Strategies to Improve Drug Tolerance in Nab Assays

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1 Strategies to Improve Drug Tolerance in Nab Assays Steven J Swanson, PhD Senior Vice President, Research ImmunoCellular Therapeutics Ltd steven.swanson@imuc.com AAPS National Biotech Conference June

2 Importance of Drug Tolerance Immunogenicity assays routinely utilize the therapeutic agent as a cornerstone, typically by immobilization of the drug Specificity of detected anti-drug antibodies is often confirmed by a reduction in binding of the ADA in the presence of excess soluble drug Circulating immune complexes can form when soluble residual drug is present in patient s blood The presence of soluble drug in the patient s serum sample for ADA analysis can inhibit the assay from detecting anti-drug antibodies 2

3 Definition of Drug Tolerance Drug tolerance describes the sensitivity of your anti-drug antibody assay in the presence of soluble drug Tolerance is determined by assessing assay sensitivity in the presence of increasing amounts of added soluble drug The positive control Ab used for sensitivity determination may NOT mimic action of patients ADA Amount of Soluble Drug Assay A Sensitivity* 3 Assay B Sensitivity* 0 mcg/ml 100 ng/ml 100 ng/ml 1 mcg/ml 1,000 ng/ml 100 ng/ml 5 mcg/ml 10,000 ng/ml 200 ng/ml 10 mcg/ml 30,000 ng/ml 500 ng/ml * Hypothetical examples for illustration only

4 Neutralizing Antibody Assays are Especially Vulnerable to Soluble Drug Interference Cell-based bioassays typically have decreased sensitivity and decreased tolerance for soluble drug when compared with immunoassays Any soluble drug in a patient s serum sample can contribute to the bioassay signal Any neutralizing antibodies in the patient s sample must neutralize the measured drug added to induce a signal and also the unmeasured residual drug 4

5 Understand Target Sensitivity After conducting a thoughtful risk-based analysis for your therapeutic, it is important to establish a target sensitivity Higher risk therapeutics may require more sensitive ADA assays Selection of your ADA assay platforms should consider drug tolerance as one of the evaluation parameters A clear knowledge of anticipated PK for your therapeutic will help you estimate how much soluble drug should be present when ADA samples are taken Determine the drug tolerance of your analytical procedure and decide if you need to develop a mitigation strategy 5

6 Strategic Approaches Delay ADA sample collection until sufficient clearance of soluble drug is achieved Disrupt circulating immune complexes Pre-treat samples to remove residual circulating drug Utilize an ADA assay that has high drug tolerance Be sure to validate that your approach does not DECREASE your ability to detect Nabs, you may have to analyze samples both with and without sample treatment 6

7 Delay ADA Sample Collection Until Sufficient Clearance of Soluble Drug is Achieved This may not be possible for drugs that have a very long ½ life mabs Pegylated drugs End of Study sample should not be taken until drug has cleared End of Study may be the only sample from the patient after receiving drug that is not compromised by circulating drug 7

8 Disrupt Circulating Immune Complexes The goal of improving drug tolerance is to detect as many of the ADA generated by the patient as possible Much of the ADA response in a patient may be bound by circulating drug in the form of small immune complexes Antibodies complexed by soluble drug will not be detected in the ADA assay Disrupting the immune complex by briefly treating the patient sample with an acid solution followed by rapid neutralization and analysis MAY allow you to detect more ADA in a sample* *Ref: Moxness et al. Clin Chem 2005; 51:

9 Drug Drug Drug Drug Drug Drug Acid Dissociation Reduces Interference + Acid + Base Ru ADA ADA + Assay Reagents + Time ADA Biotin Ref: Moxness et al. Clin Chem 2005; 51:

10 Signal/Noise Ratio (0.5 mcg/ml Ab) Acid Dissociation Reduces Interference 100 No Acid With Acid Drug Concentration (mcg/ml)

11 Treat Samples to Remove Residual Drug Use beads coated with high affinity antibodies against the therapeutic protein Construct a column with support that has covalently attached antibodies or receptor capable of binding to the therapeutic protein with high affinity For small MW therapeutic proteins: Treat sample with acid to release drug Use a size exclusion column to separate and remove soluble drug Neutralize and analyze Ab fraction for neutralizing ADA 11

12 Utilize an ADA Assay with High Drug Tolerance Non-cell based assays tend to have higher drug tolerance Using risk-based approach determine if a non-cell based assay with higher drug tolerance is preferable to a cell based assay with lower drug tolerance Consult with Regulatory Agencies if not certain 12

13 Immunogenicity Assessment Strategy Time and Effort Spent Here is Based on Risk 13

14 Importance of Biological Assays for Neutralizing Activity A biological assay can determine if detected antibodies are capable of neutralizing the biological effect of the drug.

15 Representative Bioassay Culture cells Culture cells Add drug Add drug and Ab sample Measure biological response (proliferation) Measure biological response (proliferation)

16 Biological Assay Endpoints for Detecting Neutralizing Antibodies mrna Expression Receptor PY Apoptosis Protein Expression Proliferation ADCC

17 Important Considerations If any sample manipulation is performed it is important to verify that you are not losing NAbs in the process Can be tested during assay validation Can test some samples with and without sample manipulation and compare results If using beads or resin coated with Abs against the drug, verify that there is no leaching of the Abs (it may yield false positive results) Use of Fab fragments to capture residual drug may be a good option 17