope Steepness & Dilutional Linearity Expected Relative Potency 96 hr Exposure Slope: hr Exposure m =.9992, R 2 = Hr Exposure m =.85, R 2 = hr Exposure Slope: Reference 18% Sample 2% Sample Reference 125% Sample 2% Sample Uneven Dilution Scheme S/N > 25 Even Dilution Scheme S/N drops to 5 Relative Potency (RP): Sample potency expressed as a % of reference potency RP = (Ref EC 5 *1)/ TA EC 5 1

11 5 cells /well A Multi-variable Method Development Approach Shortens Assay Development Time 92 hr 116 hr Time 92 hr 116 hr 5 cells/well Expected Potency Relative Potency % Recovery 5% 51% 11 2% 22% 12 5% 49% 98 2% 196% 98 3 cells /well Time 92 hr 116 hr 3 cells/well Expected Potency Relative Potency % Recovery 5% 58% 116 2% 231% 115 5% 59% 118 2% 242% 121 Test simulated potency samples at 5 & 2% (at lower & upper spec. limit) Both cell # & conjugate exposure time can affect % recovery (observed *1%)/ expected) Assessment of Parallelism using PLA2.: equivalence testing for slope difference 11

12 Specific Cytotoxicity Assay is Stability Indicating DP is stable at intended storage temperature DP slowly loses activity at accelerated temperature 12

13 % R e la tiv e P o te n c y Example of MAR Sensitivity for the Specific Cytotoxicity Assay Parameters % Potency Slope ± 2.71 R square.9971 P value <.1 Deviation from zero? Significant M A R MAR sensitivity in the specific cytotoxicity assay should be within the MAR specification range 13

14 Summary: Challenges for Specific / Non-specific Cytotoxicity Development Key challenges in developing robust cell based assays to measure potency of AMCs Availability of suitable cell lines (tumor, transfected) Control of slope to allow for even dilution scheme Linearity of response over specification range Assessing Parallelism Other characteristics important for a potency assay Stability indicating Sensitive to maytansinoid to antibody ratio 14

15 CONJUGATE VARIANT ACTIVITY RELATIONSHIP (CVAR) STUDIES 15

16 Utility of Bioassays in CVAR Studies: Degradation pathways and Critical Quality Attributes Forced degradation: Apply stress conditions Assess degradation pathways Critical Quality Attributes Create single attribute variants Assess affect on biological activity 16

17 In Vitro and in Vivo Bioactivity In vitro Cytotoxicity Specific (Antigen-targeted) Non-Specific (Non-targeted) Binding CDR binding to Antigen Fc binding FcR IIIa (ADCC) FcRn (PK) In vivo Efficacy Tolerability Pharmacokinetics 17

18 Cell viability signal Cell viability signal Specific and Nonspecific Cytotoxicity Assays Setup is Similar Specific Cytotoxicity Release and Stability assay Measures antigen-targeted potency Titration curve uses lower concentrations ( nm) Nonspecific Cytotoxicity Characterization assay Measures non-antigen-targeted potency Titration curve uses higher concentrations (5.2-6 nm) Result reported is % Potency Relative to Reference Linear across a 5-15% potency range EC5 ~44 pm EC5 ~7 nm Simulated 5% Simulated 5% Simulated 2% Simulated 2% Log (AMC concentration) Log (AMC concentration) 18

19 Observed %RP Fc RIIIa Binding, A45 Surrogate ADCC Assay: Binding to FC RIIIa (CD16a) Assay Schematic Assay is linear over 5-2% range TMB/STOP A45 Goat anti-human IgG F(ab ) 2 -HRP Reference 2% Sample 5% Sample AMC (or Ab ) Fc RIIIa-His Fusion [Antibody Conjugate], ng/ml 25 Mouse anti-his mab 2 15 Y= 1.21X.29 R 2 =.98 1 Conjugate Surrogate assay Quick and very accurate Low intra-assay variability (%CV <1%) Assay is linear over 5 2% range for antibody & AMC Expected %RP 19

