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EP-4740011-A1 - METHOD

EP4740011A1EP 4740011 A1EP4740011 A1EP 4740011A1EP-4740011-A1

Abstract

The present invention provides a method of determining the relative potency of a viral vector encoding a chimeric antigen receptor (CAR), the method comprising: a. providing a sample viral vector to be analysed (sample vector) and a reference viral vector of known potency (reference vector); b. transducing separate Effector cell populations with the sample vector and reference vector at various multiplicities of infection (MOIs) to form a group of transduced sample Effector cell populations and a group of transduced reference Effector cell populations; C. conducting cell impedance-based real-time cell killing assays of Target cells using the transduced sample Effector cell populations and the transduced reference cell populations; d. generating a cytotoxicity versus MOI curve for the transduced sample Effector cell populations and for the transduced reference cell populations; e. comparing parallelism of the curves generated in step (d), wherein parallel curves indicate comparable data sets; f. determining the half maximal effective concentration (EC50) of the sample Effector cell population and the reference cell population; and g. comparing the EC50 of the transduced sample Effector cell population and the transduced reference cell population to determine the relative potency of the sample viral vector.

Inventors

  • BHAUMIK, Siddhartha K.
  • WEBB, Louise
  • GASPARAVICIUTE, Agne
  • WANG, WEI

Assignees

  • Autolus Limited

Dates

Publication Date
20260513
Application Date
20240705

Claims (14)

  1. 1. A method of determining the relative potency of a viral vector encoding a chimeric antigen receptor (CAR), the method comprising: a. providing a sample viral vector to be analysed (sample vector) and a reference viral vector of known potency (reference vector); b. transducing separate Effector cell populations with the sample vector and reference vector at various multiplicities of infection (MOIs) to form a group of transduced sample Effector cell populations and a group of transduced reference Effector cell populations; c. conducting cell impedance-based real-time cell killing assays of Target cells using the transduced sample Effector cell populations and the transduced reference cell populations; d. generating a cytotoxicity versus MOI curve for the transduced sample Effector cell populations and for the transduced reference cell populations; e. comparing parallelism of the curves generated in step (d), wherein parallel curves indicate comparable data sets; f. determining the half maximal effective concentration (EC50) of the sample Effector cell population and the reference cell population; and g. comparing the EC50 of the transduced sample Effector cell population and the transduced reference cell population to determine the relative potency of the sample viral vector.
  2. 2. The method of claim 1, wherein the vector is a retroviral vector or a lentiviral vector.
  3. 3. The method of claim 1 or claim 2, wherein the CAR comprises an antigen binding domain, spacer domain, transmembrane domain, and intracellular signalling domain.
  4. 4. The method according to any one of claims 1 to 3, wherein the CAR is specific for an antigen selected from the group comprising CD19, CD20, CD22, TRBC1, TRBC2, GD2, BCMA, CD33, CD123, and CLL1.
  5. 5. The method according to claim 4, wherein the CAR is specific for CD19.
  6. 6. The method according to any one of claims 1 to 5, wherein the Effector cells are T cells or NK cells.
  7. 7. The method according to claim 6, wherein the Effector cells are CD8 positive T cells.
  8. 8. The method according to any one of claims 1 to 7, wherein the Target cells are an immobilised suspension cell line.
  9. 9. The method according to claim 8, wherein the immobilised suspension cell line is immobilised via anti-CD40 antibody coated gold plates.
  10. 10. The method according to any one of claims 1 to 9, wherein the Target cells are selected from the group comprising Raji cells, Jurkat cells, and HEK293T cells.
  11. 11. The method according to any one of claims 1 to 10, wherein the Effector to Target ratio (E:T ratio) in the cell impedance-based real-time cell killing assays is 1 :1 , 2:1 (preferred), 3:1, 4:1, 5:1, 8:1 , 10:1, or 12:1.
  12. 12. The method according to claim 11, wherein the E:T ratio is 2:1.
  13. 13. The method according to any one of claims 1 to 11, wherein transduction of Effector cells is carried out at an MOI of from 0.016 to 64.0.
  14. 14. The method according to claim 12, wherein transduction of Effector cells is carried out at an MOI of 0.016, 0.063, 0.25, 1.0, 4.0, 16.0, and/or 64.0.

