{"paper_id":"24df95a2-30a4-4b88-a21c-e4d798a21040","body_text":"Humanized anti-CD11d monoclonal antibodies suitable for basic research and therapeutic applications | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Humanized anti-CD11d monoclonal antibodies suitable for basic research and therapeutic applications Eoin N. Blythe, Christy Barreira, Corby Fink, Arthur Brown, Lynne C. Weaver, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4764783/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Immunomodulatory agents targeting the CD11d/CD18 integrin are in development for the treatment of several pathophysiologies including neurotrauma, sepsis, and atherosclerosis. Previous rodent models have successfully improved neurotrauma recovery using murine anti-CD11d therapeutic antibodies. Here, we present the progression of anti-CD11d therapy with the development of humanized anti-CD11d monoclonal antibodies. Flow cytometric analysis demonstrated that the humanized anti-CD11d-2 clone binds both human monocytes and neutrophils. Using a THP-1 model, the humanized anti-CD11d-2 clone was then determined to bind both active and inactive CD11d/CD18 conformations without inducing inflammatory cell signaling. Finally, an investigation into the impact of CK2 phosphorylation on CD11d/CD18 expression found that CK2 inhibition downregulated all β2 integrins. By developing humanized anti-CD11d monoclonal antibodies, new tools are now available to study CD11d/CD18 physiology. The subsequent characterization of these humanized anti-CD11d antibodies makes their use in therapeutic interventions possible. Biological sciences/Immunology/Immunotherapy Biological sciences/Immunology/Neuroimmunology Biological sciences/Immunology Biological sciences/Immunology/Inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Integrins are adhesion molecules involved in the recruitment and retention of leukocytes during an inflammatory response. CD11d/CD18 is a β2 integrin that promotes extravasation of leukocytes via binding to human VCAM-1 and ICAM-3, while also contributing to tissue migration via binding to an array of extracellular matrix ligands 1 . Developing novel immunomodulatory therapeutics against CD11d/CD18 is a subject of current interest. Our laboratory has pursued anti-CD11d therapeutic antibodies 2 – 5 while other laboratories have pursued small peptide inhibitors 6 . The common goal of CD11d-targeted therapeutic agents is to disrupt the accumulation of pro-inflammatory leukocytes within target tissues 1 . A perceived benefit of CD11d-targeted therapies is the low and limited expression of CD11d/CD18 that is subsequently increased during select pathophysiologies 1 . Damaging accumulation of neutrophils and monocytes is linked to the upregulation of CD11d/CD18 on these leukocytes during the acute stages of neurotrauma 7 and respiratory distress syndrome 8 , 9 . Chronic accumulation of pro-inflammatory macrophages has been associated with increased levels of CD11d in atherosclerosis 10 , obesity 11 , and non-alcoholic steatohepatitis 12 , 13 . Past work has detailed the genetic regulation of the CD11d gene 14 – 17 , but less is known regarding the post-transcriptional regulation and subsequent cell surface expression of the CD11d protein 18 . Historically, integrin-targeted immunotherapies have been limited by their broad impacts which lead to serious side effects. Efalizumab was an early integrin-targeted immunotherapy, that targeted the CD11a/CD18 β2 integrin expressed on all leukocytes. Blockage of CD11a/CD18 with Efalizumab resulted in broad immunosuppression and reactivation of the JC virus 19 . Modern approaches to integrin-targeted immunotherapies have addressed the broad immunosuppression problem by targeting integrins with restricted expression profiles. Vedolizumab was designed against the α4β7 integrin, which is primarily restricted to leukocyte recruitment within the gastro-intestinal tract 20 , 21 . Clinical use of Vedolizumab resulted in effective treatment of inflammatory bowel disease (Crohn’s and Ulcerative Colitis) without prohibitive adverse events 22 , 23 . The regulated expression profile of CD11d likewise provides an avenue to treat pathology with minimal systemic effects. Understanding the potential side effects of CD11d-targeted agents, however, cannot be determined by CD11d-expression profiles alone. Elucidating whether a CD11d-targeted agent binds the active and/or inactive conformations of CD11d/CD18 and whether it may induce or inhibit inflammatory cell signaling are important considerations. Previously, our laboratory has demonstrated that a mouse anti-human CD11d clone (217L) improves behavioral and neurological recovery within rodent neurotrauma models 2 – 5 . In the following report we showcase an array of humanized anti-CD11d clones, that can be used as a new tool in studying CD11d/CD18 mechanics. Amongst the humanized anti-CD11d clones, we identified a potential therapeutic clone (anti-CD11d-2) and characterized its binding dynamics. Finally, we uncovered an unexpected phenomenon of β2 integrin down-regulation following the blockade of CK2 phosphorylation in THP-1 cells. Results Creation of humanized anti-CD11d clones Initial anti-CD11d therapeutic antibodies were obtained from mice and tested as a treatment for acute spinal cord injury (SCI) by modulating the migration of leukocytes into the lesion area. An original murine 217L monoclonal antibody was produced that targeted the ligand-binding α-I domain of human CD11d. The murine 217L clone bound both human and rat CD11d-expressing leukocytes 24 . In rodent trials of SCI, rats treated with 217L anti-CD11d had significantly improved biochemical and behavioral recoveries 2 . A humanized anti-CD11d therapeutic clone (anti-CD11d-1) was created by combining the CDR of the murine 217L clone and the scaffolding of a human IgG4 framework. Additionally, four variants of the murine 217L CDR sequence (anti-CD11d-2, anti-CD11d-3, anti-CD11d-4, anti-CD11d-5) were produced and subsequently combined with the same human IgG4 framework. In total, five humanized IgG4 antibodies targeting human CD11d were produced and verified for specificity 18 , 25 (Fig. 1 A). The performance of the humanized anti-CD11d clones on human blood samples were tested by flow cytometry. The anti-CD11d-2 clone bound human monocytes and neutrophils at the greatest percentage and mean fluorescence intensity (MFI; Fig. 1 B,C). Subsequently, anti-CD11d-2 was used to identify the expression levels of CD11d amongst monocyte subsets. The non-classical CD14 + CD16 + monocytes expressed the greatest CD11d MFI amongst the three defined subsets (Fig. 1 D,E). Performance of humanized anti-CD11d clones in a rat model The humanized anti-CD11d clones maintained their therapeutic function within a rat SCI model. Following an experimental clip compression injury at T4, rats were treated with one of the following monoclonal antibody preparations: human IgG4 isotype control, murine 217L anti-CD11d, and the five humanized anti-CD11d variants (anti-CD11d-1, anti-CD11d-2, anti-CD11d-3, anti-CD11d-4, anti-CD11d-5) (Fig. 2 A). Spinal lesion homogenates were collected and assayed for myeloperoxidase (MPO) activity as a surrogate marker of neutrophil infiltration (Fig. 2 B). MPO activity was significantly reduced in anti-CD11d treated rats when compared to MPO activity in the isotype control-treated rats. Importantly, there was no significant difference in the MPO activity between the original murine 217L treated rats and the five humanized anti-CD11d variants (Fig. 2 B). A locomotor assessment using the BBB open field locomotor assessment 26 was then performed to determine behavioral recovery following SCI. The anti-CD11d-3 clone was chosen for use in the rat locomotor testing because it induced the greatest reduction in rat MPO levels amongst the humanized clones. Spinal cord injury rats that received anti-CD11d-3 treatment had significantly higher BBB open field locomotor scores than the IgG4 isotype control-treated rats (Fig. 2 C). The effects of murine 217L and anti-CD11d-3 were not significantly different and were very similar to our previous SCI reports 2 , 3 (Fig. 2 C). Binding affinity of the humanized anti-CD11d-2 clone The anti-CD11d-2 clone was determined to bind to the greatest percentage of human leukocytes (Fig. 1 B), and thus was chosen for further evaluation of its binding dynamics. Understanding the binding affinity of anti-CD11d-2 to different CD11d/CD18 conformations was of key interest. A THP-1 model was chosen to study the anti-CD11d binding dynamics because past genetic studies have demonstrated that PMA stimulation of THP-1 cells can dramatically increase the expression of CD11d mRNA 17 . Additional CD18 co-expression, however, is required for the transportation of functional CD11d/CD18 to the cell surface 18 . Flow cytometry was used to confirm upregulation of cell surface CD11d/CD18 expression following PMA differentiation of THP-1 cells (Fig. 3 A). The increase in cell surface expression of CD11d/CD18 in PMA differentiated THP-1 Luc2 cells was also verified with immunocytochemistry (Fig. 3 B). Establishing PMA differentiated THP-1 cells as an endogenous CD11d/CD18 model allowed for the characterization of anti-CD11d-2 binding affinity. The B max for anti-CD11d-2 was found to be 85.45 ± 3.13% (Mean ± SEM). The corresponding K d was 3.545x10 − 11 ± 0.872x10 − 11 M (Mean ± SEM) (Fig. 3 C). Treatment with Mn 2+ forces the activate β2 integrin conformation, while EDTA treatment forces the inactive confirmation 27 , 28 . The binding dynamics of anti-CD11d-2 were not significantly different in the presence of Mn 2+ or EDTA (Fig. 3 C). No intracellular signaling detected by the humanized anti-CD11d-2 clone Understanding the signaling potential of a therapeutic antibody is critical in characterizing its full mechanism of action and evaluating its potential for inducing toxic inflammatory responses. We investigated the ability of anti-CD11d-2 to induce pro-inflammatory signaling by evaluating NF-κB expression in a THP-1 model system. First, both undifferentiated and differentiated THP-1 Luc2 cells activated a robust NF-κB response following LPS treatment. Next, PMA differentiated THP-1 Luc2 cells induced NF-κB expression in response to VCAM-1 binding – a native CD11d ligand. Undifferentiated THP-1 cells did not respond to VCAM-1 (Fig. 4 A). Using VCAM-1 as a positive control, plates were then coated with anti-CD11d-2 or IgG4 isotype control antibodies. Differentiated Luc-2 THP-1 cells were subsequently added to the plates and NF-κB expression was quantified over a 24-hour period. No significant differences were found in peak NF-κB expression between all concentrations (1, 3, 5 and 10 ug/ml) of anti-CD11d-2, IgG4 isotype control, or empty untreated wells (Fig. 4 B). Outside-in integrin signaling via tyrosine phosphorylation was not observed following binding of anti-CD11d-2 to CD11d/CD18. Well-described β2 integrin signaling consists of substantial tyrosine phosphorylation, including key signal transduction by FAK following ligand binding 29 , 30 . A confluent layer of adherent PMA differentiated THP-1 cells was stimulated with soluble anti-CD11d-2 or IgG4 isotype control antibody for 1 hour. Western blot analysis then quantified general tyrosine phosphorylation and FAK phosphorylation at Tyr 397 . No significant difference in tyrosine phosphorylation between anti-CD11d-2, IgG4 isotype control, or untreated wells were observed (Fig. 4 C). Inhibition of CK2 phosphorylation modulates β2 integrin expression The unique regulation of CD11d was key to the reasoning of targeting CD11d/CD18 for therapeutic benefit. A potential CD11d CK2 phosphorylation site – unique within the set of known β2 integrins – is located on the terminal cytoplasmic tail 1 . We investigated the ability of the prospective CD11d CK2 site to contribute to the unique CD11d expression profile. Using anti-CD11d-2 as a CD11d/CD18 detection reagent, we quantified β2 integrin expression in a THP-1 model following PMA differentiation and CK2 inhibition. PMA differentiation upregulated β2 integrin expression and caused the THP-1 cells to shift from CD11a dominance to CD11b-d dominance (Fig. 5 ). Next, CK2 phosphorylation in THP-1 cells was inhibited with a CGS-CK2-1 inhibitor 31 and verified by Western blot analysis (Supplementary Fig. 