Development of Multiplex Sensitive Anti-Drug Antibody Assays for CRISPR/Cas9 Gene Therapies September 27, 2017 Junxia Wang editasmedicine.com 1
Overview of the presentation Immunogenicity Introduction Anti-Drug Antibody Assays for Cas9 Anti-Drug Antibody Assays for AAV Topic / Speaker Topic / Speaker 2
Immunogenicity Immunogenicity is defined as the propensity of the therapeutic protein product to generate immune responses to itself and to related proteins or to induce immunologically related adverse clinical events. Patient immune responses to therapeutic protein products have the potential to affect product safety and efficacy. The immunogenic response generally includes both B cell-mediated humoral (antibody) and T cell-mediated cellular arms of the immune response. The development of sensitive, specific, and selective immunogenicity assays to assess ADAs and cellular responses is a key aspect of drug product development for biologics. http://www.fda.gov/downloads/drugs/.../guidances/ucm192750.pdf 3
General principle Risk-based approach for immunogenicity evaluation The Assessment of Immunogenicity Risk Risk Mitigation Patient- related Pre-existing Abs Product-related In silico analysis In vitro non-cell assay In vitro cell-based assay Relevant animal models, mouse, rabbit, non-human primates High risk High probability Not meeting the requirement of TPP Avoidance Patient stratification De-immunized trangene De-immunized vector Reduction Change regimen Actively induce tolerance Probably meeting the requirement of target product profile (TPP) Immunogenicity Testing plan Correlation of Immunogenicity incidence and levels with safety, PK and PD (nonclinical and clinical) Monitoring Immunogenicity Throughout the Trial 4
AAV Gene Therapy and Immunity Main risks to evaluate for an AAV gene product: Pre-existing immunity to viral capsid proteins Expected immune responses to AAV (humoral and cellular immune response) Potential immune response to transgene Humoral Immune Response ADA assays Antibody response to Cas9 and AAV Cellular Immune Response PBMC T cell assays T cell responses to Cas9 and AAV 5
Humoral and cell-mediated immune responses Abbas & Lichtman: Basic immunology. 3 rd Edition. weishendopublications.com 6
Anti-Drug Antibody Assays for Cas9 editasmedicine.com 7
ADA screening and confirmatory assays Anti-drug antibody (ADA): An immunoglobulin (Ig) with specific binding properties to a drug. ADAs are typically evaluated using both screening and confirmatory assay methods using a tiered approach. In the first tier, ADA screening assays are used to detect all antibodies that bind to drug and transgene. Assays for detection of ADA facilitate understanding of the immunogenicity, safety, and efficacy. The detection of ADA is dependent on key operating parameters of the assays, such as sensitivity. The second tier comprises a confirmatory method based on a competitive inhibition with excess exogenously added drug that determines the specificity of binding antibodies to drug. http://www.fda.gov/downloads/drugs/.../guidances/ucm192750.pdf 8
Universal ADA Immunoassays for nonclinical studies Direct/Indirect ADA assay format Capture drug on surface Sample containing ADA added to well drug is captured Add detection antibody or protein G/A with tag Detection of captured ADA Detection Ab with biotin/tag Substrate G /A G /A ADA Do not require labeling of drug and is ready to use as it is. Using one assay condition across different species saves the assay development time. The streamlined immunogenicity assessment strategy speeds up the assay validation. Bautista A. C., Salimi-Moosavi H., Jawa V. Universal immunoassay applied during early development of large molecules to understand impact of immunogenicity on biotherapeutic exposure. The AAPS Journal. 2012 9
Application of the ADA assay in preclinical screening of Cas9 antibodies in human 10
