An immunologically relevant rodent model demonstrates safety of therapy using a tumour-specific IgE

Abstract

Background: Designing biologically informative models for assessing the safety of novel agents, especially for cancer immunotherapy, carries substantial challenges. The choice of an in vivo system for studies on IgE antibodies represents a major impediment to their clinical translation, especially with respect to class-specific immunological functions and safety. Fcε receptor expression and structure are different in humans and mice, so that the murine system is not informative when studying human IgE biology. By contrast, FcεRI expression and cellular distribution in rats mirror that of humans.

Methods: We are developing MOv18 IgE, a human chimeric antibody recognizing the tumour-associated antigen folate receptor alpha. We created an immunologically congruent surrogate rat model likely to recapitulate human IgE-FcεR interactions and engineered a surrogate rat IgE equivalent to MOv18. Employing this model, we examined in vivo safety and efficacy of antitumour IgE antibodies.

Results: In immunocompetent rats, rodent IgE restricted growth of syngeneic tumours in the absence of clinical, histopathological or metabolic signs associated with obvious toxicity. No physiological or immunological evidence of a "cytokine storm" or allergic response was seen, even at 50 mg/kg weekly doses. IgE treatment was associated with elevated serum concentrations of TNFα, a mediator previously linked with IgE-mediated antitumour and antiparasitic functions, alongside evidence of substantially elevated tumoural immune cell infiltration and immunological pathway activation in tumour-bearing lungs.

Conclusion: Our findings indicate safety of MOv18 IgE, in conjunction with efficacy and immune activation, supporting the translation of this therapeutic approach to the clinical arena.

Keywords: AllergoOncology; IgE; cancer; immunotherapy; rat.