1A). Unexpectedly, inhibiting CK2 phosphorylation during PMA differentiation downregulated general β2 integrin expression, but maintained the switch to CD11b-d dominance. A surface staining experiment was repeated with CK2 inhibition post-PMA differentiation and the results were analogous (data not shown). Undifferentiated cells were unaffected by CK2 inhibition (Fig. 5 ). The observed changes in β2 expression were detected by combined cell surface and intracellular staining as determined by flow cytometry. Total cell β2 integrin detection was decreased, indicating that protein expression was modulated, not integrin localization. CD18 expression was downregulated to levels observed in undifferentiated cells (Fig. 5 ). Interestingly, a significant difference between total and surface level expression of CD11d persisted with CK2 inhibition (Fig. 5 ). Discussion The humanized anti-CD11d monoclonal antibodies build on over two decades of in vivo research detailing murine clones improving neurotrauma recovery in rodent models 2 – 5 . In a rat model, the humanized anti-CD11d clones were able to maintain their therapeutic benefit and improve recovery from SCI. Thus, at least from the perspective of pre-clinical animal data, the humanized recombinant CD11d monoclonal antibodies function in vivo as expected in comparison to our previously published data using murine monoclonal antibodies 2 , 32 . Anti-CD11d-2 bound human CD11d/CD18 at the greatest percentage and MFI; therefore, anti-CD11d-2 was selected for further characterization of human CD11d binding dynamics. Performing additional binding dynamics in the presence of Mn 2+ or EDTA provides evidence that anti-CD11d-2 binds CD11d regardless of integrin conformation (Fig. 3 C). Promiscuous conformation binding allows for anti-CD11d-2 to bind inactive and active CD11d/CD18 on peripheral leukocytes and active CD11d/CD18 on tissue recruited leukocytes. Differences in conformational binding activity may explain the observed differences in human peripheral leukocyte binding amongst the anti-CD11d clones and why anti-CD11d-2 bound the greatest percentage of cells (Fig. 1 B). The use of anti-CD11d-2 to detect CD11d/CD18 expression on human peripheral leukocytes reinforces previous knowledge regarding the basal CD11d expression profile. Consistent with the literature 1 , we noted that both peripheral monocytes and neutrophils expressed CD11d/CD18. The level of CD11d/CD18 expression was consistently low across neutrophils and monocytes except for non-classical CD14 + CD16 + monocytes which expressed high levels of CD11d on the cells surface. Non-classical CD14 + CD16 + monocytes were a small proportion of total monocytes, which explains why previous studies found the overall monocyte pool to express low levels of CD11d 8 , 33 . The same previous studies were also divided about the level of CD11d expression on non-classical CD14 + CD16 + monocytes 8,33 . Our data support the conclusion that non-classical CD14 + CD16 + monocytes express relatively greater CD11d levels 33 , instead of lower levels of expression 8 . Resolving the conflicting data permits future investigation into how CD11d/CD18 may influence the unique role and migration patterns of non-classical CD14 + CD16 + monocytes. Determining the signaling potential of a monoclonal antibody-based immunotherapy is critical in understanding its full mechanism of action. A previous study described THP-1 cells releasing IL-8, IL-1β, and MCP-1 when exposed to plates coated with ICAM-3 or murine anti-CD11d clones 8 . To our knowledge, however, the mechanistic pathway for a CD11d/CD18 signaling cascade has not been described. In the absence of any literature on a known CD11d/CD18 outside-in signaling pathway, NF-κB expression and tyrosine phosphorylation were selected as broad measures of inflammatory signaling within a THP-1 cell model. Well described β2 integrin signaling cascades involve tyrosine phosphorylation and can induce NF-κB expression 29 , 30 , 34 . Here we demonstrated that only differentiated THP-1 cells can induce NF-κB expression following VCAM-1 binding (Fig. 4 A). Determining the contribution of CD11d/CD18 alone which induces signaling upon binding VCAM-1 is limited by the multitude of integrins which may also bind VCAM-1. Of note, α4β1 (VLA-4) interacts with VCAM-1 to contribute to the induction of NF-κB expression 35 , 36 . Future studies may parse out the individual contributions of CD11d/CD18 interactions towards NF-κB expression and identify a CD11d/CD18 signaling cascade upon binding VCAM-1. In the context of our current study, VCAM-1 served as a positive control for integrin induced NF-κB expression to compare against the humanized anti-CD11d-2 clone. Designed as a blocking IgG4 antibody, anti-CD11d-2 did not induce a significant inflammatory response. The absence of NF-κB expression and FAK outside-in signaling following anti-CD11d-2 binding provides initial evidence that the therapeutic clone is a blocking antibody and does not provoke a broad inflammatory response. CK2 inhibitors have long been known to modulate inflammatory responses 37 . Down-regulation of all β2 integrins following CK2 inhibition, however, was an unexpected result (Fig. 5 ). The use of an analogous CK2 inhibitor (CX-4945) in glioblastoma cells was previously shown to downregulate the β1 and α4 genes that form α4β1 and α4β7 integrins 38 . A proposed mechanism of integrin downregulation in glioblastoma cells was the inhibition of NF-κB activation 38 . Remodeling of β2 integrin expression by CK2 inhibition may result in functional changes to myeloid cell localization. A previous study found that CK2 knock-out mice have increased monocyte and neutrophil recruitment when infected with Listeria monocytogenes 39 . Integrin density is key in determining if a leukocyte will favour tissue migration or tissue retention. Adhesive forces are required for cell migration, but excessive adhesive forces induce cell immobility. Mathematic models have described the relationship between adhesive forces and cell mobility 40 , 41 , which are then observed in CD11d and CD11b models 42 – 44 . The partial downregulation of β2 integrins by CK2 inhibition may be sufficient to switch the adhesive forces from favoring cell immobility to cell migration. Future studies may investigate if CK2 inhibition can encourage leukocyte migration amongst pathophysiologies in which excessive CD11b/CD18 and/or CD11d/CD18 expression causes immobilization and harmful leukocyte accumulation. The goal of anti-CD11d therapy is to modulate the waves of leukocytes that extravasate into target tissues through CD11d/CD18 and VCAM-1 interactions. Our laboratory has extensively investigated the validity of acute anti-CD11d therapy in rodent neurotrauma models 2 – 5 . Temporary use of a blocking clone can target and prevent the extravasation of initial pro-inflammatory leukocytes immediately following neurotrauma without affecting the subsequent recruitment of wound healing leukocytes 45 . Looking beyond neurotrauma, CD11d blockade may also have a therapeutic role in acute inflammatory lung pathologies. The isolated deletion of CD11d alone can provide remarkable improvements in lung pathology and overall survival within a murine sepsis model 46 . Going beyond VCAM-1-mediated extravasation, anti-CD11d therapeutics have the potential to modulate tissue retention/migration mediated by CD11d/CD18 and extracellular matrix interactions. A small peptide inhibitor of CD11d interactions reduced macrophage retention in adipose tissue, which is observed in atherosclerosis and diabetes 6 . Indeed, CD11d −/− mice demonstrate a reduced disease burden in a murine atherosclerosis model 10 . Multiple avenues of investigation, therefore, exist to apply a temporary CD11d blockade with humanized anti-CD11d monoclonal antibodies and achieve a therapeutic goal. In conclusion, we presented the pharmacodynamics of a humanized anti-CD11d-2 clone and described a surprising pan-β2 integrin downregulation following inhibition of CK2 phosphorylation. The anti-CD11d-2 clone bound both conformations of CD11d/CD18 and did not produce an inflammatory response during in vitro assays. The activity of CK2 phosphorylation was found to have a role in the expression of all β2 integrins. Future studies may use the humanized anti-CD11d clones as a tool to propel CD11d/CD18 research when investigating the therapeutic role of CD11d/CD18 blockade in neurotrauma, sepsis, and atherosclerosis. Methods The studies reported were conducted in a manner consistent with ARRIVE guidelines. CD11d Humanized Monoclonal Antibody Derivation The original 217L mouse anti-human CD11d clone, provided by Eli Lily & Co 24 , was used as the basis for creating humanized monoclonal anti-CD11d antibodies. Complementarity-determining regions (CDR) of the original 217L clone were isolated and four subsequent CDR variants were produced. The five resulting CDR sequences were incorporated into a human IgG4 framework to create the final clones (United States Patent No. US-11873340-B2 January 16, 2024) 25 . Specificity was verified by flow cytometry using HEK293 cells transfected with expression vectors expressing human CD11d and CD18 in the absence of any other β2 integrin. The purified humanized CD11d monoclonal antibodies (anti-CD11d-1, anti-CD11d-2, anti-CD11d-3, anti-CD11d-4, anti-CD11d-5) were initially provided by Eli Lily & Co. Subsequently, the humanized CD11d monoclonal antibodies were produced in CHO cells and then purified from culture supernatants under contract with Biologics at the Human Health Therapeutics Research Center, National Research Council Canada, Montreal, Canada. Endotoxin levels were determined to be less than 0.1 EU/mg. Human Leukocyte Collection Acquiring human blood samples for this study was approved (Project ID: 7332) by the University of Western Ontario Health Science Ethics Review Board (HSERB). After obtaining informed consent, human peripheral blood was collected via venipuncture in heparin vacutainer tubes (BD, San Jose, CA, USA) as approved by the HSERB and in accordance with an approved Biosafety-Biohazard Protocol (BIO-RRI-0021) Cell Culture THP-1 (ATCC TIB-202) and THP-1 NF-kB-Luc2 (ATCC TIB-202-NFkB-Luc2) cells were all obtained from American Tissue Culture Collection (ATCC, Cedarlane). All cells were cultured with complete RPMI media supplemented with 10% FBS, 1% L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate, 10 nM HEPES, and 50 µM 2-mercaptoethanol. Additionally, 1 µg/mL of puromycin was added to the completed RPMI selection media for the THP-1 NF-kB-Luc2 cell line. All tissue culture media, plastic ware, and supplements were acquired from ThermoFisher Scientific. Cultures were grown in standing T75 flasks at 37°C and 5% CO 2 in a humidified incubator and cell density did not exceed 1x10 6 cells/mL. THP-1 cells were seeded at 1x10 6 cells/well into 6-well plates, 2x10 5 cells/well into 12-well plates, or 6x10 4 cells/well into 96-well plates and differentiated with 100nM of phorbol myristic acetate (PMA; Millipore Sigma), for up to 72 h. Flow Cytometry Rat and human primary whole blood leukocytes were stained for flow cytometry as previously described 47 . THP-1 cells were either differentiated or cultured as previously described. A 15-min incubation at 37°C and 5% CO 2 with 1 mL Versene (ThermoFisher Scientific) was used to collect adherent cells following differentiation. To block promiscuous antibody binding, THP-1 cells were resuspended in 200 µl HBSS + 0.1% BSA plus 5 µl normal goat serum (NGS) (Jackson ImmunoResearch) and 5 µl AB human serum (ThermoFisher Scientific) on ice for 20 min. Cells were then surface-stained in FACS tubes (Corning) with a combination of LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific), anti-CD18 (TS1/18) FITC (BioLegend), anti-CD11d (anti-CD11d-2) Alexa 647, anti-IgG4 Alexa 647 (Eli Lily & Co), anti-CD11a (TS2/4) PerCP (BioLegend), anti-CD11b (ICRF44) Violet 421 (BioLegend), and/or anti-CD11c (3.9) PE-Cy7 (BioLegend). Intracellular staining was performed in a 96-well U-bottom plate (Corning) after performing fixable vital dye and/or cell surface staining. Cells were washed in staining buffer (HBSS + 0.1% BSA) and then fixed and permeabilized using manufacturer’s instructions (Foxp3/Transcription Factor Staining Buffer Set, eBioscience, ThermoFisher Scientific). The cells were then spun