20 % Relative Potency % Relative Potency Preliminary Photostability: High Levels of Light 25 o C Causes Many Changes 1 Day 7 UV = 21 days of sunshine Day 7 Cool Fluorescent = 15 months of indoor light Specific Cytotoxicity (UV) Specific Cytotoxicity (FL) Non-specific Cytotoxicity (UV) Non-specific Cytotoxicity (FL) Degradation not seen under normal storage conditions Implications: careful about light exposure Photostability Stress Days Antigen Binding (UV) Antigen Binding (FL) FcyRIIIA Binding (UV) FcyRIIIA Binding (FL) Photostability Stress Days Sample Purity (SEC) (%) NR-CE (%) MAR %FM monomer HMW IgG Frag. T Day 1 UV Day 3 UV Day 7 UV T Day 1 CF Day 3 CF Day 7 CF Trend 2

21 Relative Percent Potency Relative Percent Potency Only High levels of Unconjugated Antibody (umab) Affect Potency 2 Binding titration: total protein concentration (AMC + spiked Ab) Cytotoxicity titration: conjugate concentration (AMC) % umab Binding Specific Cytotox NonSpec Cytotox % umab Binding Specific Cytotox NonSpec Cytotox Specific Cytotoxicity Results: Activity measured based on conjugate concentration No affect on potency up to 1% spike (5% mab, 5% conjugate) 2% and 4% samples showed a significant decrease in potency most likely due to blockade of receptor sites by unconjugated antibody Binding Results: Activity measured based on total antibody (mab + conjugate) concentration Slight increase in binding with increasing umab-attributable to ~1.7-fold higher affinity of antibody compared with AMC 21

22 Relative Potency (%) Aggregate Increases Non-specific Cytotoxicity Specific Cytotoxicity Non-Specific Cytotoxicity Antigen Binding Specific Cytotox Non-Specific Cytotox Fc RIIIa Binding % HMW Species 22

23 Free Maytansinoids Increase Non-specific Cytotoxicity Free May Samples Specific Cytotoxicity Assay (%RP) Non-Specific Cytotoxicity Assay (%RP) % Free May (Measured) Reference standard.7 3% Maysine 79% 91% 2.5 7% Maysine 1% 93% 4.7 3% DM1 16% 112% 3.3 7% DM1 18% 154% 6.8 3% DM1-TPA 16% 15% 3.4 7% DM1-TPA 12% 124% 7. 3% DM1 Dimer 1% 179% 3.1 7% DM1 Dimer 98% 37% 7. No increase in specific cytotoxicity with any spiked-in free may species DM1, DM1-TPA and DM1 dimer all increased conjugate toxicity in the non-specific cytotoxicity assay DM1-DM1 >DM1 > DM1-TPA > maysine 23

24 % R e la tiv e P o te n c y Relationship Between MAR and in-vitro Activity M a y ta n s in o id -A n tib o d y R a tio % R P S p e c ific C y to to x ic ity % R P N o n -S p e c ific C y to to x ic ity % D ire c t B in d in g %RP for specific and non-specific cytotoxicity increase with increasing MAR. Antigen binding increases as MAR decreases. (mab has a higher affinity than conjugate.) MAR controlled within a very narrow range within a few 1/1 th of a percent Actual range during manufacture is really quite narrow 24

25 % Relative Potency Oxidation Study Produced only Methionine Oxidation Variants 2 15 % Relative Potency vs % Oxidation *RS1-1 used as Reference H2O2 (mm) Peptide MS % Oxidation of Methionine Residues H:M99 ~2% H:M253 ~2% H:M429 ~1% HC CDR FcRN 1 5 H:M99 ~ 29% H:M253 ~ 34% H:M429 ~ 9% H2O2 (mm) 1 2 H:M99 ~ 51% H:M253 ~ 56% H:M429 ~ 18% H:M99 ~ 77% H:M253 ~ 73% H:M429 ~ 31% Specific Cytotoxicity Non-Specific Cytotoxicity Antigen Binding FcyRIIIA Binding FcRN Binding 5 H:M99 ~ 96% H:M253 ~ 97% H:M429 ~ 84% Only variant detected by analytical tests was intended one (oxidation of solvent exposed Methionine residues) Oxidation decreases Fc RIIIA and FcRN binding in a concentration dependent manner No impact on antigen binding, specific or non-specific cytotoxicity 25

