Engineering and stable production of recombinant IgE for cancer immunotherapy and AllergoOncology

Crescioli, Silvia; Chiaruttini, Giulia; Mele, Silvia; Ilieva, Kristina M.; Pellizzari, Giulia; Spencer, Daniel I. R.; Gardner, Richard A.; Lacy, Katie E.; Spicer, James; Tutt, Andrew N.J.; Wagner, Gerd K.; Karagiannis, Sophia N.

doi:10.1016/j.jaci.2017.12.986

Cited by 19 publications

(33 citation statements)

References 16 publications

(24 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We successfully generated α-CSPG4 rIgE, a rat IgE containing the variable regions of the light and heavy chains derived from the murine anti-CSPG4 clone 225.28 (Supplemental Figure 1). 31 SDS-PAGE analysis of α-CSPG4 rIgE confirmed the predicted molecular size in both non-reducing and reducing conditions ( Figure 2A). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) profiles of α-CSPG4 rIgE were comparable to those of the previously-described rat antibody MOv18 rIgE, 15 which recognizes the human FRα (Figure 2A, B).…”

Section: Generation and Functional Characterization Of α-Cspg4 Rigesupporting

confidence: 57%

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) profiles of α-CSPG4 rIgE were comparable to those of the previously-described rat antibody MOv18 rIgE, 15 which recognizes the human FRα (Figure 2A, B). Flow cytometric analysis showed that α-CSPG4 rIgE was able to bind the human CSPG4-expressing melanoma cell line A2058 in a dose-dependent manner and similarly to its human counterpart, α-CSPG4 hIgE 31 ( Figure 2C), with 50% of the maximal mean fluorescence intensity reached by the two antibodies at similar concentration (1.57 µg/ml α-CSPG4 rIgE and 1.11 µg/ml α-CSPG4 hIgE). Comparable binding of α-CSPG4 rIgE to the rat FcεRI-expressing basophilic RBL-2H3 cells and α-CSPG4 hIgE to the human FcεRI-expressing RBL-SX38 cells was observed ( Figure 2D).…”

Section: Generation and Functional Characterization Of α-Cspg4 Rigementioning

confidence: 90%

See 1 more Smart Citation

In vivo safety profile of a CSPG4-directed IgE antibody in an immunocompetent rat model

Williams

Crescioli

Sow

et al. 2019

mAbs

Self Cite

View full text Add to dashboard Cite

IgE monoclonal antibodies hold great potential for cancer therapy. Preclinical in vivo systems, particularly those in which the antibody recognizes the host species target antigen and binds to cognate Fc receptors, are often the closest approximation to human exposure and represent a key challenge for evaluating the safety of antibody-based therapies. We sought to develop an immunocompetent rat system to assess the safety of a rodent anti-tumor IgE, as a surrogate for the human therapeutic candidate. We generated a rat IgE against the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) and cross-reactive for the rat antigen. We analyzed CSPG4 distribution in normal rat and human tissues and investigated the in vivo safety of the antibody by monitoring clinical signs and molecular biomarkers after systemic administration to immunocompetent rats. Human and rat CSPG4 expression in normal tissues were comparable. Animals receiving antibody exhibited transient mild to moderate adverse events accompanied by mild elevation of serum tryptase, but not of angiotensin II or cytokines implicated in allergic reactions or cytokine storm. In the long term, repeated antibody administration was well tolerated, with no changes in animal body weight, liver and kidney functions or blood cell counts. This model provides preclinical support for the safety profiling of IgE therapeutic antibodies. Due to the comparable antigen tissue distribution in human and rat, this model may also comprise an appropriate tool for proof-of-concept safety evaluations of different treatment approaches targeting CSPG4.

show abstract

Section: Generation and Functional Characterization Of α-Cspg4 Rigesupporting

confidence: 57%

Section: Generation and Functional Characterization Of α-Cspg4 Rigementioning

confidence: 90%

In vivo safety profile of a CSPG4-directed IgE antibody in an immunocompetent rat model

Williams

Crescioli

Sow

et al. 2019

mAbs

Self Cite

View full text Add to dashboard Cite

show abstract

“…First, we engineered a mouse/human chimeric IgG1 antibody recognizing the melanoma-associated antigen CSPG4 (anti-CSPG4 IgG1), cloning the variable regions of a murine IgG2a clone (225.28s) into a human IgG1 backbone [32]. We then developed a stable expression system for rapid production of the recombinant anti-CSPG4 IgG antibody, equivalent to one previously described for the generation of an IgE class antibody [33] ( Figure 1A). We isolated the coding sequences of anti-CSPG4 IgG heavy and light chains from a previously described (pVITRO1-CSPG4 IgG1/k) vector ( Figure 1B) [32].…”