down, and each well was resuspended in a 50 µL permeabilization buffer containing BioLegend TruStain human FcX block and incubated for 5–10 min at room temperature. Without washing, cells were stained with all intracellular antibodies or isotype control antibodies for 20 min at room temperature. This was followed by two washes in 200 µL of permeabilization buffer and two washes in 200 µL of HBSS + 0.1% BSA. Cells were then resuspended in HBSS + 0.1% BSA followed by an appropriate amount of 4% paraformaldehyde (PFA) (BioShop) to allow for a final concentration of 1% PFA in each well. For saturated antibody binding curves to determine antibody affinity, blocking and surface staining occurred live in HBSS, HBSS + 1 mM EDTA or HBSS + 1 mM Mn 2+ and flow cytometry was performed on cells that were subsequently fixed. For CK2 inhibition, the SGC-CK2-1 inhibitor was a gift from Dr. David Litchfield (Western University, Canada) and can be found commercially at MedKoo Biosciences. THP-1 cells were treated with or without 5 µM of SGC-CK2-1 in the presence or absence of 100 nM PMA for 48 h. Cells were fixed and stained as described above. Flow cytometry graphs of a 48 h DMSO control treatment can be found in Supplemental Fig. 1B. A BD LSR II flow cytometer (BD Biosciences) was used for data acquisition with at least 50 000 total cells recorded per experimental condition. Data were analyzed on FlowJo, version 10.8 (BD Life Sciences) 48 . Gating strategies can be found in Supplementary Fig. 2. Experimental Spinal Cord Injury and anti-CD11d anti-inflammatory treatment All animal experiments were approved by the University of Western Ontario’s Animal Care Committee (AUP no. 2010 − 237) and conducted in accordance with the Canadian Council of Animal Care guidelines, the University Animal Care Committee’s Standard Operating Procedures and in accordance with the University’s Biosafety-Biohazard Protocol (BIO-RRI-0021). Additionally, all animal experiments were conducted in accordance with the Standards for Humane Care and Use of Laboratory Animals as approved by the Office of Laboratory Animal Welfare, Department of Health & Human Services, U.S.A. (Protocol Assurance Identification #A5527-01). Experimental spinal cord injury (SCI) was induced in female 220 gram Wistar rats (Charles River) by clip compression as previously described 49 and randomly assigned to a treatment group. To measure neutrophil infiltration into the spinal lesion using a myeloperoxidase assay, rats were given anti-CD11d clones at 2 h post-SCI for the 24 h assay time point, or at 2, 24 and 48h post-SCI for the 72h assay time point, as previously described 49 . For rats undergoing open field locomotor assessment, anti-CD11d clones were administered intravenously via the tail vein at 2, 24 and 48h post-SCI. All animals were monitored twice daily using a veterinarian approved clinical scoring sheet that monitored level of alertness and activity, hydration status (water consumption), percent weight loss, appearance of surgical wound, evidence of pain (piloerection, hunched back, discoloration around eyes), bowel movements (presence of fecal pellets in cage), bladder fullness, urine (leakage and appearance (cloudy, presence of blood). Bladders were emptied twice per day by gentle manual compressed. Myeloperoxidase assay At the 24h and 72h time points, spinal cord injured rats were euthanized by deep induction of anesthesia with 4% isoflurane followed by exsanguination (cardiac perfusion with cold PBS). Spinal cord tissue was harvested around the lesion and a homogenate was prepared as previously described 2 . A portion of the homogenates were then assayed for myeloperoxidase activity as a surrogate marker for the presence of neutrophils. Complete methods of the myeloperoxidase assay were performed as previously described 49 . Basso, Beattie and Bresnahan (BBB) Rat Open field locomotor assessment Hind limb locomotor assessment was conducted using the BBB locomotor rating scale for open field testing, as previously described 2 , 26 . The BBB assessment was conducted by four individuals experienced in the BBB open field locomotor assessment and who were blinded to the treatment each rat received until the end of the assessment. Immunocytochemistry THP-1 NF-kB-Luc2 cells were seeded onto circular cover glasses placed at the bottom of 12-well plates and differentiated with RPMI selection media containing 100 nM of PMA for 48 h at 37°C and 5% CO 2 . RPMI selection media was removed, and cells were washed once with HBSS + 0.1% BSA. In 300 µl of HBSS + 0.1%, cells were blocked with 15 µl of FcX TruStain block (BioLegend) for 20 min at 10°C. The remainder of the staining procedure occurred at room temperature in the dark. In 500 µL of fresh HBSS, anti-CD11d (anti-CD11d-2) Alexa-647 and anti-CD18 (TS1/18) FITC were applied for 20 min. Cells were washed once with HBSS and fixed in 4% PFA for 20 min. Following three washes with 1mL of HBSS, cells were permeabilized with 0.1% Triton X in HBSS for 15 min. In 300 µl of HBSS, 1:1000 DAPI stain (Millipore Sigma) was applied for 20 min. Finally, cells were washed three times with 1 mL of HBSS and imaging was performed on a Leica DMI6000 microscope (Leica Microsystems) and an EM CCO EvOLVE camera (Teledyne Photometrics). Images were analyzed on the FIJI software platform 50 . Western Blot THP-1 cells were differentiated or cultured in a 6-well plate as previously described in cell culture methods. Next, the culture media was removed, and the cells were washed twice with 1 mL PBS. The cells were incubated in 1mL of EGTA supplemented PBS for 15 min at 37°C and 5% CO 2, to minimize focal adhesion signaling. Cells were washed twice with 1 mL PBS, then stimulated in 1 mL of complete RMPI media at 37°C and 5% CO 2 . Following stimulation, cells were immediately washed in 1 mL of ice-cold PBS and lysed with radioimmunoprecipitation assay (RIPA buffer) containing Halt Protease and Halt Phosphatase inhibitor cocktails (ThermoFisher Scientific). A cell scraper was used to collect cell lysate from adherent differentiated cells and non-adherent undifferentiated cells were collected directly into Eppendorf tubes. Lysates were placed on ice for 15 min, then sonicated twice for 10 s for complete lysis. A Bradford assay determined protein concentrations using detergent compatible protein assay reagents (Bio-Rad Laboratories). The resulting supernatants were immediately stored at -80°C. For gel electrophoresis, lysate samples were mixed in 2X Tris-glycine buffer (BioLegend). Pre-made Tris-glycine gels (10%) (ThermoFisher Scientific) were loaded with 40 µL of sample or 3 µL of BLUeye protein ladder (FroggaBio). The gels were run for 150 min at 125 V, then transferred for 60 min at 100 V onto Immobilon PVDF (Bio-Rad Laboratories). In fresh 5 mL of TBS intercept blocking buffer (Li-Cor), cells were incubated overnight at 4˚C with mouse anti-phosphotyrosine (PY20) (BioLegend) and rabbit FAK protein (3285) (Cell Signaling Technology). Membranes were washed three times with 5 mL TBS + 0.1% Tween 20 (BioShop) for 5 min. In fresh 5 mL of TBS intercept blocking buffer, secondary donkey anti-rabbit 680RD and donkey anti-mouse 800CW (Li-Cor) were incubated at room temperature for 1 h. Membranes were imaged at 700 nm and 800 nm using an Odyssey Fc (Li-Cor). Blots were then stripped and re-probed overnight with rabbit anti-pTyr397 FAK (700255) (ThermoFisher Scientific) and mouse β-actin mouse (A2228) (Sigma Aldrich). Data were analyzed on Image Studio Lite, version 5.2 (Li-Cor). Raw blots can be found in Supplementary Fig. 3. For detection of CK2 phosphorylation, undifferentiated THP-1 cells were treated with 5 µM SGC-CK2-1 inhibitor for 12, 24, or 48 h. Cell lysates were collected, and gel electrophoresis occurred as described above. CK2 phosphorylation was detected by rabbit EIF2S2-pS2 51 (gifted by Dr. David Litchfield, University of Western Ontario, London, ON, Canada) and normalized with mouse β-actin (A2228). Secondary antibodies and imaging occurred as described above. Bioluminescence THP-1 NF-kB-Luc2 cells were seeded at 6x10 4 cells/well of an opaque flat 96-well plate (Corning) in 100 µl of RMPI complete media. Selected plates were differentiated with 100 nM PMA for 48 h, washed once with HBSS, then rested in serum free RPMI complete selection media for 24 h. Next, all the media was removed, replaced with 100 µl of RMPI complete media containing 150 µg/ml D-luciferin (Syd Labs, location), and treated with various conditions. The undifferentiated plates were immediately treated with 150 µg/ml D-luciferin and various conditions. Plates were incubated in a Cytation 5 imager (Agilent Technologies) at 37°C and 5% CO 2 for 24 h and bioluminescence was read every hour for 24 h. Statistical analysis Statistical analyses were performed using GibStat or GraphPad Prism, Version 9. All data were presented as the mean plus/minus the standard error of the mean. Statistical significance was detected at p < 0.05. Biological replicates were denoted by (N) whereas technical replicates were denoted by (n). One-way and two-way ANOVAs were performed with appropriate post hoc tests for multiple comparisons as noted in the figure legends. Declarations Competing Interests There are two patents awarded and one patent pending. Japanese Awarded Patent Number: 7328762Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly United States Awarded Patent Number: US 11,873,340 B2Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly Canadian Pending Patent Number: Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly Specific Aspect of the manuscript covered by the two awarded and one pending patent(s): All three patents address the design of the antibodies used in the manuscript. Eli Lilly and Company did not provide any direct funding to any of the authors of this manuscript. Eli Lilly supplied the antibodies that the authors report on in this manuscript, as an in-kind contribution to the development and functional characterization of the humanized anti-CD11d antibodies. None of the authors of this manuscript have any financial interests of any kind with Eli Lilly. Nor do any of the authors have personal or professional relations with Eli Lilly and Company or members of said company. Thus, the authors declare no competing interests. Additional Information There are two patents awarded and one patent pending. Japanese Awarded Patent Number: 7328762 Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly United States Awarded Patent Number: US 11,873,340 B2 Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly Canadian Pending Patent Number: Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly Specific Aspect of the manuscript covered by the two awarded and one pending patent(s) : All three patents address the design of the antibodies used in the manuscript. Eli Lilly and Company did not provide any direct funding to any of the authors of this manuscript. Eli Lilly supplied the antibodies that the authors report on in this manuscript, as an in-kind contribution to the development and functional characterization of the humanized anti-CD11d antibodies. None of the authors of this manuscript have any financial interests of any kind with Eli Lilly. Nor do any of the authors have personal or professional relations with Eli Lilly and Company or members of said company. Thus, the authors declare no competing interests. Funding The research reported here was supported in part by the U.S. Department of Defense CDRMP SCIRP grant SC090328P, CIHR OPG 363209 and the National Hockey League Players Associate Concussion Challenge Fund. Author Contribution The concept for this paper was that of Gregory Dekaban with contributions from Lynne Weaver and Arthur Brown. The manuscript was written by Eoin N. Blythe and edited by Drs. Gregory Dekaban, Lynne Weaver and Arthur Brown. Christy Barreira also contributed to the editing of the manuscript. Figure 1. The experimental design was devised by Gregory Dekaban and Christy Barreira. Data were acquired and analyzed by Christy Barreira. Figure 2. The experimental design was devised by Lynne Weaver, Arthur Brown and Gregory Dekaban. Figure 2B conducted by Lynne Weaver’s staff. Figure 2C conducted by Gregory Dekaban, Lynne Weaver and Dr. Weaver and Dr. Brown’s staff. Figure 3. The experimental design was devised by Eoin N. Blythe and Gregory Dekaban. Data were acquired and