mab quantification in preclinical tissue supports in vitro potency/in vivo efficacy correlations

Transcription

1 mab quantification in preclinical tissue supports in vitro potency/in vivo efficacy correlations John T. Mehl, Ph.D. Bioanalytical Research Princeton, NJ AAPS November 4, 2014 San Diego, CA 1

2 LC-MS/MS Technology for Complex Mixture Analysis Multiple-reaction monitoring (MRM) mode UPLC Electrospray ionization of peptides Parent ion Fragment ion Separation Q1 Q2 Q3 Complex mixture of digested proteins mass analyzer collision cell mass analyzer Highly specific MRM chromatogram 2

3 BMS : A bispecific molecule which targets both IL-17 and IL kda Mwt hfc, IgG4 binds IL-23 ligand binds IL-17 A, F, A/F ligand BMS targets multiple cytokines in one molecule PK and biophysical properties comparable to IgG4 antibodies Is a dual targeting molecule more efficacious than targeting individually? 3

4 In vitro studies show that BMS is a potent blocker of IL-17 and IL-23 ligands IL-17 Induced G-CSF secretion in primary airway epithelial cells Drug IC 50 (nm) BMS (IL17/IL23) Competitor #1 (IL-17R) 0.15 Competitor #2 (IL-17A) 0.19 BMS is more potent than competitor #1 & #2 in IL-17A assay. IL-23 Induced STAT3 phosphorylation in primary T-cells Drug IC 50 (nm) BMS (IL17/IL23) Stelara (IL-12/23) 0.13 Competitor #3 (IL-23) 0.54 BMS is more potent than Stelara and competitor #3 in IL-23 assay. 4

5 In Vivo mouse PD study comparing BMS with Stelara BMS Stelara BMS is equipotent with Stelara 5

6 Ear Thickness (% Change) IL-23 Acanthosis Inflammation Mouse Model IL-23 Induced ear inflammation ear Ear thickness Measurements % Inhibition vs vehicle test drug vehicle BMS Stelara BMS is less potent than Stelara in IL-23 induced inflammation model. 6

7 Ear Thickness (% Change) Acanthosis Inflammation Mouse Model IL-23 Induced ear inflammation Vehicle BMS mpk IL-23 mab 9 mpk BMS mpk Is molecular weight a factor? 240 kda 150 kda 53 kda 7

8 Whole Body Radiology Imaging Psoriasis Mouse Model Kidney Psoriasis skin Spleen Liver Muscle Image indicates that drug accumulates in the skin (dark), however it is uncertain if the signal is due to intact drug or metabolite. Is it possible to develop an orthogonal method that can confirm presence of intact drug in target tissues? 8

9 mouse tissue Anal. Chem., 2012, 84,

10 Analytical Challenges of Tissue Analysis Standard curve required for each tissue type and drug type. e.g., 4 drugs across 7 tissue types: 28 standard curves! Nanoflow is less robust and more time consuming (60min) than standard flow ( µL/min). 4 drugs x 7 tissues x 3 time points x n=3 animals: 252 samples! Standard flow not as sensitive as nanoflow 10

11 Analytical Strategy for Tissue Samples Use single serum curve for drug by homogenizing tissue directly into serum, or dilute tissue homogenate into serum. Use generic immunoaffinity to improve sensitivity and enable LC-MS/MS using standard flow rates. 11

12 Intensity, cps Immunoaffinity LC-MS/MS for mab drugs Immobilized anti-human-fc antibody (goat anti-human-fc) serum sample or tissue homogenate Capture Wash LC-MS/MS Chromatogram mab drug magnetic beads digest into peptides 12

13 Simple Immunocapture Sample Preparation (6hr) for Human mab Drugs in Animal Serum 1) 25µL beads + 25 µl serum or homogenate µl of PBST buffer, (ISTD protein) 2) Shaker plate for 1hr, or longer 96-well plate 3) Wash 3x with NH 4 HCO 3 /0.05%zwittergent ) Denature beads using 20µL acetonitrile (shake vigorously) 5) On-bead digest 3hr using 1µg trypsin in 80µL NH 4 HC0 3 (ISTD peptide) 6) Inject 30µL out of 100µL for LC-MS/MS analysis. Prepare magentic beads in advance: 2.5µg goat anti-human-fc biotin, on 0.25 mg streptavidin beads (25µL), prepare in advance, store at 4 C (4 weeks). 13

