Assessing and Controlling Potency of Vector and Drug Product for Chimeric Antigen Receptor T Cells

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1 Assessing and Controlling Potency of Vector and Drug Product for Chimeric Antigen Receptor T Cells David M, Hambly, Ph.D. Director, Analytical Development April 4th, 2016

2 Forward Looking Statements/Safe Harbor To the extent statements contained in this presentation are not descriptions of historical facts regarding Kite Pharma, Inc. ( Kite, we, us, or our ), they are forward-looking statements reflecting management s current beliefs and expectations. Forward-looking statements are subject to known and unknown risks, uncertainties, and other factors that may cause our or our industry s actual results, levels or activity, performance, or achievements to be materially different from those anticipated by such statements. You can identify forward-looking statements by words such as anticipate, believe, could, estimate, expect, intend, may, plan, potential, predict, project, should, will, would or the negative of those terms, and similar expressions that convey uncertainty of future events or outcomes. Forwardlooking statements contained in this presentation include, but are not limited to, statements regarding: (i) the success and timing of our product development activities; (ii) the ability to effectively manufacture T cell product candidates; and (iii) our and our partners ability to develop, manufacture and commercialize our product candidates and to improve the manufacturing process. Various factors may cause differences between Kite's expectations and actual results as discussed in greater detail in Kite's filings with the Securities and Exchange Commission (SEC), including without limitation in its Annual Report on Form 10-K filed with the SEC on February 29, Except as required by law, we undertake no obligation to publicly update any forward-looking statements, whether as a result of new information, future events or otherwise. This presentation shall not constitute an offer to sell or the solicitation of an offer to buy securities, nor shall there be any sale of securities in any state or jurisdiction in which such offer, solicitation or sale would be unlawful prior to registration or qualification under the securities laws of any such state or jurisdiction. 2

3 Harnessing the Power of a Patient s Own Immune System to Target and Kill Cancer Cells ENGINEERED AUTOLOGOUS CELL THERAPY (eact ) 3

4 Dual Platform Targets Both Hematological and Solid Cancers Chimeric Antigen Receptor (CAR) Targets molecules on the cell surface T Cell Receptor (TCR) Targets molecules at or below the cell surface 4

5 Overview of the Manufacturing Process Clinical Center Apheresis product Ship for Manufacturing Enrich for T cells Ficoll separation of PBMC Manufacturing Site T cell Activation Retroviral Transduction Gentle stimulation with anti-cd3 Ab Natural co-stimulatory signals from Monocytes and B cells Introduce CAR gene Clinical Center T cell Expansion Harvest / Freeze Final Product Achieve dose Test product Prepare KTE-C19 Ship to Patient 5

6 Distinct Features of T Cell Products Multi-step process Variability risk due to: Raw materials and supplies Patient cells (e.g., apheresis product) Production process Analytical methods depend on bioassay and Flow Cytometry Multiple bioassays may be required to assess the product 6

7 Final Product Phenotype is Variable Healthy Subjects Lymphoma Subjects 100% 100% Teff Tem Tcm Naïve Donor Patient % 100% CD4 CD8 Donor Patient Variability in the phenotype may impact analytical results 7

8 CAR-T Product Candidates have Distinct Mechanisms of Action Retroviral Vector Manufacturing of effective CAR T cells involves important biological functions: 1. Retroviral vector with consistent transducing potency 2. Infection, reverse transcription and integration of the CAR sequence into the cellular genome 3. Expression and localization of functional CAR in the plasma membrane 4. Antigen induced stimulation of the CAR T cell 5. Cytotoxicity directed towards antigen-positive cells 8

9 % Transduction Retroviral Vector Potency Assesses CAR Expression Measured in Transducing units/ml Potency is used to determine titer: (% CAR expressing cells) * (total number of cells) / Volume of Vector used % Transduction Titer Vector Quantity Linear Transduction = Titer Vector potency is used to measure titer at limiting dilutions 9

10 % Transduced Vector Potency Using PBMCs Assessing Potency: Donor derived PBMCs Limited number of cells Minimal dilutions Analysis replicates Donor variability may require donor qualification PBMC Method Vector Quantity An effective clinical method to assess vector potency 10

11 % Transduced Vector Potency Using a Stable T Cell Line Ideal method to assess potency: A robust, stable cell line banked to single-use vials, demonstrated intermediate precision At least 3 measurements, in replicate, across the linear range to average biological variability The capability to measure high and low titer samples Vector Quantity Banked cells can enable increased range, increased replicates, improved throughput and eliminate donor variability 11

12 CAR-T Product Candidates have Distinct Mechanisms of Action Final Product: Transduced Patient T Cells Manufacturing of effective CAR T cells involves numerous biological functions: 1. Retroviral vector with consistent transducing potency 2. Infection, reverse transcription and integration of vector RNA into the cellular genome 3. Expression and localization of functional CAR in the plasma membrane 4. Antigen induced stimulation of the CAR T cell 5. Cytotoxicity directed towards antigen-positive cells 12

13 Assessing T Cell Final Product Transduction Integrated Vector Copy Number: Quantitative PCR result in copies per cell Average of all cells in the sample Correlated to % Transduction Integration assesses product transduction 13

14 CAR Intensity CAR Intensity Final Product Potency: % CAR Expressing T Cells Flow cytometry quantitates the percentage of T cells expressing the CAR. Dose is a defined number of transduced T cells Untransduced Transduced Anti-CD3 mab fluorophore CAR specific fluorophore Utd CAR CD3 Intensity CD3 Intensity % CAR T cells x Total number of cells enables dosing 14

15 % Transduced Flow Cytometry for % Transduction: Validation Strategy Linearity and Accuracy can be assessed by mixing transduced and untransduced cells R² = LOQ can be assessed: 1. ~ N x std dev + background from untransduced cells 2. Mix transduced and untransduced at a defined level. Validate reproducible quantitation of the sample Ratio of Transduced to Untransduced Validate relevant performance parameters 15 15

16 Final Product Potency: Cytokine Release is Dependent on CAR Response to Antigen Antigen Negative Antigen Positive Antigen Negative Antigen Positive Cytokine release is widely used as a surrogate for function 16

17 Final Product Potency: Quantitative Cytokine ELISA Co-Culturing CAR and Antigen Positive Cells Negative controls demonstrate low activity without antigen, or in the absence of the CAR transduction CAR Expressing + Antigen = Strong cytokine signal CAR Expressing, no Antigen = Low signal CAR + ve CAR - ve Untransduced + Antigen = Low signal Untransduced, no Antigen = Low signal UT + ve UT - ve Potent cells release cytokines upon co-culture with antigen 17

18 Future Thoughts: Understanding Method Variability To control method performance through trending, consider external controls Example: Cytokine ELISA quantitation after culturing product and antigen positive cells ELISA uses an external control independent from the std curve Frozen qualified cytokine expressing cells to confirm the antigen positive cells are consistent Example: Flow cytometry to quantitate % transduction Stable transduced and banked cell line qualified to confirm staining and counting were controlled Ensure an ongoing supply of qualified external controls 18

19 Future Thoughts New reference standards should be qualified Use side-by-side testing in bioassays, and ideally orthogonal methods to understand the performance of the current and proposed RS External controls require qualification Consideration should be given to stability and supply Bioassays are key to the product and process control. Carefully consider the entire scope of the method lifecycle and method impacts. 19

20 Acknowledgements Product Sciences Marc Better Emily Lowe Quality Control Prentice Curry Kanti Thirumoorthy Ken Murray Translational Research John Rossi Yueh-wei Shen Heba Nowyhed 20