{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T12:45:00Z","timestamp":1763037900876,"version":"build-2065373602"},"reference-count":72,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2023,6,27]],"date-time":"2023-06-27T00:00:00Z","timestamp":1687824000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100013348","name":"Innosuisse","doi-asserted-by":"publisher","award":["40B1-0_207060","207060"],"award-info":[{"award-number":["40B1-0_207060","207060"]}],"id":[{"id":"10.13039\/501100013348","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001711","name":"the Swiss National Science Fund (SNF)","doi-asserted-by":"publisher","award":["40B1-0_207060","207060"],"award-info":[{"award-number":["40B1-0_207060","207060"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"publisher"}]},{"name":"ETH Zurich","award":["40B1-0_207060","207060"],"award-info":[{"award-number":["40B1-0_207060","207060"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule that supports T cell activation, leukocyte migration, and (lymph)angiogenesis and has been shown to contribute to the pathology of various immune-mediated disorders, including asthma and corneal graft rejection. In contrast to monoclonal antibodies (mAbs) targeting ALCAM\u2019s T cell expressed binding partner CD6, no ALCAM-targeting mAbs have thus far entered clinical development. This is likely linked with the broad expression of ALCAM on many different cell types, which increases the risk of eliciting unwanted treatment-induced side effects upon systemic mAb application. Targeting ALCAM in surface-exposed tissues, such as the lungs or the cornea, by a topical application could circumvent this issue. Here, we report the development of various stability- and affinity-improved anti-ALCAM mAb fragments with cross-species reactivity towards mouse, rat, monkey, and human ALCAM. Fragments generated in either mono- or bivalent formats potently blocked ALCAM\u2013CD6 interactions in a competition ELISA, but only bivalent fragments efficiently inhibited ALCAM\u2013ALCAM interactions in a leukocyte transmigration assay. The different fragments displayed a clear size-dependence in their ability to penetrate the human corneal epithelium. Furthermore, intranasal delivery of anti-ALCAM fragments reduced leukocyte infiltration in a mouse model of asthma, confirming ALCAM as a target for topical application in the lungs.<\/jats:p>","DOI":"10.3390\/pharmaceutics15071841","type":"journal-article","created":{"date-parts":[[2023,6,28]],"date-time":"2023-06-28T00:33:07Z","timestamp":1687912387000},"page":"1841","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Optimization and Characterization of Novel ALCAM-Targeting Antibody Fragments for Transepithelial Delivery"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-7786-9966","authenticated-orcid":false,"given":"Aline","family":"Bauer","sequence":"first","affiliation":[{"name":"Institute of Pharmaceutical Sciences, ETH Zurich, 1-5\/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3718-4873","authenticated-orcid":false,"given":"Sven","family":"Klassa","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Sciences, ETH Zurich, 1-5\/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-0725-0049","authenticated-orcid":false,"given":"Anja","family":"Herbst","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Sciences, ETH Zurich, 1-5\/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cristina","family":"Maccioni","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Sciences, ETH Zurich, 1-5\/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"William","family":"Abhamon","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Sciences, ETH Zurich, 1-5\/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noria","family":"Segueni","sequence":"additional","affiliation":[{"name":"Artimmune SAS, 13 Avenue Buffon, 45100 Orleans, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yulia","family":"Kaluzhny","sequence":"additional","affiliation":[{"name":"MatTek Corporation, 200 Homer Avenue, Ashland, MA 01721, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4403-2595","authenticated-orcid":false,"given":"Morgan","family":"Hunter","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Sciences, ETH Zurich, 1-5\/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7899-2850","authenticated-orcid":false,"given":"Cornelia","family":"Halin","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Sciences, ETH Zurich, 1-5\/10 Vladimir-Prelog-Weg, 8093 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,6,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"25783","DOI":"10.1074\/jbc.M011272200","article-title":"Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)-ALCAM