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

Hoffmann, Ricarda M.; Crescioli, Silvia; Mele, Silvia; Sachouli, Eirini; Cheung, Anthony; Chui, Connie Kin Fun; Andriollo, Paolo; Jackson, Paul J.; Lacy, Katie E.; Spicer, James; Thurston, David E.; Karagiannis, Sophia N.

doi:10.3390/cancers12041029

Cited by 26 publications

(22 citation statements)

References 51 publications

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“…Engineered SF-25 antibodies with human Fc regions recognize human malignant cell lines We cloned the murine variable region sequences of SF-25 into human IgG 1 and IgE antibody scaffolds (figure 1A) using previously established platforms. [10][11][12] High performance liquid chromatography (HPLC) analyses demonstrated high antibody purity and negligible aggregation (<5%) (figure 1B). Production of high-purity intact antibody was demonstrated in different expression systems, culture media, serum content and culture vessel conditions (online supplemental figure S1A, B).…”

Section: Resultsmentioning

confidence: 99%

“…To clone the SF-25 variable region into a human IgG 1 backbone we performed a four-fragment Polymerase Incomplete Primer Extension (PIPE) PCR: two big fragments F2 and F4 (3000-4000 bp) containing at 5' the constant regions of the light and heavy chain, respectively; and two small fragments F1 and F3 (300-400 bp) composed by SF-25 Vk and VH, respectively. [10][11][12] The PCR reactions contained 0.5 µM of each primer, 25 µL of PhusionTM Flash High-Fidelity PCR Master Mix, 10 ng of template DNA and sterile water up to 50 µL. The cycles for amplification were: 10 s at 98°C, 35 cycles of 1 s at 98°C, 5 s at 62°C, and 10 s at 72°C.…”

Section: Cloning and Production Of Recombinant Antibodiesmentioning

confidence: 99%

“…Antibodies were produced in Sp2/0 (IgE) and Expi293F (IgG) mammalian cells and purified using previously described methods (see online supplementalry materials and methods). [11][12][13] SF-25 antigen expression screening Cell lines were detached with Trypsin-EDTA treatment, counted and 2.5×10 5 cells were used per tube. 2 mL FACS buffer [Phosphate Buffer Saline (PBS); 5% Foetal Bovine Serum (FBS); 3 mM Ethylenediaminetetraacetic acid (EDTA)] was added to each tube before a 5 min centrifugation at 400rcf at 4°C.…”

Section: Cloning and Production Of Recombinant Antibodiesmentioning

confidence: 99%

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Immunotherapy using IgE or CAR T cells for cancers expressing the tumor antigen SLC3A2

Pellizzari

Martinez

Crescioli

et al. 2021

J Immunother Cancer

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BackgroundCancer immunotherapy with monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies can benefit from selection of new targets with high levels of tumor specificity and from early assessments of efficacy and safety to derisk potential therapies.MethodsEmploying mass spectrometry, bioinformatics, immuno-mass spectrometry and CRISPR/Cas9 we identified the target of the tumor-specific SF-25 antibody. We engineered IgE and CAR T cell immunotherapies derived from the SF-25 clone and evaluated potential for cancer therapy.ResultsWe identified the target of the SF-25 clone as the tumor-associated antigen SLC3A2, a cell surface protein with key roles in cancer metabolism. We generated IgE monoclonal antibody, and CAR T cell immunotherapies each recognizing SLC3A2. In concordance with preclinical and, more recently, clinical findings with the first-in-class IgE antibody MOv18 (recognizing the tumor-associated antigen Folate Receptor alpha), SF-25 IgE potentiated Fc-mediated effector functions against cancer cells in vitro and restricted human tumor xenograft growth in mice engrafted with human effector cells. The antibody did not trigger basophil activation in cancer patient blood ex vivo, suggesting failure to induce type I hypersensitivity, and supporting safe therapeutic administration. SLC3A2-specific CAR T cells demonstrated cytotoxicity against tumor cells, stimulated interferon-γ and interleukin-2 production in vitro. In vivo SLC3A2-specific CAR T cells significantly increased overall survival and reduced growth of subcutaneous PC3-LN3-luciferase xenografts. No weight loss, manifestations of cytokine release syndrome or graft-versus-host disease, were detected.ConclusionsThese findings identify efficacious and potentially safe tumor-targeting of SLC3A2 with novel immune-activating antibody and genetically modified cell therapies.

