β2AR antagonists and Β2AR gene deletion both promote skin wound repair processes

Skin wound healing is a complex process requiring the coordinated, temporal orchestration of numerous cell types and biological processes to regenerate damaged tissue. Previous work has demonstrated that a functional β-adrenergic receptor autocrine/paracrine network exists in skin, but the role of β2-adrenergic receptor (β2AR) in wound healing is unknown. A range of in vitro (single-cell migration, immunoblotting, ELISA, enzyme immunoassay), ex vivo (rat aortic ring assay), and in vivo (chick chorioallantoic membrane assay, zebrafish, murine wild-type, and β2AR knockout excisional skin wound models) models were used to demonstrate that blockade or loss of β2AR gene deletion promoted wound repair, a finding that is, to our knowledge, previously unreported. Compared with vehicle-only controls, β2AR antagonism increased angiogenesis, dermal fibroblast function, and re-epithelialization, but had no effect on wound inflammation in vivo. Skin wounds in β2AR knockout mice contracted and re-epithelialized faster in the first few days of wound repair in vivo. β2AR antagonism enhanced cell motility through distinct intracellular signalling mechanisms and increased vascular endothelial growth factor secretion from keratinocytes. β2AR antagonism promoted wound repair processes in the early stages of wound repair, revealing a possible new avenue for therapeutic intervention

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.</p

An approved <i>in vitro</i> approach to preclinical safety and efficacy evaluation of engineered T cell receptor anti-CD3 bispecific (ImmTAC) molecules - 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.</p

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.</p

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.</p

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.</p

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.</p

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.</p