analyzed by Eoin N. Blythe. Figure 4. The experimental design was devised by Eoin N. Blythe and Gregory Dekaban. Data were acquired and analyzed by Eoin N. Blythe. Figure 5. The experimental design was devised by Eoin N. Blythe, Gregory Dekaban and Corby Fink. Data acquisition and analysis were conducted by Corby Fink. Supplemental Figure 1. The experimental design was devised by Eoin N. Blythe and Gregory Dekaban. Data were acquired and analyzed by Eoin N. Blythe. Supplemental Figure 2. The experimental design was devised by Gregory Dekaban and Christy Barreira. Data were acquired and analyzed by Christy Barreira. Supplemental Figure 3. The experimental design was devised by Eoin N. Blythe and Gregory Dekaban. Data were acquired and analyzed by Eoin N. Blythe. Acknowledgement We thank Feng Bao, Todd Hryciw, Nicole Geremia and Kevin Braden from the Robarts Research Institute laboratories of Drs. Lynne C. Weaver, Arthur Brown and Gregory Dekaban, respectively, for conducting important experiments in this report. Data Availability Upon reasonable request, all data can be acquired by contacting the corresponding author. References Blythe, E. N., Weaver, L. C., Brown, A. & Dekaban, G. A. β2 Integrin CD11d/CD18: From Expression to an Emerging Role in Staged Leukocyte Migration. Front Immunol 12 , 4471 (2021). Gris, D. et al. Transient blockade of the CD11d/CD18 integrin reduces secondary damage after spinal cord injury, improving sensory, autonomic, and motor function. J Neurosci 24 , 4043–4051 (2004). Geremia, N. M. et al. CD11d Antibody Treatment Improves Recovery in Spinal Cord-Injured Mice. J Neurotrauma 29 , 539–550 (2012). Utagawa, A. et al. Transient blockage of the CD11d/CD18 integrin reduces contusion volume and macrophage infiltration after traumatic brain injury in rats. Brain Res 1207 , 155–163 (2008). Shultz, S. R., Bao, F., Weaver, L. C., Cain, D. P. & Brown, A. Treatment with an anti-CD11d integrin antibody reduces neuroinflammation and improves outcome in a rat model of repeated concussion. J Neuroinflammation 10 , 26 (2013). Cui, K. et al. Inhibition of integrin αDβ2–mediated macrophage adhesion to end product of docosahexaenoic acid (DHA) oxidation prevents macrophage accumulation during inflammation. J Biol Chem 294 , 14370–14382 (2019). Bao, F. et al. Human spinal cord injury causes specific increases in surface expression of β integrins on leukocytes. J Neurotrauma 28 , 269–280 (2011). Miyazaki, Y. et al. Integrin αDβ2 (CD11d/CD18) is expressed by human circulating and tissue myeloid leukocytes and mediates inflammatory signaling. PLoS ONE 9 , e112770 (2014). Shanley, T. P. et al. Requirements for alpha d in IgG immune complex-induced rat lung injury. J Immunol 160 , 1014–1020 (1998). Aziz, M. H. et al. The Upregulation of Integrin αDβ2 (CD11d/CD18) on Inflammatory Macrophages Promotes Macrophage Retention in Vascular Lesions and Development of Atherosclerosis. J Immunol 198 , 4855–4867 (2017). Thomas, A. P., Dunn, T. N., Oort, P. J., Grino, M. & Adams, S. H. Inflammatory phenotyping identifies CD11d as a gene markedly induced in white adipose tissue in obese rodents and women. J Nutr 141 , 1172–1180 (2011). Shen, Z. et al. Expansion of macrophage and liver sinusoidal endothelial cell subpopulations during non-alcoholic steatohepatitis progression. iScience 26 , 106572 (2023). Koelsch, N. et al. The crosstalking immune cells network creates a collective function beyond the function of each cellular constituent during the progression of hepatocellular carcinoma. Sci Rep 13 , 12630 (2023). Noti, J. D., Johnson, A. K. & Dillon, J. D. Structural and Functional Characterization of the Leukocyte Integrin Gene CD11d: ESSENTIAL ROLE OF Sp1 AND Sp3*. J Biol Chem 275 , 8959–8969 (2000). Noti, J. D. Expression of the myeloid-specific leukocyte integrin gene CD11d during macrophage foam cell differentiation and exposure to lipoproteins. Int J Mol Med 10 , 721–727 (2002). Noti, J. D., Johnson, A. K. & Dillon, J. D. The zinc finger transcription factor transforming growth factor beta-inducible early gene-1 confers myeloid-specific activation of the leukocyte integrin CD11d promoter. J Biol Chem 279 , 26948–26958 (2004). Noti, J. D., Johnson, A. K. & Dillon, J. D. The leukocyte integrin gene CD11d is repressed by gut-enriched Kruppel-like factor 4 in myeloid cells. J Biol Chem 280 , 3449–3457 (2005). McKillop, W. M., Barrett, J. W., Pasternak, S. H., Chan, B. M. C. & Dekaban, G. A. The extracellular domain of CD11d regulates its cell surface expression. Journal of Leukocyte Biology 86 , 851–862 (2009). Schwab, N. et al. Fatal PML associated with efalizumab therapy: insights into integrin αLβ2 in JC virus control. Neurology 78 , 458–467 (2012). DeNucci, C. C., Pagan, A. J., Mitchell, J. S. & Shimizu, Y. CONTROL OF α4β7 INTEGRIN EXPRESSION AND CD4 T CELL HOMING BY THE β1 INTEGRIN SUBUNIT. J Immunol 184 , 2458–2467 (2010). Schneider, I. et al. Expression and function of α4β7 integrin predict the success of vedolizumab treatment in inflammatory bowel disease. Translational Research 253 , 8–15 (2023). Qiu, B., Liang, J.-X. & Li, C. Efficacy and safety of vedolizumab for inflammatory bowel diseases: A systematic review and meta-analysis of randomized controlled trials. Medicine (Baltimore) 101 , e30590 (2022). Ha, C. & Kornbluth, A. Vedolizumab as a Treatment for Crohn’s Disease and Ulcerative Colitis. Gastroenterol Hepatol (N Y) 10 , 793–800 (2014). Grayson, M. H. et al. alphadbeta2 integrin is expressed on human eosinophils and functions as an alternative ligand for vascular cell adhesion molecule 1 (VCAM-1). J Exp Med 188 , 2187–2191 (1998). Dekaban; Gregory et al. Anti-CD11D antibodies and uses thereof. 22 (2024). Basso, D. M., Beattie, M. S. & Bresnahan, J. C. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12 , 1–21 (1995). Ye, F., Kim, C. & Ginsberg, M. H. Reconstruction of integrin activation. Blood 119 , 26–33 (2012). Zhang, K. & Chen, J. The regulation of integrin function by divalent cations. Cell Adh Migr 6 , 20–29 (2012). Hogg, N., Patzak, I. & Willenbrock, F. The insider’s guide to leukocyte integrin signalling and function. Nat Rev Immunol 11 , 416–426 (2011). Chang, Y.-C. et al. Molecular basis for autoinhibition of RIAM regulated by FAK in integrin activation. Proc Natl Acad Sci U S A 116 , 3524–3529 (2019). Menyhart, D. et al. Comparison of CX-4945 and SGC-CK2-1 as inhibitors of CSNK2 using quantitative phosphoproteomics: Triple SILAC in combination with inhibitor-resistant CSNK2. Current Research in Chemical Biology 3 , 100041 (2023). Saville, L. R. et al. A monoclonal antibody to CD11d reduces the inflammatory infiltrate into the injured spinal cord: a potential neuroprotective treatment. J Neuroimmunol 156 , 42–57 (2004). Steppich, B. et al. Selective mobilization of CD14(+)CD16(+) monocytes by exercise. Am J Physiol Cell Physiol 279 , C578-586 (2000). Kim, C. H. et al. Aggregation of beta2 integrins activates human neutrophils through the IkappaB/NF-kappaB pathway. J Leukoc Biol 75 , 286–292 (2004). Mussbacher, M. et al. Cell Type-Specific Roles of NF-κB Linking Inflammation and Thrombosis. Front Immunol 10 , 85 (2019). Kong, D.-H., Kim, Y. K., Kim, M. R., Jang, J. H. & Lee, S. Emerging Roles of Vascular Cell Adhesion Molecule-1 (VCAM-1) in Immunological Disorders and Cancer. Int J Mol Sci 19 , 1057 (2018). Hong, H. & Benveniste, E. N. The Immune Regulatory Role of Protein Kinase CK2 and Its Implications for Treatment of Cancer. Biomedicines 9 , 1932 (2021). Zheng, Y. et al. Targeting Protein Kinase CK2 Suppresses Prosurvival Signaling Pathways and Growth of Glioblastoma. Clinical Cancer Research 19 , 6484–6494 (2013). Larson, S. R. et al. Myeloid Cell CK2 Regulates Inflammation and Resistance to Bacterial Infection. Frontiers in Immunology 11 , (2020). DiMilla, P. A., Barbee, K. & Lauffenburger, D. A. Mathematical model for the effects of adhesion and mechanics on cell migration speed. Biophys J 60 , 15–37 (1991). Palecek, S. P., Loftus, J. C., Ginsberg, M. H., Lauffenburger, D. A. & Horwitz, A. F. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385 , 537–540 (1997). Yakubenko, V. P. et al. The Role of Integrin αDβ2 (CD11d/CD18) in Monocyte/Macrophage Migration. Exp Cell Res 314 , 2569–2578 (2008). Lishko, V. K., Yakubenko, V. P. & Ugarova, T. P. The interplay between integrins alphaMbeta2 and alpha5beta1 during cell migration to fibronectin. Exp Cell Res 283 , 116–126 (2003). Cui, K., Ardell, C. L., Podolnikova, N. P. & Yakubenko, V. P. Distinct Migratory Properties of M1, M2, and Resident Macrophages Are Regulated by αDβ2 and αMβ2 Integrin-Mediated Adhesion. Front Immunol 9 , 2650 (2018). Thawer, S. G. et al. Temporal changes in monocyte and macrophage subsets and microglial macrophages following spinal cord injury in the lys-egfp-ki mouse model. J Neuroimmunol 261 , 7–20 (2013). Koutsogiannaki, S. et al. αDβ2 as a novel target of experimental polymicrobial sepsis. Frontiers in Immunology 13 , (2022). Fink, C. et al. Fluorine-19 Cellular MRI Detection of In Vivo Dendritic Cell Migration and Subsequent Induction of Tumor Antigen-Specific Immunotherapeutic Response. Mol Imaging Biol 22 , 549–561 (2020). FlowJo TM Software Mac, v10.8. Becton, Dickinson and Company. Bao, F., Chen, Y., Dekaban, G. A. & Weaver, L. C. Early anti-inflammatory treatment reduces lipid peroxidation and protein nitration after spinal cord injury in rats. J Neurochem 88 , 1335–1344 (2004). Fiji: an open-source platform for biological-image analysis | Nature Methods. https://www.nature.com/articles/nmeth.2019. Wells, C. I. et al. Development of a potent and selective chemical probe for the pleiotropic kinase CK2. Cell Chem Biol 28 , 546-558.e10 (2021). Additional Declarations Competing interest reported. There are two patents awarded and one patent pending. Japanese Awarded Patent Number: 7328762 Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly United States Awarded Patent Number: US 11,873,340 B2 Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly Canadian Pending Patent Number: Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly Specific Aspect of the manuscript covered by the two awarded and one pending patent(s): All three patents address the design of the antibodies used in the manuscript. Eli Lilly and Company did not provide any direct funding to any of the authors of this manuscript. Eli Lilly supplied the antibodies that the authors report on in this manuscript, as an in-kind contribution to the development and functional characterization of the humanized anti-CD11d antibodies. None of the authors of this manuscript have any financial interests of any kind with Eli Lilly. Nor do any of the authors have personal or professional relations with Eli Lilly and Company or members of said company. Thus, the authors declare no competing interests. Supplementary Files SupplementalFiguresFinal.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4764783\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":341478124,\"identity\":\"9ad85d3a-cfe8-4e7d-8090-eee87f6c53d9\",\"order_by\":0,\"name\":\"Eoin N. Blythe\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Microbiology \\u0026 Immunology, University of Western Ontario\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Eoin\",\"middleName\":\"N.\",\"lastName\":\"Blythe\",\"suffix\":\"\"},{\"id\":341478125,\"identity\":\"717692e1-35c0-4609-833a-fad79b994e48\",\"order_by\":1,\"name\":\"Christy Barreira\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Molecular Medicine Research Laboratories, Robarts Research Institute\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Christy\",\"middleName\":\"\",\"lastName\":\"Barreira\",\"suffix\":\"\"},{\"id\":341478126,\"identity\":\"a88ee5c4-e47d-405c-8dae-0e9f01890dcd\",\"order_by\":2,\"name\":\"Corby Fink\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Microbiology \\u0026 Immunology, University of Western Ontario\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Corby\",\"middleName\":\"\",\"lastName\":\"Fink\",\"suffix\":\"\"},{\"id\":341478127,\"identity\":\"7f508db1-2989-47c6-8c78-c7ee8951f57f\",\"order_by\":3,\"name\":\"Arthur Brown\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Anatomy \\u0026 Cell Biology, University of Western Ontario\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Arthur\",\"middleName\":\"\",\"lastName\":\"Brown\",\"suffix\":\"\"},{\"id\":341478128,\"identity\":\"99c3e56c-a633-4090-80b8-4e1bd6624dda\",\"order_by\":4,\"name\":\"Lynne C. Weaver\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Physiology \\u0026 Pharmacology, University of Western Ontario\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Lynne\",\"middleName\":\"C.