Section: Anti-cspg4 Monoclonal Antibody Generation and Evaluation Of mentioning

confidence: 99%

“…The pVITRO1-CSPG4-IgG/k vector ( Figure 1B) was previously developed in our group [32]. The dual UCOE-vector system (UCOE-CSPG4-HC[ε] and UCOE-CSPG4-LC[κ]; Figure 1C) was developed by using PIPE cloning, as described previously for the IgE version of this antibody [33]. A stable anti-CSPG4 IgG-expressing Expi293F cell line has been developed as previously described for the anti-CSPG4 IgE [33].…”

Section: Cspg4 Igg Expressionmentioning

confidence: 99%

“…The dual UCOE-vector system (UCOE-CSPG4-HC[ε] and UCOE-CSPG4-LC[κ]; Figure 1C) was developed by using PIPE cloning, as described previously for the IgE version of this antibody [33]. A stable anti-CSPG4 IgG-expressing Expi293F cell line has been developed as previously described for the anti-CSPG4 IgE [33]. Anti-CSPG4 IgG used in this study was purified into PBS using Protein A columns (Thermo Fisher Scientific, 20356) according the manufacturer's instructions, followed by analytic size exclusion chromatography as described below.…”

Section: Cspg4 Igg Expressionmentioning

confidence: 99%

See 1 more Smart Citation

A Novel Antibody-Drug Conjugate (ADC) Delivering a DNA Mono-Alkylating Payload to Chondroitin Sulfate Proteoglycan (CSPG4)-Expressing Melanoma

Hoffmann

Crescioli

Mele

et al. 2020

Cancers

Self Cite

View full text Add to dashboard Cite

Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo.

show abstract

IgE re-programs alternatively-activated human macrophages towards pro-inflammatory anti-tumoural states

et al. 2019

Self Cite

View full text Add to dashboard Cite

Background Antibody Fc-driven engagement of macrophages is critical for evoking cellular activation and effector functions and influencing tumour-associated macrophage (TAM) recruitment. We previously reported that IgE class antibodies promote restriction of cancer growth in rodent models associated with significant TAM infiltration. However, the human macrophage-associated IgE-Fc Receptor (FcεR) axis remains unexplored. We investigated the effects of anti-tumour IgE stimulation on human macrophage activation. Methods Human blood monocyte-differentiated quiescent (M0), classically-(M1) and alternatively-(M2) activated macrophages were crosslinked with IgE and polyclonal antibodies to mimic immune complex formation. We examined surface marker expression, cytokine secretion, protein kinase phosphorylation and gene expression in IgE-stimulated macrophages and IgE antibody-dependent macrophage-mediated cytotoxicity (ADCC) against tumour cells. Findings A proportion (40%) of M2 and (<20%) M0 and M1 macrophages expressed the high-affinity IgE receptor FcεRI. IgE crosslinking triggered upregulation of co-stimulatory CD80, increased TNFα, IFNγ, IL-1β, IL-12, IL-10, IL-13, CXCL9, CXCL11 and RANTES secretion by M0 and M2 and additionally enhanced MCP-1 by M2 macrophages. IgE-stimulated M1 macrophages retained secretion of pro-inflammatory cytokines. IgE crosslinking enhanced the FcεRI-dependent signalling pathway, including phosphorylation of the Lyn kinase, ERK1/2 and p38 in M2 macrophages and upregulated Lyn gene expression by M1 and M2 macrophages. Anti-tumour IgE engendered ADCC of cancer cells by all macrophage subsets. Interpretation IgE can engage and re-educate alternatively-activated macrophages towards pro-inflammatory phenotypes and prime all subsets to mediate anti-tumour functions. This points to IgE-mediated cascades with potential to activate immune stroma and may be significant in the clinical development of strategies targeting tumour-resident macrophages.

show abstract

Engineering and stable production of recombinant IgE for cancer immunotherapy and AllergoOncology

Cited by 19 publications

References 16 publications

In vivo safety profile of a CSPG4-directed IgE antibody in an immunocompetent rat model

In vivo safety profile of a CSPG4-directed IgE antibody in an immunocompetent rat model

A Novel Antibody-Drug Conjugate (ADC) Delivering a DNA Mono-Alkylating Payload to Chondroitin Sulfate Proteoglycan (CSPG4)-Expressing Melanoma

IgE re-programs alternatively-activated human macrophages towards pro-inflammatory anti-tumoural states

Contact Info

Product

Resources

About