interaction","volume":"276","author":"Nelissen","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"55315","DOI":"10.1074\/jbc.M407776200","article-title":"Molecular isolation and characterization of a soluble isoform of activated leukocyte cell adhesion molecule that modulates endothelial cell function","volume":"279","author":"Ikeda","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3212","DOI":"10.1182\/blood-2005-09-3881","article-title":"Long-term engagement of CD6 and ALCAM is essential for T-cell proliferation induced by dendritic cells","volume":"107","author":"Zimmerman","year":"2006","journal-title":"Blood"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"978","DOI":"10.1096\/fj.12-217844","article-title":"Novel role for ALCAM in lymphatic network formation and function","volume":"27","author":"Iolyeva","year":"2013","journal-title":"FASEB J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2558","DOI":"10.1016\/j.ajpath.2017.07.008","article-title":"Stromal Expression of Activated Leukocyte Cell Adhesion Molecule Promotes Lung Tumor Growth and Metastasis","volume":"187","author":"Willrodt","year":"2017","journal-title":"Am. J. Pathol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2134","DOI":"10.1182\/blood.V98.7.2134","article-title":"ALCAM (CD166): Its role in hematopoietic and endothelial development","volume":"98","author":"Ohneda","year":"2001","journal-title":"Blood"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"759","DOI":"10.3389\/fimmu.2019.00759","article-title":"ALCAM Mediates DC Migration through Afferent Lymphatics and Promotes Allospecific Immune Reactions","volume":"10","author":"Willrodt","year":"2019","journal-title":"Front. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2637","DOI":"10.1016\/j.febslet.2006.04.013","article-title":"Activated leukocyte cell adhesion molecule is a component of the endothelial junction involved in transendothelial monocyte migration","volume":"580","author":"Masedunskas","year":"2006","journal-title":"FEBS Lett."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2894","DOI":"10.1177\/0271678X16678639","article-title":"ALCAM (CD166) is involved in extravasation of monocytes rather than T cells across the blood-brain barrier","volume":"37","author":"Lyck","year":"2017","journal-title":"J. Cereb. Blood Flow. Metab."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"994","DOI":"10.1164\/rccm.201703-0532OC","article-title":"Activated Leukocyte Cell Adhesion Molecule Stimulates the T-Cell Response in Allergic Asthma","volume":"197","author":"Kim","year":"2018","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2687","DOI":"10.1073\/pnas.1615253114","article-title":"CD6 as a potential target for treating multiple sclerosis","volume":"114","author":"Li","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"898","DOI":"10.1038\/cmi.2017.119","article-title":"Genetic and experimental evidence for the involvement of the CD6 lymphocyte receptor in psoriasis","volume":"15","author":"Julia","year":"2018","journal-title":"Cell. Mol. Immunol."},{"doi-asserted-by":"crossref","unstructured":"Chalmers, S.A., Ayilam Ramachandran, R., Garcia, S.J., Der, E., Herlitz, L., Ampudia, J., Chu, D., Jordan, N., Zhang, T., and Parodis, I. (2022). The CD6\/ALCAM pathway promotes lupus nephritis via T cell-mediated responses. J. Clin. Investig., 132.","key":"ref_13","DOI":"10.1172\/JCI147334"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"677","DOI":"10.4168\/aair.2019.11.5.677","article-title":"Activated Leukocyte Cell Adhesion Molecule Modulates Th2 Immune Response in Atopic Dermatitis","volume":"11","author":"Oh","year":"2019","journal-title":"Allergy Asthma Immunol. Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1111\/cei.13104","article-title":"Activated leucocyte cell adhesion molecule (ALCAM\/CD166) regulates T cell responses in a murine model of food allergy","volume":"192","author":"Kim","year":"2018","journal-title":"Clin. Exp. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1026521","DOI":"10.3389\/fmed.2022.1026521","article-title":"The dual role of CD6 as a therapeutic target in cancer and autoimmune disease","volume":"9","author":"Fox","year":"2022","journal-title":"Front. Med."