\",\"lastName\":\"Weaver\",\"suffix\":\"\"},{\"id\":341478129,\"identity\":\"cc61ee3a-ad46-4561-8f68-7455a2f842a8\",\"order_by\":5,\"name\":\"Gregory A. Dekaban\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYDCCA2wMzEBKjnQtxqRrSWwgWgff8bbEzwU1d9I33Eg+wPCjhggtkmeOHZaecexZ7oYbaQmMPceI0GJwI71BmoftcO6G2zkGzAxsxGi5/7z5N8+/w+kGYC3/iLKF7Zg0b9vhBLAWxjYitEieSUuzntl32HDm/WcJB3v7iNDCd/yY8e2Cb4fl+c4cPvjgxzcitKCAA6RqGAWjYBSMglGAAwAAzhA8hmUx6ngAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Department of Microbiology \\u0026 Immunology, University of Western Ontario\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Gregory\",\"middleName\":\"A.\",\"lastName\":\"Dekaban\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-07-18 19:24:30\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4764783/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4764783/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":62858707,\"identity\":\"38f422bd-0a69-4b24-9ad6-4dcaf32ad236\",\"added_by\":\"auto\",\"created_at\":\"2024-08-20 09:59:44\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2200411,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCreation of humanized anti-CD11d monoclonal antibodies.\\u003c/strong\\u003e (A) Conceptual diagram of the components which were combined to create the humanized anti-CD11d clones. The murine 217L clone CDR sequence was isolated and inserted into a human IgG4 framework. Four variants of the 217L CDR sequence were subsequently made and inserted into the same human IgG4 framework. (B) Percent bound and (C) quantified MFI flow cytometric analysis of anti-CD11d antibodies binding to primary human leukocytes (N=5). (D) Flow cytometry diagram and (E) quantified MFI analysis of CD11d/CD18 expression amongst primary human monocyte subsets as determined by the humanized anti-CD11d-2 clone (N=4). Error bars represent standard error. Significance was calculated by one-way ANOVA and a Tukey’s multiple comparisons test (*p\\u0026lt;0.05), (**p\\u0026lt;0.01), (***p\\u0026lt;0.001), (****p\\u0026lt;0.0001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4764783/v1/3b6236ad9ff9ea64ee2040e9.jpg\"},{\"id\":62858711,\"identity\":\"6f30b145-7a35-4c92-8a0b-cc2b5d00f5c9\",\"added_by\":\"auto\",\"created_at\":\"2024-08-20 09:59:44\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1130860,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eHumanized anti-CD11d clones improve biochemical and behavioural recovery in a rat spinal cord injury model.\\u003c/strong\\u003e (A) Conceptual diagram of spinal cord compression injury and treatment with theraupetic anti-CD11d antibodies. (B) Myeloperoxidase assay of spinal cord lesion homogenates as a surregate for neutrophil infiltration (N=6). Note an uninjured control was only performed for the 24 hr timepoint. Error bars represent standard error. Significance was calculated by one-way ANOVA and a Tukey’s multiple comparisons test (*p\\u0026lt;0.05), (**p\\u0026lt;0.01), (***p\\u0026lt;0.001), (****p\\u0026lt;0.0001). (C) BBB open field locomotor scores in murine 217L (N=4), anti-CD11d-3 treated mice (N=9), and IgG4 isotype control treated mice (N=10). Error bars represent standard error. Two-way ANOVA and Newman Kuhl post-hoc test demonstrated a significant treatment effect (P=0.0029), a significant effect of time (P\\u0026lt;0.0001) and a significant interaction of treatment and time (P=0.0006). The only time that is not significant is at 1 week when a difference is not expected. From week 2 until 10 weeks, they are all significant at either P\\u0026lt;0.01 or P\\u0026lt;0.05. Note there was no data obtained in week 9.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4764783/v1/d0651a3612ec84f974ea2ac8.jpg\"},{\"id\":62858712,\"identity\":\"6361a001-7390-4d8c-a9af-853398a6f8e7\",\"added_by\":\"auto\",\"created_at\":\"2024-08-20 09:59:44\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2565235,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eHumanized anti-CD11d-2 binding dynamics in a THP-1 model.\\u003c/strong\\u003e (A) Flow cytometric analysis gated on live THP-1 cells differentiated with 100 nM PMA for up to 72 h (N=3). (B) Immunohistochemistry of 100 nM PMA differentiated THP-1 Luc2 cells for 72 h, then stained in the presence of human TruStain FcX block. Images are a representation of multiple fields of view (n=5) across several independent repeats (N=3). (C) Binding dynamics of anti-CD11d-2 to endogenous CD11d on 100 nM PMA differentiated THP-1 cells as determined by flow cytometry (N=3). Blocking and cell surface staining occurred live to allow for conformation change in HBSS, HBSS + 1mM EDTA or HBSS + 1mM Mn\\u003csup\\u003e2+\\u003c/sup\\u003e. Cells were subsequently fixed for analysis. The binding curve is presented using a break in the x axis. Error bars represent standard error. Significance was calculated by one-way ANOVA and a Tukey’s multiple comparisons test (*p\\u0026lt;0.05), (**p\\u0026lt;0.01), (****p\\u0026lt;0.0001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4764783/v1/b9c7f4328b13ad387b699fa0.jpg\"},{\"id\":62859778,\"identity\":\"f39114bf-e4c2-434b-b7e9-0246d922c780\",\"added_by\":\"auto\",\"created_at\":\"2024-08-20 10:07:44\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1785295,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAbsence of humanized anti-CD11d-2 inducing pro-inflammatory β2 integrin signaling.\\u003c/strong\\u003e (A) NF-κB expression was detected by a luciferase assay in THP-1 Luc2 cells. Following a 48 h culture in the presence or absence of 100 nM PMA, THP-1 Luc2 cells were collected and blocked with 5% HSA. Blocked THP-1 Luc2 cells were dropped onto untreated wells, LPS (25 ng/ml) containing wells, or VCAM-1 (5 ug/ml) coated wells. The plates were incubated and NF-κB expression was measured in triplet every hour for 24 h (N=3). (B) Luciferase NF-κB assay following anti-CD11d-2 stimulation in 4 h PMA differentiated THP-1 Luc2 cells. Cells blocked by 5% HSA were dropped onto plates coated with VCAM-1 (5 µg/ml) or various concentrations (μg/ml) of antibodies. The plates were incubated and NF-κB expression was measured in triplet every hour for 24 h (N=3). Peak NF-κB expression was calculated as the mean value between 4-8 h post stimulation and normalized to VCAM-1 (N=3). Significance was calculated by one-way ANOVA and a Tukey’s multiple comparisons test (p\\u0026lt;0.05), (****p\\u0026lt;0.0001). (C) Western blot analysis of 72 h 100 nM PMA differentiated THP-1 cells stimulated with soluble anti-CD11d-2 (μg/ml) or IgG4 isotype control (μg/ml). Blots were performed in duplicate and normalized to untreated wells (N=3). The representative images are from the same blot that was probed first for P-Tyr and FAK (Supplemental Fig. 3A), then re-probed for P-FAK and β-actin (Supplemental Fig. 3B). Monochrome cropped singlet rows of the same columns are displayed for each protein target. Error bars represent standard error. Significance was calculated by one-way ANOVA and a Tukey’s multiple comparisons test (p\\u0026lt;0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4764783/v1/0cd5bd281a5867767772e5f0.jpg\"},{\"id\":62858709,\"identity\":\"9c25eda7-4fc0-41f6-a3c6-44240509be8a\",\"added_by\":\"auto\",\"created_at\":\"2024-08-20 09:59:44\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1508135,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eModulation of β2 integrin expression by CK2 inhibition.\\u003c/strong\\u003e Flow cytometric analysis of β2 integrin expression in THP-1 cells treated with combinations of 100nM PMA and CGS-CK2-1 inhibitor (5ug/ml) for 48 h. Surface level and internal level integrin expression was recorded to determine the surface level only (white) and total (grey) β2 integrin expression. Error bars represent standard error, (N=3). Significance between surface and total expression within a treatment group was calculated by a two-way ANOVA and a Tukey’s multiple comparisons (**p\\u0026lt;0.01), (***p\\u0026lt;0.001), (****p\\u0026lt;0.0001). Additional total β2 integrin expression analysis was then performed between treatment groups. Error bars represent standard error, (N=3). Significance in levels of total expression between treatment groups were calculated by one-way ANOVA and a Tukey’s multiple comparisons (**p\\u0026lt;0.01), (***p\\u0026lt;0.001), (****p\\u0026lt;0.0001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4764783/v1/4584c04140630376484748ae.jpg\"},{\"id\":63426685,\"identity\":\"e306760f-328e-4545-85cc-7d3e464edaba\",\"added_by\":\"auto\",\"created_at\":\"2024-08-28 04:00:41\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":10056692,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4764783/v1/6276e614-5516-4a63-b6ba-b47e19b368bf.pdf\"},{\"id\":62858708,\"identity\":\"ca752b3d-92ed-4b97-bd3e-0811b50342e4\",\"added_by\":\"auto\",\"created_at\":\"2024-08-20 09:59:44\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":535448,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementalFiguresFinal.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4764783/v1/9ddf6cb42f0aa486fe4e35e9.pdf\"}],\"financialInterests\":\"Competing interest reported. There are two patents awarded and one patent pending. \\nJapanese Awarded Patent Number: 7328762\\nPatentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; \\nInventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. \\nPatentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. \\nInventors: Barrett Allan, Kristine Kikly \\nUnited States Awarded Patent Number: US 11,873,340 B2\\nPatentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; \\nInventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. \\nPatentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. \\nInventors: Barrett Allan, Kristine Kikly \\nCanadian Pending Patent Number: \\nPatentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; \\nInventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. \\nPatentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. \\nInventors: Barrett Allan, Kristine Kikly \\nSpecific Aspect of the manuscript covered by the two awarded and one pending patent(s): All three patents address the design of the antibodies used in the manuscript. \\nEli Lilly and Company did not provide any direct funding to any of the authors of this manuscript. Eli Lilly supplied the antibodies that the authors report on in this manuscript, as an in-kind contribution to the development and functional characterization of the humanized anti-CD11d antibodies. \\nNone of the authors of this manuscript have any financial interests of any kind with Eli Lilly. Nor do any of the authors have personal or professional relations with Eli Lilly and Company or members of said company. Thus, the authors declare no competing interests.