26 Is antibody-mediated effector function an important contributor to ADC activity? Compare anti-tumor activity of non-binding antibody Fc mutant conjugate and wild-type antibody conjugate 2 examples Parent antibody has no activity in xenograft models Parent antibody has activity in xenograft models 26

27 Effect of D265A Fc mutation on Fc RIIIa and Antigen binding Fc Mutant Antibody Fc Mutant AMC Fc RIIIa Binding Reference Control Fc mutant mab Fc mutant AMC Antigen Binding Fc Mutant AMC Sample FcyRIIIA Binding %RP Test of Parallelism Antigen Binding %RP Test of Parallelism Ctl mab Control 99 Pass 98 Pass TA1 Fc Mutant mab N/A fail 11 Fail CTL AMC Control 95 Pass 93 Pass TA2 Fc Mutant AMC N/A fail 12 Fail Fc mutant version of naked antibody and conjugate no longer binds to Fc RIIIA No impact on antigen binding or specific & non-specific cytotoxicity 27

28 Tumor Volume (mm 3 ) Tumor Volume (mm 3 ) Median Tumor Volume (mm 3 ) Tumor Volume (mm 3 ) Tumor Volume (mm 3 ) Activity of Fc Mutant (D265A) AMC is Comparable to wild type AMC Xenograft model in SCID mice Vehicle AMC 15 µg/kg DM1 AMC 3 µg/kg DM1 FcMutant AMC 15 µg/kg DM1 FcMutant AMC 3 µg/kg DM1 2 doses 1 week apart Days Post Inoculation Treatment T/C PR CR Result AMC, 75 99% /6 /6 Inactive AMC, 15 26% /6 /6 Active AMC, 3 8% 5/6 4/6 Highly Active AMC, Fc Mutant, 75 7% /6 /6 Inactive AMC, Fc Mutant, 15 66% /6 /6 Inactive AMC, Fc Mutant, 3 % 5/6 4/6 Highly Active AMC 15 µg/kg qw x Days Post Inoculation Fc Mutant AMC 15 µg/kg qw x Days Post Inoculation AMC 3 µg/kg qw x Days Post Inoculation Fc Mutant AMC 3 µg/kg qw x Days Post Inoculation The FcR IIIa binding mutant (D265A) AMC has similar activity to the wild type AMC, suggesting that ADCC is not important for the mechanism of action of this AMC 28

29 Tumor volume (mm 3 ) mean SEM Major Mechanism of AMC Action is through Maytansinoid Directed anti-tumor activity 1 8 Control Single dose, 1 mg/kg iv Days post tumor implant N297A_12425 Antibody N297A naked Ab Antibody naked Ab Antibody activity greatly decreased in absence of effector function AMC N297A_12425-SMCC-DM1 Conjugate activity only SMCC-DM1 AMC slightly decreased in absence of effector function N297A = Non-glycosylated mutant No effector function 29

30 Conclusions from CVAR Studies Degradation pathways starting to emerge Increased aggregates, fragments, free maytansinoids Decreased MAR, monomer, Increased non-specific cytotoxicity Decreased specific cytotoxicity, and sometimes antigen binding Critical Quality Attributes (CQA) Aggregates generally considered a CQA for potential impact on immunogenicity Aggregates increase non-specific cytotoxicity (mechanism and significance unknown) MAR influences specific cytotoxicity ADCC appears to be a less important MOA To be determined on a case-by-case basis Free maytansinoid impurities have no effect at AMC doses which are potent in cytotoxicity assays. Unconjugated antibody has no effect on cytotoxicity until present at high levels 3

31 Acknowledgements Thank you to all of the ImmunoGen Departments which provided data and materials Analytical and Pharmaceutical Science BioAnalytical Science Process Science and Engineering Antibody Engineering Chemistry Pharmacology Quality Control Operations 31