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Section: Resultsmentioning

confidence: 99%

Section: Cloning and Production Of Recombinant Antibodiesmentioning

confidence: 99%

Section: Cloning and Production Of Recombinant Antibodiesmentioning

confidence: 99%

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Immunotherapy using IgE or CAR T cells for cancers expressing the tumor antigen SLC3A2

Pellizzari

Martinez

Crescioli

et al. 2021

J Immunother Cancer

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“…Lee et al developed cisplatin-loaded CS-binding liposomes, that effectively inhibited tumor growth in liver metastasis murine model (Lee et al, 2002). Similarly, antibodydrug conjugate targeting CSPG4 triggered melanoma regression in vivo (Hoffmann et al, 2020). Several similar approaches that utilize HSPGs have been proposed for treatment of breast (Khatun et al, 2013) and liver cancers (Longmuir et al, 2009).…”

Section: Proteoglycans In Cancer Therapy and Diagnosismentioning

confidence: 99%

Proteoglycans as Mediators of Cancer Tissue Mechanics

Barkovskaya

Buffone

Žídek

et al. 2020

Front. Cell Dev. Biol.

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Proteoglycans are a diverse group of molecules which are characterized by a central protein backbone that is decorated with a variety of linear sulfated glycosaminoglycan side chains. Proteoglycans contribute significantly to the biochemical and mechanical properties of the interstitial extracellular matrix where they modulate cellular behavior by engaging transmembrane receptors. Proteoglycans also comprise a major component of the cellular glycocalyx to influence transmembrane receptor structure/function and mechanosignaling. Through their ability to initiate biochemical and mechanosignaling in cells, proteoglycans elicit profound effects on proliferation, adhesion and migration. Pathologies including cancer and cardiovascular disease are characterized by perturbed expression of proteoglycans where they compromise cell and tissue behavior by stiffening the extracellular matrix and increasing the bulkiness of the glycocalyx. Increasing evidence indicates that a bulky glycocalyx and proteoglycan-enriched extracellular matrix promote malignant transformation, increase cancer aggression and alter anti-tumor therapy response. In this review, we focus on the contribution of proteoglycans to mechanobiology in the context of normal and transformed tissues. We discuss the significance of proteoglycans for therapy response, and the current experimental strategies that target proteoglycans to sensitize cancer cells to treatment.

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“…Although CSPG4 received much attention as a potential therapeutic agent in recent years (16,17,(26)(27)(28)(29)(30)(31), there is little information concerning the mechanisms that regulate the expression of this proteoglycan (32). To the best of our knowledge, no study has investigated whether CSPG4 expression is altered by inhibiting signaling through the MAPK pathway.…”

Section: Introductionmentioning

confidence: 99%

Expression of chondroitin sulfate proteoglycan 4 (CSPG4) in melanoma cells is downregulated upon inhibition of BRAF

et al. 2021

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Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth-factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF-mutant CSPG4-positive or -negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient-derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4-positive cells as compared to pre-therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug-resistance in melanoma cells.

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

Cited by 26 publications

References 51 publications

Immunotherapy using IgE or CAR T cells for cancers expressing the tumor antigen SLC3A2

Immunotherapy using IgE or CAR T cells for cancers expressing the tumor antigen SLC3A2

Proteoglycans as Mediators of Cancer Tissue Mechanics

Expression of chondroitin sulfate proteoglycan 4 (CSPG4) in melanoma cells is downregulated upon inhibition of BRAF

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