final poster 151202 (1)
Post on 12-Apr-2017
115 Views
Preview:
TRANSCRIPT
Development of the MSD-based assay for determining the PD-1 Antibody concentration in mouse serum and tumorKhushbu Bhatt, Tatiana Tolstykh, Lakshmi Srinivasan, Michael Lampa, Yu-an Zhang, Mikhail Levit, Andrew Hebert, Dmitri Wiederschain, Timothy Wagenaar
Sanofi Oncology, 640 Memorial Drive, Cambridge, MA 02138
Abstract
IntroductionPD-1 (Programmed cell death protein 1/ CD279) is an immune-inhibitory cell-surface receptor expressed on T-cells. Its ligands PD-L1 and PD-L2 (Programmed death ligand 1/2) (CD274) are present on the tumor cells and various antigen presenting cells (APCs) such as dendritic cells and macrophages. Upon binding to its ligands PD-1 initiates a signaling cascade within the T cell to attenuate the T cell response which contributes to immune evasion of the tumor cells. PDL-1 is highly expressed in melanoma, renal cell carcinoma (RCC) and non-small cell lung cancer (NSCLC). Thus expression of PD-1 on TILs (tumor infiltrating lymphocytes) and its interaction with PDL-1 and PDL-2 on tumor and other immune cells suppresses the recognition of tumor antigen by the T-cells. Therein lies the therapeutic rationale that by blocking the interaction of PD-1 with its ligands the anti-tumor immune response can stimulated.
Results
ConclusionMSD based assay with high specificity and sensitivity to analyze the PD-1 antibody concentration as low as 0.5 ng, with minimal background and matrix effect was developed and validated. This assay was used to determine the antibody levels at various time points in serum and tumor.
Acknowledgement I would like to thank my mentor Tatiana Tolstykh and my team members Lakshmi Srinivasan, Timothy Wagenaar, Michael Lampa, Yu-an Zhang for training me in the lab and helping me with the project. Thanks to Mikhail Levit and Andrew Hebert for helping me out with the execution of MSD assay. I would also like to thank the Section Head Dmitri Wiederschain for his continued motivation, guidance, support and patience during my time in Sanofi.
Materials and MethodsMSD (Meso Scale Discovery) Meso Scale Discovery’s MULTI-ARRAY®
technology is an unique immunoassay platform which enables detection of protein markers in single and multiplex format utilizing the electro-chemiluminescence technique.
MSD Assay’s improved sensitivity, expandable dynamic range, multiplexing ability and no matrix effect gives an edge over the other traditional techniques like ELISA, RIA.
Simple WES : Protein Simple Simple Western is an automated size-based
Western analysis which separates and detects proteins as large as 440 kD in as little as 3 hours.
Total Protein Assay from Protein Simple was used to analyze the total protein content in the Pd-1 antibody in which target proteins are separated by size, labelled with biotin reagent and are then detected by chemiluminescence using Streptavidin-HRP
Purity/Total Protein Concentration of PD-1 (RMP1-14) antibody Evaluation of binding of PD-1 antibody to PD-1 MSD Assay Development -Optimizing variables: 1. PD-1 antigen capture level 2. Detection antibody concentration -Standard curve generation -Effect of serum on the assay -In-vivo validation of the assay PK-PD Study to evaluate PD-1 antibody in mouse Determining the kinetics of PD-1 antibody in serum and tumor Target Engagement using FACS
Assay Development Outline
MSD Assay Development
Low background, high signal to noise ratios, 3 log dynamic range (0.5 ng-10,000 ng), high sensitivity, low matrix (serum) effect Optimized Variables: PD-1 coating concentration-2 ug/ml , Detection antibody concentration- 0.5 ug/ml Minimum PD-1 antibody detection level: 0.5 ng/ml
MSD Kit
Results
PK/PD study to evaluate mouse PD1 antibody
Cancer Immunology: PD-1 and Beyond. (n.d.). Retrieved from https://www.smartpatients.com/pathways/pd-1
The PD-1 receptor and its ligands PDL-1 and PDL-2 are important inhibitory molecules on T cells that contribute to tumor immune evasion. Checkpoint blocking antibodies targeting PD-1 and PD-L1 have shown significant anti-tumor activity in a range of human cancer types. To investigate the effect of checkpoint blockade in murine tumor models several surrogate antibodies targeting PD-1 have been described in the literature. To identify an optimal dosing regimen of the PD-1 antibody RMP1-14 a more thorough understanding of the antibody pharmacokinetics was required. To this end, a Meso Scale Discovery (MSD) assay was developed to determine the concentration of PD-1 antibody in mice serum and tumor. Different variables like PD-1 plate coating concentration and detection antibody concentration were optimized to yield an assay with three-log dynamic range, low background, minimal matrix effect and high signal to noise ratio. The optimized MSD assay was used to evaluate RMP1-14 pharmacokinetics after a single IV injection in tumor bearing mice. The MSD assay indicated dose proportional expose of RMP1-14 with a strong correlation of serum and tumor antibody levels.
Protein Simple WES PD-1ISOTYPE
Typical dose-response obtained when RMP1-14 was serially diluted
Normalized with BSA as standard
Purity/Total Protein Concentration of RMP1-14 (PD-1 antibody)
Binding of PD-1 antibody to PD-1 confirmed using FACS
EL4 cell line which constitutively express high levels of PD-1 was used to confirm antibody binding
PD-1 antigen capture level Detection antibody concentration Standard curve generation and effect of serum (33% ) on the assay
Dose proportional increase in serum concentration PK supports dosing every 3 to 4 day intervals Correlation in tumor and serum PK of PD-1 antibody Significant decrease in MFI of PD-1 on intratumoral CD8 T cells at
336 hrs.
Serum and Tumor PK of PD-1 antibodySerumTumor
- Time points when mice were taken down
Tumor PK Data
Time (hrs)
RM
P1-
14, n
g/m
g o
f to
tal p
rote
in
6 168 3360
50
100
150
200PBS5 mg/kg10 mg/kg20 mg/kg
Serum PK Data
Time, hrs
RM
P1-
14, u
g/m
l
6 168 3360
500
1000
1500PBS5 mg/kg10 mg/kg20 mg/kg
Serum and Tumor PK of PD-1 antibodyTumorSerum
Target Engagement confirmed by FACS
top related