\",\"formattedTitle\":\"Humanized anti-CD11d monoclonal antibodies suitable for basic research and therapeutic applications\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eIntegrins are adhesion molecules involved in the recruitment and retention of leukocytes during an inflammatory response. CD11d/CD18 is a β2 integrin that promotes extravasation of leukocytes via binding to human VCAM-1 and ICAM-3, while also contributing to tissue migration via binding to an array of extracellular matrix ligands\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Developing novel immunomodulatory therapeutics against CD11d/CD18 is a subject of current interest. Our laboratory has pursued anti-CD11d therapeutic antibodies\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR3 CR4\\\" citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e while other laboratories have pursued small peptide inhibitors\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. The common goal of CD11d-targeted therapeutic agents is to disrupt the accumulation of pro-inflammatory leukocytes within target tissues\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eA perceived benefit of CD11d-targeted therapies is the low and limited expression of CD11d/CD18 that is subsequently increased during select pathophysiologies\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Damaging accumulation of neutrophils and monocytes is linked to the upregulation of CD11d/CD18 on these leukocytes during the acute stages of neurotrauma\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e and respiratory distress syndrome\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e. Chronic accumulation of pro-inflammatory macrophages has been associated with increased levels of CD11d in atherosclerosis\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e, obesity\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e, and non-alcoholic steatohepatitis\\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. Past work has detailed the genetic regulation of the CD11d gene\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR15 CR16\\\" citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e, but less is known regarding the post-transcriptional regulation and subsequent cell surface expression of the CD11d protein\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eHistorically, integrin-targeted immunotherapies have been limited by their broad impacts which lead to serious side effects. Efalizumab was an early integrin-targeted immunotherapy, that targeted the CD11a/CD18 β2 integrin expressed on all leukocytes. Blockage of CD11a/CD18 with Efalizumab resulted in broad immunosuppression and reactivation of the JC virus\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. Modern approaches to integrin-targeted immunotherapies have addressed the broad immunosuppression problem by targeting integrins with restricted expression profiles. Vedolizumab was designed against the α4β7 integrin, which is primarily restricted to leukocyte recruitment within the gastro-intestinal tract\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. Clinical use of Vedolizumab resulted in effective treatment of inflammatory bowel disease (Crohn\\u0026rsquo;s and Ulcerative Colitis) without prohibitive adverse events\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. The regulated expression profile of CD11d likewise provides an avenue to treat pathology with minimal systemic effects. Understanding the potential side effects of CD11d-targeted agents, however, cannot be determined by CD11d-expression profiles alone. Elucidating whether a CD11d-targeted agent binds the active and/or inactive conformations of CD11d/CD18 and whether it may induce or inhibit inflammatory cell signaling are important considerations.\\u003c/p\\u003e \\u003cp\\u003ePreviously, our laboratory has demonstrated that a mouse anti-human CD11d clone (217L) improves behavioral and neurological recovery within rodent neurotrauma models\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR3 CR4\\\" citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. In the following report we showcase an array of humanized anti-CD11d clones, that can be used as a new tool in studying CD11d/CD18 mechanics. Amongst the humanized anti-CD11d clones, we identified a potential therapeutic clone (anti-CD11d-2) and characterized its binding dynamics. Finally, we uncovered an unexpected phenomenon of β2 integrin down-regulation following the blockade of CK2 phosphorylation in THP-1 cells.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCreation of humanized anti-CD11d clones\\u003c/h2\\u003e \\u003cp\\u003eInitial anti-CD11d therapeutic antibodies were obtained from mice and tested as a treatment for acute spinal cord injury (SCI) by modulating the migration of leukocytes into the lesion area. An original murine 217L monoclonal antibody was produced that targeted the ligand-binding α-I domain of human CD11d. The murine 217L clone bound both human and rat CD11d-expressing leukocytes\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. In rodent trials of SCI, rats treated with 217L anti-CD11d had significantly improved biochemical and behavioral recoveries\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. A humanized anti-CD11d therapeutic clone (anti-CD11d-1) was created by combining the CDR of the murine 217L clone and the scaffolding of a human IgG4 framework. Additionally, four variants of the murine 217L CDR sequence (anti-CD11d-2, anti-CD11d-3, anti-CD11d-4, anti-CD11d-5) were produced and subsequently combined with the same human IgG4 framework. In total, five humanized IgG4 antibodies targeting human CD11d were produced and verified for specificity\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). The performance of the humanized anti-CD11d clones on human blood samples were tested by flow cytometry. The anti-CD11d-2 clone bound human monocytes and neutrophils at the greatest percentage and mean fluorescence intensity (MFI; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB,C). Subsequently, anti-CD11d-2 was used to identify the expression levels of CD11d amongst monocyte subsets. The non-classical CD14\\u003csup\\u003e+\\u003c/sup\\u003e CD16\\u003csup\\u003e+\\u003c/sup\\u003e monocytes expressed the greatest CD11d MFI amongst the three defined subsets (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD,E).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePerformance of humanized anti-CD11d clones in a rat model\\u003c/h2\\u003e \\u003cp\\u003eThe humanized anti-CD11d clones maintained their therapeutic function within a rat SCI model. Following an experimental clip compression injury at T4, rats were treated with one of the following monoclonal antibody preparations: human IgG4 isotype control, murine 217L anti-CD11d, and the five humanized anti-CD11d variants (anti-CD11d-1, anti-CD11d-2, anti-CD11d-3, anti-CD11d-4, anti-CD11d-5) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). Spinal lesion homogenates were collected and assayed for myeloperoxidase (MPO) activity as a surrogate marker of neutrophil infiltration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). MPO activity was significantly reduced in anti-CD11d treated rats when compared to MPO activity in the isotype control-treated rats. Importantly, there was no significant difference in the MPO activity between the original murine 217L treated rats and the five humanized anti-CD11d variants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). A locomotor assessment using the BBB open field locomotor assessment\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e was then performed to determine behavioral recovery following SCI. The anti-CD11d-3 clone was chosen for use in the rat locomotor testing because it induced the greatest reduction in rat MPO levels amongst the humanized clones. Spinal cord injury rats that received anti-CD11d-3 treatment had significantly higher BBB open field locomotor scores than the IgG4 isotype control-treated rats (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). The effects of murine 217L and anti-CD11d-3 were not significantly different and were very similar to our previous SCI reports\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBinding affinity of the humanized anti-CD11d-2 clone\\u003c/h2\\u003e \\u003cp\\u003eThe anti-CD11d-2 clone was determined to bind to the greatest percentage of human leukocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB), and thus was chosen for further evaluation of its binding dynamics. Understanding the binding affinity of anti-CD11d-2 to different CD11d/CD18 conformations was of key interest. A THP-1 model was chosen to study the anti-CD11d binding dynamics because past genetic studies have demonstrated that PMA stimulation of THP-1 cells can dramatically increase the expression of CD11d mRNA\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e. Additional CD18 co-expression, however, is required for the transportation of functional CD11d/CD18 to the cell surface\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e. Flow cytometry was used to confirm upregulation of cell surface CD11d/CD18 expression following PMA differentiation of THP-1 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). The increase in cell surface expression of CD11d/CD18 in PMA differentiated THP-1 Luc2 cells was also verified with immunocytochemistry (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eEstablishing PMA differentiated THP-1 cells as an endogenous CD11d/CD18 model allowed for the characterization of anti-CD11d-2 binding affinity. The B\\u003csub\\u003emax\\u003c/sub\\u003e for anti-CD11d-2 was found to be 85.45\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.13% (Mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SEM). The corresponding K\\u003csub\\u003ed\\u003c/sub\\u003e was 3.545x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;11\\u003c/sup\\u003e \\u0026plusmn; 0.872x10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;11\\u003c/sup\\u003e M (Mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SEM) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). Treatment with Mn\\u003csup\\u003e2+\\u003c/sup\\u003e forces the activate β2 integrin conformation, while EDTA treatment forces the inactive confirmation\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e. The binding dynamics of anti-CD11d-2 were not significantly different in the presence of Mn\\u003csup\\u003e2+\\u003c/sup\\u003e or EDTA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eNo intracellular signaling detected by the humanized anti-CD11d-2 clone\\u003c/h2\\u003e \\u003cp\\u003eUnderstanding the signaling potential of a therapeutic antibody is critical in characterizing its full mechanism of action and evaluating its potential for inducing toxic inflammatory responses. We investigated the ability of anti-CD11d-2 to induce pro-inflammatory signaling by evaluating NF-κB expression in a THP-1 model system. First, both undifferentiated and differentiated THP-1 Luc2 cells activated a robust NF-κB response following LPS treatment. Next, PMA differentiated THP-1 Luc2 cells induced NF-κB expression in response to VCAM-1 binding \\u0026ndash; a native CD11d ligand. Undifferentiated THP-1 cells did not respond to VCAM-1 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). Using VCAM-1 as a positive control, plates were then coated with anti-CD11d-2 or IgG4 isotype control antibodies. Differentiated Luc-2 THP-1 cells were subsequently added to the plates and NF-κB expression was quantified over a 24-hour period. No significant differences were found in peak NF-κB expression between all concentrations (1, 3, 5 and 10 ug/ml) of anti-CD11d-2, IgG4 isotype control, or empty untreated wells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eOutside-in integrin signaling via tyrosine phosphorylation was not observed following binding of anti-CD11d-2 to CD11d/CD18. Well-described β2 integrin signaling consists of substantial tyrosine phosphorylation, including key signal transduction by FAK following ligand binding\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. A confluent layer of adherent PMA differentiated THP-1 cells was stimulated with soluble anti-CD11d-2 or IgG4 isotype control antibody for 1 hour. Western blot analysis then quantified general tyrosine phosphorylation and FAK phosphorylation at Tyr\\u003csub\\u003e397\\u003c/sub\\u003e. No significant difference in tyrosine phosphorylation between anti-CD11d-2, IgG4 isotype control, or untreated wells were observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eInhibition of CK2 phosphorylation modulates β2 integrin expression\\u003c/h2\\u003e \\u003cp\\u003eThe unique regulation of CD11d was key to the reasoning of targeting CD11d/CD18 for therapeutic benefit. A potential CD11d CK2 phosphorylation site \\u0026ndash; unique within the set of known β2 integrins \\u0026ndash; is located on the terminal cytoplasmic tail\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. We investigated the ability of the prospective CD11d CK2 site to contribute to the unique CD11d expression profile. Using anti-CD11d-2 as a CD11d/CD18 detection reagent, we quantified β2 integrin expression in a THP-1 model following PMA differentiation and CK2 inhibition. PMA differentiation upregulated β2 integrin expression and caused the THP-1 cells to shift from CD11a dominance to CD11b-d dominance (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). Next, CK2 phosphorylation in THP-1 cells was inhibited with a CGS-CK2-1 inhibitor\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e and verified by Western blot analysis (Supplementary Fig.