ELISA to Luminex Technology ELISA Basic principle behind all immunoassays Plate-bound assay format with color absorbance detection A single analyte approach Requires large sample volume 50 ml More drug needed to coat plates Sensitivity: ng/ml Luminex bead assays Same principle as ELISA using beads with specific detection Large dynamic range: Typical 4.5 logs Multiplex capability up to 500 analytes per run Small sample volume 10 ml or less Customizable and automated High sensitivity: pg/ml 21 CFR Part 11 compliant 11
Response ADA Luminex assays for AAV/Cas9 gene therapy Luminex bead system Incubate drug coated beads with sample containing ADAs Detection Each of 500 beads can be identified by its unique proportion of component dyes. ADAs Secondary antibody conjugate or protein G/A with biotin - Biotin Streptavidin -- PE 100000 10000 1000 100 SpCas9 SaCas9 AAV5 (custom made for Editas) Anti-SpCas9 (Commercial) Anti-SaCas9 (Made in house) Anti-AAV 10 10-2 10-1 10 0 10 1 10 2 10 3 Positive control antibody (ng/ml) 12
% Maximum Signal Platform comparison (Sensitivity) 1000 100 ELISA Luminex 10 1 0.1 0.01 10-4 10-3 10-2 10-1 10 0 10 1 10 2 10 3 10 4 Positive control antibody (ng/ml) 13
Evaluation of serum matrix interference in Cas9 ADA assay (non-human primates) MFI (Mean with SD) 50000 40000 No serum 1 to 10 1 to 20 30000 1 to 40 20000 10000 0 10-3 10-2 10-1 10 0 10 1 10 2 10 3 10 4 10 5 Positive control (ng/ml) Assay summary (5% Cyno serum): Sensitivity: 2 ng/ml Minimum Required Dilution (MRD): 20 Analytical range: 2 ng/ml to 1000 ng/ml 14
SNR Minimum Required Dilution (MRD) MRD is the sample dilution that yields a signal close to that of the assay diluent and allows for the highest signal-to-noise ratio. 100 10 1 to 10 1 to 20 1 to 40 MRD = 10 SNR = 2 1 10-3 10-2 10-1 10 0 10 1 10 2 10 3 10 4 Positive control (ng/ml) 15
MFI Implementation of Cas9 ADA screening assay in preclinical non-human primate studies Reproducibility 20000 15000 10000 5000 July August QC-1 QC-2 QC-3 Blank 0 1 234 5 678 9 101112 13 141516 17 181920 21 222324 25 2627 Animal ID 16
Response Assay principle to dissect ADA specificity SaCas9 ADA confirmatory assay ADA preincubation with excess Drug Incubate drug coated beads with sample containing ADAs Screening positive ADA Detection Secondary antibody conjugate or protein G/A with biotin - Biotin Streptavidin -- PE No binding 100000 10000 1000 100 10 10-2 10-1 10 0 10 1 10 2 10 3 Positive control antibody (ng/ml) 17
MFI (Mean with SD) Determination of the specificity of binding antibodies to drug by competition SaCas9 ADA confirmatory assay 25000 20000 15000 10000 No drug Excess Drug SpCas9 5000 0 Negative sample High Medium QC Low Blank Positive samples I16468 I16471 I16472 I16453 I16464 Animal ID # 18
Anti-Drug Antibody Assays for AAV editasmedicine.com 19
Evaluation of Cyno serum matrix interference in ADA screening assay for AAV MFI (Mean with SD) 40000 30000 20000 No serum 1 to 5 (A) 1 to 10 (A) 1 to 20 (A) 10000 0 10 0 10 1 10 2 10 3 10 4 10 5 Postive control (ng/ml) 20
Implementation of AAV ADA screening assay in preclinical non-human primate studies MFI 40000 21 35000 30000 25000 20000 15000 10000 5 8 17 QC-1 QC-2 QC-3 Blank 5000 0 1 2 3 4 5 6 7 8 9 10 1112 13 1415 16 1718 Animal ID # Animal # 19 2021 22 23 21
Summary AAV vectors can elicit efficient humoral and cellular responses against the vector and transgene product. Therefore it is important to develop immunogenicity assays in the early stage of drug development to understand the factors which influence PK, efficacy of the drug or induce unwanted side effects. A sensitive Luminex-based method has been developed and implemented in preclinical non-human primates and human studies to screen for antibodies against AAV and transgene Cas9. The greater sensitivity allows for the detection of low levels of ADAs. The flexibility of the luminex xmap technology allows multiplex ADA or other protein analysis which could be important for small samples, such as tear, mouse tail blood, eye vitreous and aqueous humor. It could also save on time and resources. 22