Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Parekh, Bhavin S.; Berger, E.; Sibley, Sharon; Cahya, Suntara; Liqun, Xiao; LaCerte, Melinda Ann; Vaillancourt, Peter; Wooden, Scott K.; Gately, Dennis

doi:10.4161/mabs.19873

Cited by 81 publications

(84 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…ADCC is quantified through NFAT pathway induced luminescence. 15 Assay equivalency was demonstrated using purified human peripheral NK cells in Ramos and KG-1 cells (Supporting Information Methods).…”

Section: Antibody-dependent Cellular Cytotoxicity (Adcc) and Complemementioning

confidence: 99%

Antitumor activity of an anti‐CD98 antibody

et al. 2015

View full text Add to dashboard Cite

CD98 is expressed on several tissue types and specifically upregulated on fast-cycling cells undergoing clonal expansion. Various solid (e.g., nonsmall cell lung carcinoma) as well as hematological malignancies (e.g., acute myeloid leukemia) overexpress CD98. We have identified a CD98-specific mouse monoclonal antibody that exhibits potent preclinical antitumor activity against established lymphoma tumor xenografts. Additionally, the humanized antibody designated IGN523 demonstrated robust tumor growth inhibition in leukemic cell-line derived xenograft models and was as efficacious as standard of care carboplatin in patient-derived nonsmall lung cancer xenografts. In vitro studies revealed that IGN523 elicited strong ADCC activity, induced lysosomal membrane permeabilization and inhibited essential amino acid transport function, ultimately resulting in caspase-3 and -7-mediated apoptosis of tumor cells. IGN523 is currently being evaluated in a Phase I clinical trial for acute myeloid leukemia (NCT02040506). Furthermore, preclinical data support the therapeutic potential of IGN523 in solid tumors.CD98 is a heterodimeric protein that comprises a heavy and light chain. The CD98 heavy chain is a type II transmembrane glycoprotein that forms a heterodimer via covalent linkage to one of 6 amino acid transporters. 1,2 CD98 is overexpressed on the cell surface of almost all tumor cells, regardless of tissue origin and increased expression of a CD98-light chain, L-type amino acid transporter 1 (LAT-1) occurs in many types of human cancers, including breast, colon, oral, ovarian, esophageal, glioma and leukemia. 3,4 Increased uptake of amino acids supports the high growth rate of cancer cells by providing the building blocks for protein synthesis. [4][5][6] Moreover, the higher expression of CD98 heavy chain and LAT-1 in metastatic vs. primary tumors suggests that over-expression of CD98/LAT-1 may facilitate progression and metastasis of human cancers.CD98 also regulates integrin signaling by association with integrin b-subunits, thereby controlling cell proliferation, survival, migration, epithelial adhesion and polarity. 3,7 The function of CD98 in regulating both amino acid transport and integrin signaling can contribute to the rapid proliferation and clonal expansion of lymphocytes and tumor cells. 3 The expression pattern and pleiotropic function of CD98 heavy and light chains suggest these proteins are promising targets for treatment of a variety of human cancers. Although small molecule inhibitors of LAT-1 activity have demonstrated preclinical antitumor activity in a number of cancer cell types, including NSCLC, colon cancer, oral cancer and breast cancer, development of antibodies against CD98 heavy chain has received less focus. 5,[8][9][10] Murine monoclonal antibodies to CD98 inhibit lymphocyte proliferation and the growth of bladder cancer, lymphoma, glioma, prostate and colon cancer cells in preclinical models. [11][12][13] To identify therapeutic anti-CD98 antibodies with significant antitumor activity, we ...

show abstract

Section: Antibody-dependent Cellular Cytotoxicity (Adcc) and Complemementioning

confidence: 99%

Antitumor activity of an anti‐CD98 antibody

et al. 2015

View full text Add to dashboard Cite

show abstract

“…The ADCC is an important biological function attributed to the mechanism of action of several therapeutic antibodies, in particular for oncology targeting mAbs (Parekh et al. 2012). …”

Section: Introductionmentioning

confidence: 99%

Molecular modeling of antibodies for the treatment of TNFα‐related immunological diseases

