A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2)

Boshuizen, Ronald S.; Marsden, Catherine J.; Turkstra, J.A.; Rossant, Christine; Slootstra, Jerry W.; Copley, C G; Schwamborn, Klaus

doi:10.4161/mabs.36237

Cited by 22 publications

(10 citation statements)

References 51 publications

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“…Thus, panning of phages against NadA-gd A33−K69 or NadA-cc L121−K158 was used to isolate target specific VHH-phage clones. Similar peptide-based panning strategy has been used to isolate VHH against breast carcinoma proteins–Muc-1 (Rahbarizadeh et al, 2004 ), CD44 (Kavousipour et al, 2018 ), and ScFv against human angiotensin-I (Cobaugh et al, 2008 ) and CXC chemokine receptor-2 (Boshuizen et al, 2014 ). Besides, it was reported that the homogenization of protein with adjuvants may denature some protein molecules before immunization (Friguet et al, 1984 ), and the elicited antibody pool may contain, target as well as off-target (against unfolded or degraded antigens) antibodies.…”

Section: Discussionmentioning

confidence: 99%

Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

Kulkarni

Mochnáčová

Majerová

et al. 2020

Front. Mol. Biosci.

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Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A33 to K69) and the first coiled-coil domain (L121 to K158). Here, the phage display was used to develop a variable heavy chain domain (VHH) that can block receptor binding sites of recombinant NadA (rec-NadA). A phage library displaying VHH was panned against synthetic peptides (NadA-gdA33−K69 or NadA-ccL121−K158), gene encoding VHH was amplified from bound phages and re-cloned in the expression vector, and the soluble VHHs containing disulfide bonds were overexpressed in the SHuffle E. coli. From the repertoire of 96 clones, two VHHs (VHHF3–binding NadA-gdA33−K69 and VHHG9–binding NadA-ccL121−K158) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHHF3 and VHHG9 significantly reduced the adhesion of NM on hBMECs in situ and hindered the traversal of NM across the in-vitro BBB model. The work presents a phage display pipeline with a single-round of panning to select receptor blocking VHHs. It also demonstrates the production of soluble and functional VHHs, which blocked the interaction between NadA and its receptor, decreased adhesion of NM on hBMECs, and reduced translocation of NM across BBB in-vitro. The selected NadA blocking VHHs could be promising molecules for therapeutic translation.

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Section: Discussionmentioning

confidence: 99%

Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

Kulkarni

Mochnáčová

Majerová

et al. 2020

Front. Mol. Biosci.

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“…Treatment with a CXCR2 neutralizing antibody significantly inhibited ELR(+)-CXC-chemokine-induced proliferation, invasion, and tube formation of human umbilical vein endothelial cells (HUVEC) in vitro and reduced tumor volume, Ki-67 proliferation index, and microvessel density in a pancreatic cancer mouse model 319 . A CXCR2 monoclonal antibody discovered using a combination of in vitro techniques specifically inhibited CXCL8 and Gro-α-induced ß-arrestin recruitment in CXCR2 overexpressing cells with IC 50 values of 0.3 and 0.2 nM, respectively 477 . In spite of several successful preclinical studies using CXCR1/2 neutralizing antibodies in the treatment of cancer and COPD, to date none of the antibodies have been advanced to clinical trials for these diseases.…”

Section: Antibody Therapymentioning

confidence: 99%

Role of the CXCL8-CXCR1/2 Axis in Cancer and Inflammatory Diseases

Ha¹,

Debnath²,

Neamati³

2017

Theranostics

554

464

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The chemokine receptors CXCR1/2 and their ligand CXCL8 are essential for the activation and trafficking of inflammatory mediators as well as tumor progression and metastasis. The CXCL8-CXCR1/2 signaling axis is involved in the pathogenesis of several diseases including chronic obstructive pulmonary diseases (COPD), asthma, cystic fibrosis and cancer. Interaction between CXCL8 secreted by select cancer cells and CXCR1/2 in the tumor microenvironment is critical for cancer progression and metastasis. The CXCL8-CXCR1/2 axis may play an important role in tumor progression and metastasis by regulating cancer stem cell (CSC) proliferation and self-renewal. During the past two decades, several small-molecule CXCR1/2 inhibitors, CXCL8 releasing inhibitors, and neutralizing antibodies against CXCL8 and CXCR1/2 have been reported. As single agents, such inhibitors are expected to be efficacious in various inflammatory diseases. Several preclinical studies suggest that combination of CXCR1/2 inhibitors along with other targeted therapies, chemotherapies, and immunotherapy may be effective in treating select cancers. Currently, several of these inhibitors are in advanced clinical trials for COPD, asthma, and metastatic breast cancer. In this review, we provide a comprehensive analysis of the role of the CXCL8-CXCR1/2 axis and select genes co-expressed in this pathway in disease progression. We also discuss the latest progress in developing small-molecule drugs targeting this pathway.

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“…We chose CXCR2 to determine whether our cell adjuvant could be applied in multi-pass membrane proteins. CXCR2, a GPCR with 7 transmembrane domains, was identified as a key factor in tumor growth, angiogenesis, and metastasis 2 , making it an attractive target for Ab drug development 15 . We then transiently expressed CXCR2 on the cell surface of 3T3/mGM-CSF to generate 3T3/mGM-CSF/CXCR2 cells.…”

Section: Resultsmentioning

confidence: 99%

Use of syngeneic cells expressing membrane-bound GM-CSF as an adjuvant to induce antibodies against native multi-pass transmembrane protein

Huang

Cheng

Hsieh

et al. 2019

Sci Rep

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Membrane antigens (mAgs) are important targets for the development of antibody (Ab) drugs. However, native mAgs are not easily prepared, causing difficulties in acquiring functional Abs. In this study, we present a platform in which human mAgs were expressed in native form on cell adjuvants made with membrane-bound cytokines that were then used immunize syngeneic mice directly. The membrane-bound cytokines were used as immune stimulators to enhance specific Ab responses against the desired mAgs. Then, mAgs-expressing xenogeneic cells were used for Ab characterization to reduce non-specific binding. We established cell adjuvants by expressing membrane-bound cytokines (mIL-2, mIL-18, or mGM-CSF) on BALB/3T3 cells, which were effective in stimulating splenocyte proliferation in vitro . We then transiently expressed ecotropic viral integration site 2B (EVI2B) on the adjuvants and used them to directly immunize BALB/c mice. We found that 3T3/mGM-CSF cells stimulated higher specific anti-EVI2B Ab response in the immunized mice than the other cell adjuvants. A G-protein coupled receptor (GPCR), CXCR2, was then transiently expressed on 3T3/mGM-CSF cell adjuvant to immunize mice. The immune serum exhibited relatively higher binding to xenogeneic 293 A/CXCR2 cells than 293 A cells (~3.5-fold). Several hybridoma clones also exhibited selective binding to 293 A/CXCR2 cells. Therefore, the cell adjuvant could preserve the native conformation of mAgs and exhibit anti-mAg Ab stimulatory ability, providing a more convenient and effective method to generate functional Abs, thus possibly accelerating Ab drug development.

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A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2)

Cited by 22 publications

References 51 publications

Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

Role of the CXCL8-CXCR1/2 Axis in Cancer and Inflammatory Diseases

Use of syngeneic cells expressing membrane-bound GM-CSF as an adjuvant to induce antibodies against native multi-pass transmembrane protein

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