},{"doi-asserted-by":"crossref","unstructured":"Bughani, U., Saha, A., Kuriakose, A., Nair, R., Sadashivarao, R.B., Venkataraman, R., Patel, S., Deshchougule, A.T., Montero, E., and Pai, H.V. (2017). T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab. PLoS ONE, 12.","key":"ref_17","DOI":"10.1371\/journal.pone.0180088"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"999","DOI":"10.1080\/19420862.2016.1196521","article-title":"Therapeutic monoclonal antibodies for respiratory diseases: Current challenges and perspectives, 31 March\u20131 April 2016, Tours, France","volume":"8","author":"Desoubeaux","year":"2016","journal-title":"MAbs"},{"doi-asserted-by":"crossref","unstructured":"Liang, W., Pan, H.W., Vllasaliu, D., and Lam, J.K.W. (2020). Pulmonary Delivery of Biological Drugs. Pharmaceutics, 12.","key":"ref_19","DOI":"10.3390\/pharmaceutics12111025"},{"doi-asserted-by":"crossref","unstructured":"Loscher, M., Seiz, C., Hurst, J., and Schnichels, S. (2022). Topical Drug Delivery to the Posterior Segment of the Eye. Pharmaceutics, 14.","key":"ref_20","DOI":"10.3390\/pharmaceutics14010134"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1167\/iovs.08-2372","article-title":"Efficient intraocular penetration of topical anti-TNF-alpha single-chain antibody (ESBA105) to anterior and posterior segment without penetration enhancer","volume":"50","author":"Ottiger","year":"2009","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1046\/j.1365-2249.2002.01808.x","article-title":"Penetration of engineered antibody fragments into the eye","volume":"128","author":"Thiel","year":"2002","journal-title":"Clin. Exp. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"8718","DOI":"10.1167\/iovs.11-7871","article-title":"Corneal penetration of topical and subconjunctival bevacizumab","volume":"52","author":"Dastjerdi","year":"2011","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"910","DOI":"10.1038\/sj.eye.6701669","article-title":"Topically applied antibody fragments penetrate into the back of the rabbit eye","volume":"19","author":"Williams","year":"2005","journal-title":"Eye"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1513\/pats.200409-049TA","article-title":"The lungs as a portal of entry for systemic drug delivery","volume":"1","author":"Patton","year":"2004","journal-title":"Proc. Am. Thorac. Soc."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1038\/nrd2153","article-title":"Inhaling medicines: Delivering drugs to the body through the lungs","volume":"6","author":"Patton","year":"2007","journal-title":"Nat. Rev. Drug. Discov."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.jconrel.2014.05.021","article-title":"PEGylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract","volume":"187","author":"Koussoroplis","year":"2014","journal-title":"J. Control. Release"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"109006","DOI":"10.1016\/j.exer.2022.109006","article-title":"Topical ocular drug delivery systems: Innovations for an unmet need","volume":"218","author":"Yang","year":"2022","journal-title":"Exp. Eye Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1625","DOI":"10.1007\/s13346-021-00967-w","article-title":"Inhaled antibodies: Formulations require specific development to overcome instability due to nebulization","volume":"11","author":"Mayor","year":"2021","journal-title":"Drug. Deliv. Transl. Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1515","DOI":"10.1242\/jcs.02280","article-title":"Internalization and recycling of ALCAM\/CD166 detected by a fully human single-chain recombinant antibody","volume":"118","author":"Piazza","year":"2005","journal-title":"J. Cell Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.jprot.2014.01.022","article-title":"A comprehensive surface proteome analysis of myeloid leukemia cell lines for therapeutic antibody development","volume":"99","author":"Strassberger","year":"2014","journal-title":"J. Proteom."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1007\/978-1-4939-2272-7_12","article-title":"Site-specific biotinylation of purified proteins using BirA","volume":"1266","author":"Fairhead","year":"2015","journal-title":"Methods Mol. Biol."},{"doi-asserted-by":"crossref","unstructured":"Cousin, N., Bartel, S., Scholl, J., Tacconi, C., Egger, A., Thorhallsdottir, G., Neri, D., Dieterich, L.C., and Detmar, M. (2022). Antibody-Mediated Delivery of VEGF-C Promotes Long-Lasting Lymphatic Expansion That Reduces Recurrent Inflammation. Cells, 12.","key":"ref_33","DOI":"10.3390\/cells12010172"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1126\/science.1948029","article-title":"X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil","volume":"254","author":"Klemm","year":"1991","journal-title":"Science"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1021\/bi981891e","article-title":"Extremely fast folding of a very stable leucine zipper with a strengthened hydrophobic core and lacking electrostatic interactions between helices","volume":"38","author":"Durr","year":"1999","journal-title":"Biochemistry"},{"unstructured":"Tissot, K., Ewert, S., Auf Der Maur, A., Barberis, A., and Escher, D. (2003). Immunoglobulin