Elucidation of B-cell specific drug immunogenicity liabilities via a novel ex vivo assay

Abstract

The advent of large molecule therapeutics has revolutionized treatment options for previously unmet medical needs. This advent has also led to an increased impact of immunogenicity on drug efficacy and safety. In order to maximize the potential of large molecule therapeutics, immunogenicity-related liabilities must be identified as early in development as possible, using an integrated risk assessment that takes into account the various cell types and processes involved. Here, we describe the development of an ex vivo B-cell immunogenicity assay, to capture a key component of the immune response that has been missing from previously published ex vivo immunogenicity assays. Plasmablasts/plasma cells were preferentially expanded in this assay, a subset of which were drug-specific and presented drug-specific peptides on MHC Class II. This assay represents an important tool in the immunogenicity risk assessment toolkit, to allow liabilities to be identified and mitigated early in the drug development process.

Keywords: anti-drug antibodies; assay development; biotherapeutics; immunogenicity; in vitro B-cell assay.

Figure 1

Ex vivo B-cell immunogenicity assay results in generation of plasmablasts/plasma cells after 7 days of stimulation. Schematic - PBMCs are isolated from healthy donors on a Ficoll gradient, then incubated with LLME to reduce TCs and NKs. Depleted PBMCs are then incubated with Class A CpG, IL-2, IL-4, and antigen. Cells are restimulated at Day 4 with Class B CpG, IL-2, and IL-4. Flow cytometric and/or confocal analysis is performed at Day 7 (A). Representative plot of CD38 vs CD27 on healthy donor PBMCs before stimulation (B) vs after 7 days of stimulation with an immunogenic drug (C). Samples were gated for lymphocytes on FSC/SSC, for single cells on SSC-A/SSC-H, CD45⁺ cells, then CD3^negCD19^+/lo.

Figure 2

Flow cytometric and confocal analysis of drug-specific B-cells. PBMCs were stimulated with no exogenous antigen, KLH, or a set of drugs for 7 days. Drug specific B-cell frequency was determined by anti-PGLALA staining or incubation with fluorophore-conjugated drug, and replicate experiments plotted (A). Confocal images of B-cells isolated from Day 7 ex vivo immunogenicity cultures after stimulation with no antigen (B) or CEA-IL2v (C, D). Cells were stained with a nuclear Hoechst stain (blue), an anti-CD19 Alexa Fluor 488 (green), and an anti-CD79b PE (orange). Antigen was visualized either by an anti-PGLALA Alexa Fluor 647 (C) or direct conjugation of antigen to Alexa Fluor 647 (D) (red). Images are representative of cells acquired on an Opera Phenix using a 63× water immersion lens (maximum projection of 3 μm stack).

Figure 3

CEA-IL2v does not promote immunogenicity in trans. PMBCs were stimulated with durvalumab alone, or durvalumab + CEA-IL2v. Drugs were conjugated to different fluorophores (CEA-IL2v Alexa Fluor 647, durvalumab Alexa Fluor 680) to enable visualization of antigen-specific cells for the two drugs in the same well. Representative flow plots of durvalumab-stimulated cells (A) and CEA-IL2v + durvalumab-stimulated cells (B), and percentages of durvalumab-positive B-cells in various stimulation conditions (C) are shown.

Figure 4

T-cell phenotype after stimulation. T_C/T_H T-cell profile and CD25 and CD69 expression on these subsets was assessed by flow cytometry after 7 days of stimulation with various drugs. (A), T_C as a % of total CD3⁺ lymphocytes; (B) CD69⁺ as a % of T_C; (C) CD69⁺ as a % of T_H; (D) CD25⁺ as a % of T_C; (E) CD25⁺ as a % of T_H.

Figure 5

T-cell cytokine profiles after stimulation. After 7 days of stimulation with various drugs, PBMCs were incubated for 4 hours with Brefelden A, then stained intracellularly for the cytokines IL-2 (A), IFNγ (B), IL-4 (C), and IL-6 (D).

Figure 6

MAPPs of KLH⁺ B-cells demonstrate presentation of KLH-derived peptides. After 7 days of stimulation with KLH, antigen-specific B-cells and moDCs were isolated via FACS, lysed, MHC Class II-DR immunoprecipitated, and MHC Class II-DR -associated peptides identified via mass spectrometry. Peptides were mapped to KLH by PEAKS software.

Figure 7

Spearman correlation between WBA and clinical response rates (A), optimizing the correlation between assay and clinical response rates by varying the % positive B cell threshold to call a specific donor´s assay readout a positive response. A ROC curve for the empirically found ‘optimal setting’ (FC cutoff 5, ADA rate cutoff 15%) was generated (B). A random permutation approach explored if any arbitrary assignment of clinical responses could be ‘predicted’ with comparable accuracy, for the empirical AUC (C) and response rate correlation (D).