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. 2018 Oct 15;13(10):e0205491.
doi: 10.1371/journal.pone.0205491. eCollection 2018.

An Approved in Vitro Approach to Preclinical Safety and Efficacy Evaluation of Engineered T Cell Receptor anti-CD3 Bispecific (ImmTAC) Molecules

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Free PMC article

An Approved in Vitro Approach to Preclinical Safety and Efficacy Evaluation of Engineered T Cell Receptor anti-CD3 Bispecific (ImmTAC) Molecules

Jane Harper et al. PLoS One. .
Free PMC article

Abstract

Robust preclinical testing is essential to predict clinical safety and efficacy and provide data to determine safe dose for first-in-man studies. There are a growing number of examples where the preclinical development of drugs failed to adequately predict clinical adverse events in part due to their assessment with inappropriate preclinical models. Preclinical investigations of T cell receptor (TCR)-based immunotherapies prove particularly challenging as these biologics are human-specific and thus the conventional testing in animal models is inadequate. As these molecules harness the full force of the immune system, and demonstrate tremendous potency, we set out to design a preclinical package that would ensure adequate evaluation of these therapeutics. Immune Mobilising Monoclonal TCR Against Cancer (ImmTAC) molecules are bi-specific biologics formed of an affinity-enhanced TCR fused to an anti-CD3 effector function. ImmTAC molecules are designed to activate human T lymphocytes and target peptides within the context of a human leukocyte antigen (HLA), thus require an intact human immune system and peptidome for suitable preclinical screening. Here we draw upon the preclinical testing of four ImmTAC molecules, including IMCgp100, the first ImmTAC molecule to reach the clinic, to present our comprehensive, informative and robust approach to in vitro preclinical efficacy and safety screening. This package comprises a broad range of cellular and molecular assays using human tissues and cultured cells to test efficacy, safety and specificity, and hence predict human responses in clinical trials. We propose that this entirely in vitro package offers a potential model to be applied to screening other TCR-based biologics.