Figure 1

Rat MOv18 IgE and IgG2b antibody engineering, purification and characterization. A, Schematic representations of the cloned rat rMOv18 IgE and IgG2b chimeric antibodies with mouse variable domains specific for the human folate receptor α (FRα) and rat constant domains (light green: human Cε, dark green: rat Cε, dark orange: human Cγ, light orange: rat Cγ, light grey: human Cκ, dark grey: rat C_L, purple: mouse V_H, brown: mouse V_L). B, The heavy and light chains of each rMOv18 antibody were cloned into a single mammalian expression vector per antibody (pVITRO1). This resulted in the production of pVITRO‐Rat MOv18 IgE and IgG2b. C, Purification of rMOv18 IgE with MEP HyperCel™ resin and 25% isopropanol elution buffer (MW: molecular weight marker, F1‐F8: fractions 1‐8). D, E, non‐reducing SDS‐PAGE (D) and HPLC‐SEC elution profiles (E) of HPLC‐purified hMOv18 IgE and IgG1, and rMOv18 IgE and IgG2b demonstrate intact monomeric antibodies of purities >85%. F, rMOv18 IgE and IgG2b bind to rat primary monocytes, RBL‐2H3 rat mast cells, FRα‐expressing rat colon adenocarcinoma CC531tFR cells and human ovarian carcinoma IGROV1 cells, but not to non‐FRα‐expressing human melanoma A375 cells. G, hMOv18 IgE shows weak binding, while rMOv18 IgE demonstrates significant binding to rat FcεRI‐expressing RBL‐2H3 cells. Human and rat MOv18 IgE display similar binding to human FRα‐expressing CC531tFR cells [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Establishment of the tumour‐bearing surrogate rat model and antibody‐mediated tumour growth restricting potencies. A, Schematic representation of immunocompetent model design: rMOv18 IgE engages native rat FcεR‐expressing immune effector cells to target syngeneic rat tumour cells expressing human folate receptor α (FRα). B, FRα expression of CC531tFR cells immediately prior to in vivo tumour challenge. C, Determination of optimum i.v. CC531tFR tumour challenge. Representative images of Indian ink‐stained lungs reveal white tumour lesions (n = 5). D, Rat MOv18 IgE demonstrates superior tumour growth restriction. Percentage (%) tumour occupancy and number of metastases/cm² quantified following two doses (every 14 days) of PBS, or rMOv18 IgE and IgG2b at 10 mg/kg (n = 6). rMOv18 IgE compared with rMOv18 IgG2b (mean % tumour occupancy: 8.5% vs 20.7%, P = .003; mean number of lung metastases/cm²: 14.7 vs 31.4, P = .0049) or with PBS (mean % tumour occupancy: 33.3%, P < .0001; mean number of lung metastases/cm²: 45.2, P < .0001). E, Dosing regimen following tumour challenge was on days 1 and 14 over a 30‐day period [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Rat MOv18 IgE and IgG induce comparable mild toxicities only. A, Percentage of rats in each treatment group experiencing the 3 detected toxicities (piloerection, hunching and reduced activity). B, Percentage of animals within each treatment group to experience 1, 2 or 3 toxicities. C, Histopathological evaluations of tissues 30 days after administration of two doses (every 14 days) of rMOv18 IgE or IgG2b at 5 mg/kg and 50 mg/kg or PBS rats (n = 10 per group). Sections were stained with H&E and observed at brain = 25 × magnification; colon, kidney, liver, mesenteric lymph nodes (LN), tail and thymus = 50 × magnification; spleen = 100 × magnification [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Absence of rMOv18 IgE‐associated changes in body weight or blood haematological or biochemical parameters. A, Weekly weights of rats following weekly treatment with rMOv18 IgE at 1 mg/kg (n = 10), 10 mg/kg (n = 10) and 50 mg/kg (n = 10) or PBS (n = 10). B‐E, Blood biochemical and haematological parameters were measured immediately before necropsy on day 30 following 4 weekly doses of rMOv18 IgE at 1 mg/kg (n = 10), 10 mg/kg (n = 10) and 50 mg/kg (n = 10) or PBS (n = 10). Dotted lines indicate normal range values for each parameter determined in healthy Wistar Albino Glaxo (WAG)/RijCrl.24 F‐G, Rat anti‐human folate receptor α (FRα) antibodies and rat antidrug antibodies (ADA) measured in rat serum following weekly treatment with PBS, or rMOv18 IgE at 1 mg/kg, 10 mg/kg and 50 mg/kg (n = 10 per group) [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Rat MOv18 IgE induces immune cell infiltration and immune activating signatures. A, B, Percentage (%) tumour occupancy and number of metastases/cm² quantified following four (weekly) doses of PBS, or rMOv18 IgE at 1 mg/kg, 10 mg/kg and 50 mg/kg (n = 10 per group). C, Representative images of Indian ink‐stained lungs from PBS‐ and rMOv18 IgE‐treated rats (n = 10 per group). D, Representative images of paraffin‐embedded lung sections from PBS‐ and rMOv18 IgE‐treated rats stained with H&E (n = 10 per group). Magnification 200x. Black arrow: glandular tumour islet; white arrow: immune and stromal cells. E, GSEA analyses showing enrichment of immune‐associated signatures (red) in rMOv18 IgE‐treated (two doses, 2 weeks apart) tumour‐bearing rat lungs and cell cycle‐associated signatures (yellow) in PBS‐treated tumour‐bearing rat lungs. Other signatures are shown in blue. The y‐axis corresponds to the normalized enrichment score (NES). F, Heat maps showing the differential expression of all genes that constitute the IL‐12 and NK cells signature between rMOv18 IgE‐ and PBS‐treated rat lungs [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Rat MOv18 IgE triggers TNFα but no evidence of cytokine storm or allergic response. A, Cytokine/chemokine production measured in rat serum following weekly treatment with PBS, or rMOv18 IgE at 1 mg/kg, 10 mg/kg and 50 mg/kg (n = 10 per group). B‐D, Concentrations of selected analytes in rat sera