Frameworks which Demonstrate Enhanced Stability in the Intracellular Environment and Methods of Identifying Same. (WO\/2003\/097697), Internation Patent Number.","key":"ref_36"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.ymeth.2004.04.007","article-title":"Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering","volume":"34","author":"Ewert","year":"2004","journal-title":"Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1093\/nar\/29.1.207","article-title":"IMGT, the international ImMunoGeneTics database","volume":"29","author":"Lefranc","year":"2001","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"4025","DOI":"10.1073\/pnas.1810576116","article-title":"Five computational developability guidelines for therapeutic antibody profiling","volume":"116","author":"Raybould","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2405","DOI":"10.1002\/ijc.23408","article-title":"A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo","volume":"122","author":"Villa","year":"2008","journal-title":"Int. J. Cancer"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1002\/eji.1830240409","article-title":"A directory of human germ-line V kappa segments reveals a strong bias in their usage","volume":"24","author":"Cox","year":"1994","journal-title":"Eur. J. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1016\/0022-2836(92)90223-7","article-title":"The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops","volume":"227","author":"Tomlinson","year":"1992","journal-title":"J. Mol. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"480","DOI":"10.1016\/S0076-6879(00)26071-0","article-title":"Design and use of phage display libraries for the selection of antibodies and enzymes","volume":"326","author":"Viti","year":"2000","journal-title":"Methods Enzymol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/0890-8508(87)90008-9","article-title":"A novel tumour marker related to the c-myc oncogene product","volume":"1","author":"Chan","year":"1987","journal-title":"Mol. Cell. Probes"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1182\/blood-2010-12-326447","article-title":"Tissue inflammation modulates gene expression of lymphatic endothelial cells and dendritic cell migration in a stimulus-dependent manner","volume":"118","author":"Vigl","year":"2011","journal-title":"Blood"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1243","DOI":"10.1038\/ni1139","article-title":"Visualizing dendritic cell networks in vivo","volume":"5","author":"Lindquist","year":"2004","journal-title":"Nat. Immunol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/S0022-1759(98)00204-X","article-title":"An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow","volume":"223","author":"Lutz","year":"1999","journal-title":"J. Immunol. Methods"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2089","DOI":"10.1084\/jem.180.6.2089","article-title":"Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen","volume":"180","author":"Zal","year":"1994","journal-title":"J. Exp. Med."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"21769","DOI":"10.1074\/jbc.273.34.21769","article-title":"Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel","volume":"273","author":"Pini","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2880","DOI":"10.1167\/iovs.18-23944","article-title":"New Human Organotypic Corneal Tissue Model for Ophthalmic Drug Delivery Studies","volume":"59","author":"Kaluzhny","year":"2018","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"107867","DOI":"10.1016\/j.exer.2019.107867","article-title":"Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model","volume":"190","author":"Kaluzhny","year":"2020","journal-title":"Exp. Eye Res."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1016\/j.jcmgh.2016.12.010","article-title":"Cell Adhesion Molecule CD166\/ALCAM Functions Within the Crypt to Orchestrate Murine Intestinal Stem Cell Homeostasis","volume":"3","author":"Smith","year":"2017","journal-title":"Cell. Mol. Gastroenterol. Hepatol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"3437","DOI":"10.1210\/jc.2002-021888","article-title":"Human blastocysts and endometrial epithelial cells express activated leukocyte cell adhesion molecule (ALCAM\/CD166)","volume":"88","author":"Fujiwara","year":"2003","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1563","DOI":"10.1084\/jem.181.4.1563","article-title":"Identification and characterization of a 100-kD ligand for CD6 on human thymic epithelial cells","volume":"181","author":"Patel","year":"1995","journal-title":"J. Exp. Med."