\\u0026nbsp;1A). Unexpectedly, inhibiting CK2 phosphorylation during PMA differentiation downregulated general β2 integrin expression, but maintained the switch to CD11b-d dominance. A surface staining experiment was repeated with CK2 inhibition post-PMA differentiation and the results were analogous (data not shown). Undifferentiated cells were unaffected by CK2 inhibition (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). The observed changes in β2 expression were detected by combined cell surface and intracellular staining as determined by flow cytometry. Total cell β2 integrin detection was decreased, indicating that protein expression was modulated, not integrin localization. CD18 expression was downregulated to levels observed in undifferentiated cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). Interestingly, a significant difference between total and surface level expression of CD11d persisted with CK2 inhibition (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe humanized anti-CD11d monoclonal antibodies build on over two decades of \\u003cem\\u003ein vivo\\u003c/em\\u003e research detailing murine clones improving neurotrauma recovery in rodent models\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR3 CR4\\\" citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. In a rat model, the humanized anti-CD11d clones were able to maintain their therapeutic benefit and improve recovery from SCI. Thus, at least from the perspective of pre-clinical animal data, the humanized recombinant CD11d monoclonal antibodies function \\u003cem\\u003ein vivo\\u003c/em\\u003e as expected in comparison to our previously published data using murine monoclonal antibodies\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eAnti-CD11d-2 bound human CD11d/CD18 at the greatest percentage and MFI; therefore, anti-CD11d-2 was selected for further characterization of human CD11d binding dynamics. Performing additional binding dynamics in the presence of Mn\\u003csup\\u003e2+\\u003c/sup\\u003e or EDTA provides evidence that anti-CD11d-2 binds CD11d regardless of integrin conformation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). Promiscuous conformation binding allows for anti-CD11d-2 to bind inactive and active CD11d/CD18 on peripheral leukocytes and active CD11d/CD18 on tissue recruited leukocytes. Differences in conformational binding activity may explain the observed differences in human peripheral leukocyte binding amongst the anti-CD11d clones and why anti-CD11d-2 bound the greatest percentage of cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003eThe use of anti-CD11d-2 to detect CD11d/CD18 expression on human peripheral leukocytes reinforces previous knowledge regarding the basal CD11d expression profile. Consistent with the literature\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e, we noted that both peripheral monocytes and neutrophils expressed CD11d/CD18. The level of CD11d/CD18 expression was consistently low across neutrophils and monocytes except for non-classical CD14\\u003csup\\u003e+\\u003c/sup\\u003eCD16\\u003csup\\u003e+\\u003c/sup\\u003e monocytes which expressed high levels of CD11d on the cells surface. Non-classical CD14\\u003csup\\u003e+\\u003c/sup\\u003eCD16\\u003csup\\u003e+\\u003c/sup\\u003e monocytes were a small proportion of total monocytes, which explains why previous studies found the overall monocyte pool to express low levels of CD11d\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. The same previous studies were also divided about the level of CD11d expression on non-classical CD14\\u003csup\\u003e+\\u003c/sup\\u003eCD16\\u003csup\\u003e+\\u003c/sup\\u003e monocytes\\u003csup\\u003e8,33\\u003c/sup\\u003e. Our data support the conclusion that non-classical CD14\\u003csup\\u003e+\\u003c/sup\\u003eCD16\\u003csup\\u003e+\\u003c/sup\\u003e monocytes express relatively greater CD11d levels\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e, instead of lower levels of expression\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. Resolving the conflicting data permits future investigation into how CD11d/CD18 may influence the unique role and migration patterns of non-classical CD14\\u003csup\\u003e+\\u003c/sup\\u003eCD16\\u003csup\\u003e+\\u003c/sup\\u003e monocytes.\\u003c/p\\u003e \\u003cp\\u003eDetermining the signaling potential of a monoclonal antibody-based immunotherapy is critical in understanding its full mechanism of action. A previous study described THP-1 cells releasing IL-8, IL-1β, and MCP-1 when exposed to plates coated with ICAM-3 or murine anti-CD11d clones\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. To our knowledge, however, the mechanistic pathway for a CD11d/CD18 signaling cascade has not been described. In the absence of any literature on a known CD11d/CD18 outside-in signaling pathway, NF-κB expression and tyrosine phosphorylation were selected as broad measures of inflammatory signaling within a THP-1 cell model. Well described β2 integrin signaling cascades involve tyrosine phosphorylation and can induce NF-κB expression\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e. Here we demonstrated that only differentiated THP-1 cells can induce NF-κB expression following VCAM-1 binding (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). Determining the contribution of CD11d/CD18 alone which induces signaling upon binding VCAM-1 is limited by the multitude of integrins which may also bind VCAM-1. Of note, α4β1 (VLA-4) interacts with VCAM-1 to contribute to the induction of NF-κB expression\\u003csup\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e. Future studies may parse out the individual contributions of CD11d/CD18 interactions towards NF-κB expression and identify a CD11d/CD18 signaling cascade upon binding VCAM-1. In the context of our current study, VCAM-1 served as a positive control for integrin induced NF-κB expression to compare against the humanized anti-CD11d-2 clone. Designed as a blocking IgG4 antibody, anti-CD11d-2 did not induce a significant inflammatory response. The absence of NF-κB expression and FAK outside-in signaling following anti-CD11d-2 binding provides initial evidence that the therapeutic clone is a blocking antibody and does not provoke a broad inflammatory response.\\u003c/p\\u003e \\u003cp\\u003eCK2 inhibitors have long been known to modulate inflammatory responses\\u003csup\\u003e\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e. Down-regulation of all β2 integrins following CK2 inhibition, however, was an unexpected result (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). The use of an analogous CK2 inhibitor (CX-4945) in glioblastoma cells was previously shown to downregulate the β1 and α4 genes that form α4β1 and α4β7 integrins\\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e. A proposed mechanism of integrin downregulation in glioblastoma cells was the inhibition of NF-κB activation\\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e. Remodeling of β2 integrin expression by CK2 inhibition may result in functional changes to myeloid cell localization. A previous study found that CK2 knock-out mice have increased monocyte and neutrophil recruitment when infected with \\u003cem\\u003eListeria monocytogenes\\u003c/em\\u003e\\u003csup\\u003e\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e. Integrin density is key in determining if a leukocyte will favour tissue migration or tissue retention. Adhesive forces are required for cell migration, but excessive adhesive forces induce cell immobility. Mathematic models have described the relationship between adhesive forces and cell mobility\\u003csup\\u003e\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e, which are then observed in CD11d and CD11b models\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR43\\\" citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e. The partial downregulation of β2 integrins by CK2 inhibition may be sufficient to switch the adhesive forces from favoring cell immobility to cell migration. Future studies may investigate if CK2 inhibition can encourage leukocyte migration amongst pathophysiologies in which excessive CD11b/CD18 and/or CD11d/CD18 expression causes immobilization and harmful leukocyte accumulation.\\u003c/p\\u003e \\u003cp\\u003eThe goal of anti-CD11d therapy is to modulate the waves of leukocytes that extravasate into target tissues through CD11d/CD18 and VCAM-1 interactions. Our laboratory has extensively investigated the validity of acute anti-CD11d therapy in rodent neurotrauma models\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR3 CR4\\\" citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. Temporary use of a blocking clone can target and prevent the extravasation of initial pro-inflammatory leukocytes immediately following neurotrauma without affecting the subsequent recruitment of wound healing leukocytes\\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e. Looking beyond neurotrauma, CD11d blockade may also have a therapeutic role in acute inflammatory lung pathologies. The isolated deletion of CD11d alone can provide remarkable improvements in lung pathology and overall survival within a murine sepsis model\\u003csup\\u003e\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u003c/sup\\u003e. Going beyond VCAM-1-mediated extravasation, anti-CD11d therapeutics have the potential to modulate tissue retention/migration mediated by CD11d/CD18 and extracellular matrix interactions. A small peptide inhibitor of CD11d interactions reduced macrophage retention in adipose tissue, which is observed in atherosclerosis and diabetes\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. Indeed, CD11d\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e mice demonstrate a reduced disease burden in a murine atherosclerosis model\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e. Multiple avenues of investigation, therefore, exist to apply a temporary CD11d blockade with humanized anti-CD11d monoclonal antibodies and achieve a therapeutic goal.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, we presented the pharmacodynamics of a humanized anti-CD11d-2 clone and described a surprising pan-β2 integrin downregulation following inhibition of CK2 phosphorylation. The anti-CD11d-2 clone bound both conformations of CD11d/CD18 and did not produce an inflammatory response during \\u003cem\\u003ein vitro\\u003c/em\\u003e assays. The activity of CK2 phosphorylation was found to have a role in the expression of all β2 integrins. Future studies may use the humanized anti-CD11d clones as a tool to propel CD11d/CD18 research when investigating the therapeutic role of CD11d/CD18 blockade in neurotrauma, sepsis, and atherosclerosis.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003eThe studies reported were conducted in a manner consistent with ARRIVE guidelines.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCD11d Humanized Monoclonal Antibody Derivation\\u003c/h2\\u003e \\u003cp\\u003eThe original 217L mouse anti-human CD11d clone, provided by Eli Lily \\u0026amp; Co\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e, was used as the basis for creating humanized monoclonal anti-CD11d antibodies. Complementarity-determining regions (CDR) of the original 217L clone were isolated and four subsequent CDR variants were produced. The five resulting CDR sequences were incorporated into a human IgG4 framework to create the final clones (United States Patent No. US-11873340-B2 January 16, 2024)\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e. Specificity was verified by flow cytometry using HEK293 cells transfected with expression vectors expressing human CD11d and CD18 in the absence of any other β2 integrin. The purified humanized CD11d monoclonal antibodies (anti-CD11d-1, anti-CD11d-2, anti-CD11d-3, anti-CD11d-4, anti-CD11d-5) were initially provided by Eli Lily \\u0026amp; Co. Subsequently, the humanized CD11d monoclonal antibodies were produced in CHO cells and then purified from culture supernatants under contract with Biologics at the Human Health Therapeutics Research Center, National Research Council Canada, Montreal, Canada. Endotoxin levels were determined to be less than 0.1 EU/mg.