Pierri

Bossis

Punzi

et al. 2016

Pharmacology Res & Perspec

View full text Add to dashboard Cite

Therapeutic monoclonal antibodies (mAbs) have high efficacy in treating TNF α‐related immunological diseases. Other than neutralizing TNF α, these IgG1 antibodies exert Fc receptor‐mediated effector functions such as the complement‐dependent cytotoxicity (CDC) and antibody‐dependent cell cytotoxicity (ADCC). The crystallizable fragment (Fc) of these IgG1 contains a single glycosylation site at Asn 297/300 that is essential for the CDC and ADCC. Glycosylated antibodies lacking core fucosylation showed an improved ADCC. However, no structural data are available concerning the ligand‐binding interaction of these mAbs used in TNF α‐related diseases and the role of the fucosylation. We therefore used comparative modeling for generating complete 3D mAb models that include the antigen‐binding fragment (Fab) portions of infliximab, complexed with TNF α (4G3Y.pdb), the Fc region of the human IGHG1 fucosylated (3SGJ) and afucosylated (3SGK) complexed with the Fc receptor subtype Fcγ RIIIA, and the Fc region of a murine immunoglobulin (1IGT). After few thousand steps of energy minimization on the resulting 3D mAb models, minimized final models were used to quantify interactions occurring between Fcγ RIIIA and the fucosylated/afucosylated Fc fragments. While fucosylation does not affect Fab‐TNF α interactions, we found that in the absence of fucosylation the Fc–mAb domain and Fcγ RIIIA are closer and new strong interactions are established between G129 of the receptor and S301 of the Chimera 2 Fc mAb; new polar interactions are also established between the Chimera 2 Fc residues Y299, N300, and S301 and the Fcγ RIIIA residues K128, G129, R130, and R155. These data help to explain the reduced ADCC observed in the fucosylated mAbs suggesting the specific AA residues involved in binding interactions.

show abstract

“…28 The ADCC reporter gene assay, which is equivalent to LDH ADCC bioassay in testing ADCC activity, was then used for evaluating the pathway activation by therapeutic antibody in an ADCC mechanism of action. 29 We chose a Jurkat cell line that stably expresses the FcgRIIIa complex and the luciferase reporter gene under the control of the NFAT response elements as the effector cells. A431 cells were used as target cells.…”

Section: Pan Exhibits Superior Adcc Activity Compared With Panitumumabmentioning

confidence: 99%

“…29 A431 cells, which overexpressed EGFR, were seeded at 10,000 cells per well in a 96-well opaque tissue culture plate. Cetuximab, panitumumab or Pan were serially diluted and incubated with the A431 cells for approximately 1 h at 37 C, 5% CO 2 .…”

Section: Adcc-reporter Gene Bioassaymentioning

confidence: 99%

Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency

Yang

Guo

Mao³

et al. 2015

mAbs

View full text Add to dashboard Cite

These authors contributed equally to this work.Keywords: monoclonal antibody, EGFR, panitumumab, ADCC, target-selective activation, Probody TM Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; mAb, monoclonal antibody; EGFR, epidermal growth factor receptor; uPA, urokinase-type plasminogen activator; TKI, tyrosine kinase inhibitor; IgG, Immunoglobulin G; ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; CI, confidence interval; LC, light chain; HC, heavy chain; EGFR VIII, EGFR Type III Variant; CRC, colorectal cancer; ECD, extracellular domain; SEC, size exclusion chromatography; CCK-8, Cell Counting Kit 8YunPanitumumab, as a commercially available antibody, is an effective anticancer therapeutic against epidermal growth factor receptor (EGFR), although it exerts weak antibody-dependent cell-mediated cytotoxicity (ADCC) activity owing to its IgG2 nature. Here, we firstly engineered panitumumab by grafting its variable region into an IgG1 backbone. The engineered panitumumab (denoted as Pan) retained binding activity identical to the parental antibody while exhibiting stronger ADCC activity in vitro and more potent antitumor effect in vivo. To further enhance the target selectivity of Pan, we generated Pan-P by tethering an epitope-blocking peptide to Pan via a tumor-specific protease selective linker. Pan-P showed almost 40-fold weaker affinity compared with Pan, but functional activity was restored to a similar extent as Pan when Pan-P was selectively activated by urokinase-type plasminogen activator (uPA). More importantly, targeted localization of Pan-P was observed in tumor samples from colorectal cancer (CRC) patients and tumor-bearing nude mice, strongly indicating that specific activation also existed ex vivo and in vivo. Furthermore, Pan-P also exhibited effective in vivo antitumor potency similar to Pan. Taken together, our data evidence the enhanced antitumor potency and excellent target selectivity of Pan-P, suggesting its potential use for minimizing on-target toxicity in anti-EGFR therapy.

show abstract

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Cited by 81 publications

References 51 publications

Antitumor activity of an anti‐CD98 antibody

Antitumor activity of an anti‐CD98 antibody

Molecular modeling of antibodies for the treatment of TNFα‐related immunological diseases

Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency

Contact Info

Product

Resources

About