14 Design of the animal tissue study C57BL/6 mice were injected (5 µg) in right ear with IL-23 to induce inflammation. IL-23 ear Four test drugs were administered sc. into abdomen at molar equivalent doses to four different animal group (n=9): Stelara 150 kda, IgG1 (anti-il12/23) BMS-MAB1: 150 kda, IgG1 (anti-il17) BMS-MAB2: 150 kda, IgG4 (anti-il23) BMS : 240 kda, IgG4 (anti-17/23) test drug Tissues were collected on days 4, 7, 11 (n=3) and homogenized. 14

15 Light Chain Stelara (Ustekinumab) Sequence DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPYTFGQGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy Chain EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWLGWVRQMPGKGLDWIGIMSVDSDIRYSP SFQGQVTMSVDKSITTAYLQWNSLKASDTAMYYCARRRPGQGYFDFWGQGTLVTVSSSSTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 15

16 Immunoaffinity-LC-MS/MS of Stelara from Mouse Serum 2000 ng/ml Max. 1.7e5 cps. Intensity, cps 2.0e5 1.9e5 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 EVQLVQSGAEVK SLIYAASSLQSGVPSR 1.98 YSPSFQGQVTMSVDK SIL-Peptide ISTD ASQGISSWLAWYQQKPEK

17 Peptides from Stelara at 20 ng/ml (LLOQ) in Mouse Serum Light chain and Heavy Chain Peptides Are Monitored Max cps. Max cps. Intensity, cps EVQLVQSGAEVK from heavy chain BLK serum Intensity, cps ASQGISSWLAWYQQKPEK from light chain 2.76 BLK serum Max cps. XIC of +MRM (8 pairs): / Da ID: SLI y7 from Sample 13 (Stellara Curve1 STD 20ng/ml) of TissuePenet_DS557_Stellara_ Max cps. Intensity, cps YSPSFQGQVTMSVDK from heavy chain BLK serum Intensity, cps SLIYAASSLQSGVPSR from light chain BLK serum Sleczka, BG; Mehl, JT; Shuster, DJ; et al., Bioanalysis 6(13)

18 Max cps Intensity, cps Homogenized Tissue QC Samples for BMS-MAB Overlay of QC and blank homogenate serum STD 20 ng/ml Intensity, cps Max cps. skin QC 40 ng/ml Intensity, cps 2.51 Max cps lung QC 40 ng/ml Intensity, cps 2.51 Max cps liver QC 40 ng/ml Intensity, cps 2.51 Max cps spleen QC 40 ng/ml Intensity, cps 2.52 Max cps. colon QC 40 ng/ml

19 Tissue Homogenization QCs for BMS-MAB1 Peptide 1 QC 40 ng/ml Tissue mean % accuracy %CV Ear Skin Lung Liver Spleen Colon QC 400 ng/ml mean % accuracy %CV Drug level in tissue homogenate was calculated using serum standard curve Results indicate that there is no matrix effect due to the presence of tissue homogenate 19

20 Change in Stelara (ustekinumab) mouse tissue levels within each tissue type, with time by Immunoaffinity LC-MS/MS 20

21 Tissue/Serum Ratio AUC Tissue/Serum AUC Ratios of Various Proteins in Ear from Mouse Inflammation Model (inflamed) (control) BMS Stelara BMS-MAB1 BMS-MAB2 Comparable tissue penetration observed for all four drugs into inflamed right ear 21

22 Summary Immunoaffinity LC-MS/MS is a powerful approach for determining tissue levels of human mab drug in animal models. A generic immunoaffinity enrichment step removes matrix affects and enables highly sensitive quantification. Homogenation of tissues directly to control serum enables calibration using a common serum curve, significantly reducing the number of required calibration curves. This approach can provide valuable information about mab distribution without the need for radiolabel or molecular tags. The results indicate that the BMS bispecific drug has equal penetration into inflamed ear tissue as the comparator drugs. 22

23 Acknowledgement Bogdan Sleczka (LC-MS assay) Yongxin Zhu Lorell Discenza Celia D Arienzo Gene Ciccimaro Tim Olah Adrienne Tymiak Ragini Vuppugalla (PK data analysis) Paul Mangan Katherine Lewis Robin Moore (tissue work) Dave Shuster (animal dosing) Kim McIntyre Alex Kozhich Surendran Rajendran Dieter Drexler Joseph Cantone 23