Description

METHOD FIELD OF THE INVENTION The present invention provides a method for determining the potency of a viral vector preparation. More specifically, the present invention provides a method of determining the potency of a viral vector encoding a chimeric antigen receptor using a cell impedance-based real-time assay. BACKGROUND TO THE INVENTION Adoptive cell therapy (ACT) is a personalised therapy that involves administration to the subject of immune cells with activity directed against a specific disease related antigen. In recent years, the development of chimeric antigen receptors (CARs) has led to the approval of several adoptive cell therapies targeting cancers. Viral vectors are typically used in the manufacture of ACT drug products to transduce cells. Although vector batches are manufactured using standardised processes, there remains the possibility that individual vector lots may possess different biological activities. Vector potency is therefore a critical attribute that must be tested prior to the release of a batch of vector. Many existing vector potency methods rely on the inclusion of marker genes that are otherwise unconnected to the activity of the final drug product. This is not ideal in a clinical product intended for use in humans. Other methods rely on indirect measurement of activity, such as cytokine release. There is therefore a need for alternative methods for determining the potency of viral vector preparations. DESCRIPTION OF THE FIGURES Figure 1 - CD8 Cells transduced with higher concentrations of vector are better able to kill Target cells, up until a maximum percentage cytolysis is reached. CD8 cells were serially transduced, on method day 1, with high to low concentrations of vector (i.e., at different Multiplicity of infections (MOIs)). On method day 7, 100,000 total CD8 cells (Effectors) were seeded into 3 separate xCELLigence plates that had been pre-seeded on method day 6 with 50,000 Targets. The percentage cytolysis was normalised to Targets and non-transduced Effectors and assessed at the 24 hour co-culture time-point. Since transduction was carried out using 1 million CD8 cells per 1 mL of viral supernatant, the MOI shown is numerically equivalent to the vector’s infectious titre in million Tll/mL. The graph shows the average ± standard deviation (SD) when data points from all 3 plates are combined. Figure 2 - A 4PL model fits the vector dose response curve. Two batches of vector, PDE-B20042 and 21079/B were used to transduce CD8 cells. For each vector, two separate but identical transduction series (T 1 and T2) were performed. (A) The percentage transduction efficiency was assessed on method day 7 for Effectors transduced on day 1 at different MOIs. Non-linear regression was used to fit curves to the data points. (B) The CAR density per CD8 cell was assessed on method day 7. Linear regression was used to fit lines through the data points. (C) 100,000 total Effector cells per well were seeded, on method day 7, into two separate xCELLigence plates pre-seeded on method day 6 with 50,000 Targets per well. In plate 1 vector PDE-B20042 was used both as the reference standard and as a test sample, together with 21079/B. In plate 2, 21079/B provided the reference standard and a test sample, together with PDE-B20042. The percentage cytolysis at 24 hours co-culture was assessed, with data normalised to targets and non-transduced Effectors (mock). Non-linear regression was used to fit curves to the data points. Figure 3 - The vector potency method can be used for stability testing. CD8 cells were transduced with vector batch PDE-B20042 or 21079/B. In both runs, PDE- B20042 acted as the reference standard. Vector 21079/B acted as the test sample in both runs. For stability testing, vector 21079/B on day 1 was either: not treated, freeze-thawed four times, treated at 60 °C for 1 hour, 60 °C for 6 hours, 40 °C for 30 minutes, 40 °C for 1 hour, 40 °C for 2 hours, or 40 °C for 3 hours. (A) The percentage transduction efficiency was assessed on method day 7 for all transduction series. The graph inserted at top right of the right hand graph shows a magnification of the percentage transduction efficiency for the top two vector concentrations. (B) Six days post transduction, 100,000 transduced Effectors were seeded into an XCELLigence plate pre-seeded with 50,000 Target cells the day before. The percentage cytolysis of the Targets was normalised to Targets and non-transduced Effectors (mock) and assessed over time. Each stability run consisted of two plates. Each data point shows the average ± SD of three replicates within the plate. SUMMARY OF ASPECTS OF THE INVENTION The present inventors provide herein a method for determining the potency of a viral vector preparation. More specifically, there is provided herein a method of determining the potency of a virus encoding chimeric antigen receptor. The method involves the use of a cell impedance-based real-time cell killing assay. As such