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"500","DOI":"10.1111\/j.1742-481X.2011.00823.x","article-title":"Activated leukocyte cell adhesion molecule impacts on clinical wound healing and inhibits HaCaT migration","volume":"8","author":"Sanders","year":"2011","journal-title":"Int. Wound J."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"1927","DOI":"10.3389\/fmicb.2020.01927","article-title":"Solubility, Stability, and Avidity of Recombinant Antibody Fragments Expressed in Microorganisms","volume":"11","author":"Kang","year":"2020","journal-title":"Front. Microbiol."},{"unstructured":"Honegger, A. (2008). Handbook of Experimental Pharmacology, Springer.","key":"ref_57"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.biochi.2020.08.019","article-title":"Antibody stability: A key to performance\u2014Analysis, influences and improvement","volume":"177","author":"Ma","year":"2020","journal-title":"Biochimie"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"9030","DOI":"10.1074\/jbc.RA118.002538","article-title":"Linker engineering in anti-TAG-72 antibody fragments optimizes biophysical properties, serum half-life, and high-specificity tumor imaging","volume":"293","author":"Long","year":"2018","journal-title":"J. Biol. Chem."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"989","DOI":"10.1093\/protein\/6.8.989","article-title":"An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability","volume":"6","author":"Whitlow","year":"1993","journal-title":"Protein Eng."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1038\/gt.2017.3","article-title":"Balanced secretion of anti-CEA x anti-CD3 diabody chains using the 2A self-cleaving peptide maximizes diabody assembly and tumor-specific cytotoxicity","volume":"24","author":"Molgaard","year":"2017","journal-title":"Gene Ther."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"e28810","DOI":"10.4161\/onci.28810","article-title":"Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications","volume":"3","author":"Compte","year":"2014","journal-title":"Oncoimmunology"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"874","DOI":"10.1111\/resp.13052","article-title":"Mouse models of severe asthma: Understanding the mechanisms of steroid resistance, tissue remodelling and disease exacerbation","volume":"22","author":"Maltby","year":"2017","journal-title":"Respirology"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"105189","DOI":"10.1016\/j.phrs.2020.105189","article-title":"Why do some asthma patients respond poorly to glucocorticoid therapy?","volume":"160","author":"Henderson","year":"2020","journal-title":"Pharmacol. Res."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"e13901","DOI":"10.1111\/eci.13901","article-title":"Molecular mechanisms of glucocorticoid resistance","volume":"53","author":"Huang","year":"2023","journal-title":"Eur. J. Clin. Investig."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"921967","DOI":"10.3389\/fmed.2022.921967","article-title":"Challenges in severe asthma: Do we need new drugs or new biomarkers?","volume":"9","author":"Adatia","year":"2022","journal-title":"Front. Med."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"1900588","DOI":"10.1183\/13993003.00588-2019","article-title":"Management of severe asthma: A European Respiratory Society\/American Thoracic Society guideline","volume":"55","author":"Holguin","year":"2020","journal-title":"Eur. Respir. J."},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"1394","DOI":"10.1016\/j.jaip.2019.03.022","article-title":"New Targeted Therapies for Uncontrolled Asthma","volume":"7","author":"Corren","year":"2019","journal-title":"J. Allergy Clin. Immunol. Pract."},{"doi-asserted-by":"crossref","unstructured":"Calzetta, L., Aiello, M., Frizzelli, A., Pistocchini, E., Ritondo, B.L., Rogliani, P., and Chetta, A. (2022). Investigational Treatments in Phase I and II Clinical Trials: A Systematic Review in Asthma. Biomedicines, 10.","key":"ref_69","DOI":"10.3390\/biomedicines10092330"},{"doi-asserted-by":"crossref","unstructured":"Fieux, M., Le Quellec, S., Bartier, S., Coste, A., Louis, B., Giroudon, C., Nourredine, M., and Bequignon, E. (2021). FcRn as a Transporter for Nasal Delivery of Biologics: A Systematic Review. Int. J. Mol. Sci., 22.","key":"ref_70","DOI":"10.3390\/ijms22126475"},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1016\/j.jconrel.2014.10.003","article-title":"Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system","volume":"196","author":"Guilleminault","year":"2014","journal-title":"J. Control. Release"},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/bs.pmch.2022.04.001","article-title":"Advances in the design of new types of inhaled medicines","volume":"61","author":"Czechtizky","year":"2022","journal-title":"Prog. Med. 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