Conflict of interest statement

All authors are/were employees of Immunocore Ltd and the study was entirely funded by this organisation. IMCgp100 biologic is an investigational new drug produced by Immunocore Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1
Schematic representation of a systematic pre-clinical package to assess the (A) efficacy, (B) safety and (C) specificity of ImmTAC molecules for clinical development. (A) The efficacy of ImmTAC molecules against a wide range of indication relevant cells presenting target peptide-HLA is assessed. These cellular assays include both patient primary tumour cells and patient T cells relevant to the indication of interest. Cytokine and chemokine analysis form a key element of efficacy measurements. (B) The safety profile of ImmTAC molecules is measured using an array of cellular assays that screen a large panel of normal and specialised antigen-positive and antigen-negative cells. ImmTAC-induced cytokine release and platelet activation is measured in whole blood. Allo-reactivity assays test cross-reactivity against different HLA-subtypes. (C) A thorough peptide screening package is used to assess potential peptide cross-reactivity, incorporating computational BLAST searches and alanine- and x-scanning peptide mutagenesis. Potential off-target peptides that are identified as closely related to the target peptide (peptide ‘missmatch’) are further screened in cellular assays to assess cross-reactivity.
Fig 2
Fig 2. ImmTAC-mediated T cell activation and cytotoxicity.
Effector cells (E = PBMCs) obtained from a melanoma patient (for ImmTAC-gp100) or healthy donors (for ImmTAC-nybr1, ImmTAC-mageA3 and ImmTAC-nyeso) were incubated with Ag+ cell lines that present target peptide-HLA or Ag- cell lines that are HLA-relevant but do not present target peptide. Cells were incubated in the presence or absence of ImmTAC molecules. IFNγ release was assessed by ELISpot assay. Ag+ cells: Mel526, CAMA1 A2b2m, EJM and IM9 for ImmTAC-gp100, ImmTAC-nybr1, ImmTAC-mageA3 and ImmTAC-nyeso respectively. Ag- cells: A375, MDA MB 231, Colo205 and Mel526 for ImmTAC-gp100, ImmTAC-nybr1, ImmTAC-mageA3 and ImmTAC-nyeso, respectively. Statistical differences between Ag+ and Ag- cells in the presence of effector cells (E) + ImmTAC was measured using a Two-way ANOVA where *** p<0.0001, **p<0.01. If unmarked, results were not significant.
Fig 3
Fig 3. ImmTAC-mediated killing capacity is dependent on the levels of target antigen presentation.
(A) gp100 mRNA levels measured in specified cell lines using qRT-PCR. Expression is presented as Normalised Relative Quantity (NRQ) relative to housekeeping genes (RPL32 and HPRT1). NRQ = (RQ target gene/geometric mean RQ housekeeping genes) x 104, RQ = Efficiency-CT. n = 3, 4, 5, 3 and 2 for Mel526, Mel624, MeWo, WM266-4 and A375, respectively. (B) Levels of HLA-A*02 protein on the cell surface of specified cell lines measured by flow cytometry (mean fluorescence intensity (MFI) adjusted to the isotype control). n = 8, 9, 4 and 3 for Mel526, Mel624, MeWo, VM266-4 and A375, respectively. (C) Killing capacity of ImmTAC-gp100-redirected T cells assessed using the IncuCyte assay. A range of cell lines expressing different levels of gp100-HLA-A*02 complexes were incubated with decreasing concentrations of ImmTAC-gp100. The apoptotic index was determined by calculating the % ratio of apoptotic cells to the total number of target melanoma cells at the experimental endpoint (52 hours). n = 2–3. Statistical differences in apoptotic index between Ag+ (Mel 526, Mel624, MeWo and WM266-4) and Ag- (A375) cell lines were individually measured at each concentration of ImmTAC molecule using an unpaired T-test where *** p<0.0001, **p<0.01 and *p<0.05. If unmarked, results were not significant.
Fig 4
Fig 4. On-target, off-tumour activity of ImmTAC-gp100 against skin melanocytes.
IFNγ release was measured by ELISpot assay from normal human epidermal melanocytes (NHEMs) from four HLA-A*02-positive healthy donors (Melanocyte Donors 1–4), gp100 +ve (Ag+) melanoma cells (Mel526 cell line) and gp100 –ve (Ag-) control melanoma cells (A375 cell line) incubated with polyclonal (non-tumour specific) PBMCs effector cells (E) in the presence or absence of increasing concentrations of gp100-specific ImmTAC molecule (ImmTAC-gp100). Results presented represent the most reactive PBMC donor tested. Statistical differences in IFN γ release between donor NHEM cells and Ag- melanoma cells in the presence of effector cells (E) + ImmTAC molecule was measured using a Two-way ANOVA where *** p<0.0001, **p<0.01. If marked, results were not significant.
Fig 5
Fig 5. Cellular safety and specificity of ImmTAC-nybr1 in whole blood.
Whole blood freshly isolated from four healthy HLA-A*02-positive donors was incubated with increasing concentrations (0.1 nM -10 nM) of NYBR1-specific ImmTAC molecule (ImmTAC-nybr1) and the release of cytokines (IL-2, IL-6, IFNγ and TNFα) was measured using the Meso Scale Discovery (MSD) assay. Whole blood incubated with an anti-CD3 antibody alone or in combination with anti-CD28 antibody were used as positive controls. Representative results from one donor are presented. Statistical differences in cytokine release in the presence or absence of ImmTAC molecule at each concentration tested (0.1 nM—10 nM) were measured using a One-way ANOVA where *** p<0.0001. If unmarked, results were not significant.
Fig 6
Fig 6. Alloreactivity assessment of ImmTAC molecules.
Alloreactivity potential of ImmTAC-mageA3 recognising MAGE-A3 peptide in the context of HLA-A*01 was measured across cells from donors (D1-25) bearing a range of HLA types using the IFNγ ELISpot assay. Donor cells were isolated and incubated with polyclonal CD8+ T cells in the presence or absence of 0.2 nM ImmTAC-mageA3. IFNγ-release recorded in the absence of ImmTAC-mageA3 was subtracted from measurements (n = 3) and plotted as means. EJM (Ag+ & HLA-A*01+) and Colo205 (Ag- & HLA-A*01+) cell lines were used as controls. No statistical differences in IFNγ-release were observed across donor cell types when compared to IFNγ-release in the absence of ImmTAC molecule, measured using paired, one-tailed t-tests.
Fig 7
Fig 7. Molecular analysis of ImmTAC specificity for NY-ESO-1 peptide (SLLMWITQC).
(A) Each amino acid in the target peptide sequence was individually replaced by alanine (alanine-scanning mutagenesis) and each of the 9 single-mutated peptides were pulsed onto T2 HLA-A*02+ cells. Pulsed T2 cells presenting peptide were incubated with PBMCs from three healthy donors with 0.1 nM ImmTAC-nyeso molecule and IFNγ-release was measured by ELISpot assay. Representative results from one donor are presented. Dotted lines indicate 20% of cognate peptide reactivity (B) Each amino acid in the peptide sequence was exchanged by each of the 19 amino acids (X-scanning mutagenesis). Activation of PBMCs by peptide pulsed T2 cells in the presence of 0.1 nM ImmTAC-nyeso was measured by IFNγ ELISpot assay.

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Grant support

The study was entirely funded by Immunocore Ltd. All authors were funded by Immunocore Ltd at the time of contribution to the manuscript. The funder was involved in study design, data collection and analysis, decision to publish and preparation of the manuscript.
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