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eHuman Leukocyte Collection\\u003c/h2\\u003e \\u003cp\\u003e Acquiring human blood samples for this study was approved (Project ID: 7332) by the University of Western Ontario Health Science Ethics Review Board (HSERB). After obtaining informed consent, human peripheral blood was collected via venipuncture in heparin vacutainer tubes (BD, San Jose, CA, USA) as approved by the HSERB and in accordance with an approved Biosafety-Biohazard Protocol (BIO-RRI-0021)\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCell Culture\\u003c/h2\\u003e \\u003cp\\u003eTHP-1 (ATCC TIB-202) and THP-1 NF-kB-Luc2 (ATCC TIB-202-NFkB-Luc2) cells were all obtained from American Tissue Culture Collection (ATCC, Cedarlane). All cells were cultured with complete RPMI media supplemented with 10% FBS, 1% L-glutamine, 100 U/mL penicillin, 100 \\u0026micro;g/mL streptomycin, 1 mM sodium pyruvate, 10 nM HEPES, and 50 \\u0026micro;M 2-mercaptoethanol. Additionally, 1 \\u0026micro;g/mL of puromycin was added to the completed RPMI selection media for the THP-1 NF-kB-Luc2 cell line. All tissue culture media, plastic ware, and supplements were acquired from ThermoFisher Scientific. Cultures were grown in standing T75 flasks at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e in a humidified incubator and cell density did not exceed 1x10\\u003csup\\u003e6\\u003c/sup\\u003e cells/mL. THP-1 cells were seeded at 1x10\\u003csup\\u003e6\\u003c/sup\\u003e cells/well into 6-well plates, 2x10\\u003csup\\u003e5\\u003c/sup\\u003e cells/well into 12-well plates, or 6x10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well into 96-well plates and differentiated with 100nM of phorbol myristic acetate (PMA; Millipore Sigma), for up to 72 h.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFlow Cytometry\\u003c/h2\\u003e \\u003cp\\u003eRat and human primary whole blood leukocytes were stained for flow cytometry as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e\\u003c/sup\\u003e. THP-1 cells were either differentiated or cultured as previously described. A 15-min incubation at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e with 1 mL Versene (ThermoFisher Scientific) was used to collect adherent cells following differentiation. To block promiscuous antibody binding, THP-1 cells were resuspended in 200 \\u0026micro;l HBSS\\u0026thinsp;+\\u0026thinsp;0.1% BSA plus 5 \\u0026micro;l normal goat serum (NGS) (Jackson ImmunoResearch) and 5 \\u0026micro;l AB human serum (ThermoFisher Scientific) on ice for 20 min. Cells were then surface-stained in FACS tubes (Corning) with a combination of LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific), anti-CD18 (TS1/18) FITC (BioLegend), anti-CD11d (anti-CD11d-2) Alexa 647, anti-IgG4 Alexa 647 (Eli Lily \\u0026amp; Co), anti-CD11a (TS2/4) PerCP (BioLegend), anti-CD11b (ICRF44) Violet 421 (BioLegend), and/or anti-CD11c (3.9) PE-Cy7 (BioLegend).\\u003c/p\\u003e \\u003cp\\u003eIntracellular staining was performed in a 96-well U-bottom plate (Corning) after performing fixable vital dye and/or cell surface staining. Cells were washed in staining buffer (HBSS\\u0026thinsp;+\\u0026thinsp;0.1% BSA) and then fixed and permeabilized using manufacturer\\u0026rsquo;s instructions (Foxp3/Transcription Factor Staining Buffer Set, eBioscience, ThermoFisher Scientific). The cells were then spun down, and each well was resuspended in a 50 \\u0026micro;L permeabilization buffer containing BioLegend TruStain human FcX block and incubated for 5\\u0026ndash;10 min at room temperature. Without washing, cells were stained with all intracellular antibodies or isotype control antibodies for 20 min at room temperature. This was followed by two washes in 200 \\u0026micro;L of permeabilization buffer and two washes in 200 \\u0026micro;L of HBSS\\u0026thinsp;+\\u0026thinsp;0.1% BSA. Cells were then resuspended in HBSS\\u0026thinsp;+\\u0026thinsp;0.1% BSA followed by an appropriate amount of 4% paraformaldehyde (PFA) (BioShop) to allow for a final concentration of 1% PFA in each well.\\u003c/p\\u003e \\u003cp\\u003eFor saturated antibody binding curves to determine antibody affinity, blocking and surface staining occurred live in HBSS, HBSS\\u0026thinsp;+\\u0026thinsp;1 mM EDTA or HBSS\\u0026thinsp;+\\u0026thinsp;1 mM Mn\\u003csup\\u003e2+\\u003c/sup\\u003e and flow cytometry was performed on cells that were subsequently fixed.\\u003c/p\\u003e \\u003cp\\u003eFor CK2 inhibition, the SGC-CK2-1 inhibitor was a gift from Dr. David Litchfield (Western University, Canada) and can be found commercially at MedKoo Biosciences. THP-1 cells were treated with or without 5 \\u0026micro;M of SGC-CK2-1 in the presence or absence of 100 nM PMA for 48 h. Cells were fixed and stained as described above. Flow cytometry graphs of a 48 h DMSO control treatment can be found in Supplemental Fig.\\u0026nbsp;1B.\\u003c/p\\u003e \\u003cp\\u003eA BD LSR II flow cytometer (BD Biosciences) was used for data acquisition with at least 50 000 total cells recorded per experimental condition. Data were analyzed on FlowJo, version 10.8 (BD Life Sciences)\\u003csup\\u003e\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u003c/sup\\u003e. Gating strategies can be found in Supplementary Fig.\\u0026nbsp;2.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eExperimental Spinal Cord Injury and anti-CD11d anti-inflammatory treatment\\u003c/h2\\u003e \\u003cp\\u003e All animal experiments were approved by the University of Western Ontario\\u0026rsquo;s Animal Care Committee (AUP no. 2010\\u0026thinsp;\\u0026minus;\\u0026thinsp;237) and conducted in accordance with the Canadian Council of Animal Care guidelines, the University Animal Care Committee\\u0026rsquo;s Standard Operating Procedures and in accordance with the University\\u0026rsquo;s Biosafety-Biohazard Protocol (BIO-RRI-0021). Additionally, all animal experiments were conducted in accordance with the Standards for Humane Care and Use of Laboratory Animals as approved by the Office of Laboratory Animal Welfare, Department of Health \\u0026amp; Human Services, U.S.A. (Protocol Assurance Identification #A5527-01).\\u003c/p\\u003e \\u003cp\\u003eExperimental spinal cord injury (SCI) was induced in female 220 gram Wistar rats (Charles River) by clip compression as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u003c/sup\\u003e and randomly assigned to a treatment group. To measure neutrophil infiltration into the spinal lesion using a myeloperoxidase assay, rats were given anti-CD11d clones at 2 h post-SCI for the 24 h assay time point, or at 2, 24 and 48h post-SCI for the 72h assay time point, as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u003c/sup\\u003e. For rats undergoing open field locomotor assessment, anti-CD11d clones were administered intravenously via the tail vein at 2, 24 and 48h post-SCI. All animals were monitored twice daily using a veterinarian approved clinical scoring sheet that monitored level of alertness and activity, hydration status (water consumption), percent weight loss, appearance of surgical wound, evidence of pain (piloerection, hunched back, discoloration around eyes), bowel movements (presence of fecal pellets in cage), bladder fullness, urine (leakage and appearance (cloudy, presence of blood). Bladders were emptied twice per day by gentle manual compressed.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMyeloperoxidase assay\\u003c/h2\\u003e \\u003cp\\u003eAt the 24h and 72h time points, spinal cord injured rats were euthanized by deep induction of anesthesia with 4% isoflurane followed by exsanguination (cardiac perfusion with cold PBS). Spinal cord tissue was harvested around the lesion and a homogenate was prepared as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. A portion of the homogenates were then assayed for myeloperoxidase activity as a surrogate marker for the presence of neutrophils. Complete methods of the myeloperoxidase assay were performed as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBasso, Beattie and Bresnahan (BBB) Rat Open field locomotor assessment\\u003c/h2\\u003e \\u003cp\\u003eHind limb locomotor assessment was conducted using the BBB locomotor rating scale for open field testing, as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. The BBB assessment was conducted by four individuals experienced in the BBB open field locomotor assessment and who were blinded to the treatment each rat received until the end of the assessment.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunocytochemistry\\u003c/h2\\u003e \\u003cp\\u003eTHP-1 NF-kB-Luc2 cells were seeded onto circular cover glasses placed at the bottom of 12-well plates and differentiated with RPMI selection media containing 100 nM of PMA for 48 h at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. RPMI selection media was removed, and cells were washed once with HBSS\\u0026thinsp;+\\u0026thinsp;0.1% BSA. In 300 \\u0026micro;l of HBSS\\u0026thinsp;+\\u0026thinsp;0.1%, cells were blocked with 15 \\u0026micro;l of FcX TruStain block (BioLegend) for 20 min at 10\\u0026deg;C. The remainder of the staining procedure occurred at room temperature in the dark. In 500 \\u0026micro;L of fresh HBSS, anti-CD11d (anti-CD11d-2) Alexa-647 and anti-CD18 (TS1/18) FITC were applied for 20 min. Cells were washed once with HBSS and fixed in 4% PFA for 20 min. Following three washes with 1mL of HBSS, cells were permeabilized with 0.1% Triton X in HBSS for 15 min. In 300 \\u0026micro;l of HBSS, 1:1000 DAPI stain (Millipore Sigma) was applied for 20 min. Finally, cells were washed three times with 1 mL of HBSS and imaging was performed on a Leica DMI6000 microscope (Leica Microsystems) and an EM CCO EvOLVE camera (Teledyne Photometrics). Images were analyzed on the FIJI software platform\\u003csup\\u003e\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eWestern Blot\\u003c/h2\\u003e \\u003cp\\u003eTHP-1 cells were differentiated or cultured in a 6-well plate as previously described in cell culture methods. Next, the culture media was removed, and the cells were washed twice with 1 mL PBS. The cells were incubated in 1mL of EGTA supplemented PBS for 15 min at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2,\\u003c/sub\\u003e to minimize focal adhesion signaling. Cells were washed twice with 1 mL PBS, then stimulated in 1 mL of complete RMPI media at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. Following stimulation, cells were immediately washed in 1 mL of ice-cold PBS and lysed with radioimmunoprecipitation assay (RIPA buffer) containing Halt Protease and Halt Phosphatase inhibitor cocktails (ThermoFisher Scientific). A cell scraper was used to collect cell lysate from adherent differentiated cells and non-adherent undifferentiated cells were collected directly into Eppendorf tubes. Lysates were placed on ice for 15 min, then sonicated twice for 10 s for complete lysis. A Bradford assay determined protein concentrations using detergent compatible protein assay reagents (Bio-Rad Laboratories). The resulting supernatants were immediately stored at -80\\u0026deg;C.\\u003c/p\\u003e \\u003cp\\u003eFor gel electrophoresis, lysate samples were mixed in 2X Tris-glycine buffer (BioLegend). Pre-made Tris-glycine gels (10%) (ThermoFisher Scientific) were loaded with 40 \\u0026micro;L of sample or 3 \\u0026micro;L of BLUeye protein ladder (FroggaBio). The gels were run for 150 min at 125 V, then transferred for 60 min at 100 V onto Immobilon PVDF (Bio-Rad Laboratories). In fresh 5 mL of TBS intercept blocking buffer (Li-Cor), cells were incubated overnight at 4˚C with mouse anti-phosphotyrosine (PY20) (BioLegend) and rabbit FAK protein (3285) (Cell Signaling Technology). Membranes were washed three times with 5 mL TBS\\u0026thinsp;+\\u0026thinsp;0.1% Tween 20 (BioShop) for 5 min. In fresh 5 mL of TBS intercept blocking buffer, secondary donkey anti-rabbit 680RD and donkey anti-mouse 800CW (Li-Cor) were incubated at room temperature for 1 h. Membranes were imaged at 700 nm and 800 nm using an Odyssey Fc (Li-Cor). Blots were then stripped and re-probed overnight with rabbit anti-pTyr397 FAK (700255) (ThermoFisher Scientific) and mouse β-actin mouse (A2228) (Sigma Aldrich). Data were analyzed on Image Studio Lite, version 5.2 (Li-Cor). Raw blots can be found in Supplementary Fig.\\u0026nbsp;3.\\u003c/p\\u003e \\u003cp\\u003eFor detection of CK2 phosphorylation, undifferentiated THP-1 cells were treated with 5 \\u0026micro;M SGC-CK2-1 inhibitor for 12, 24, or 48 h. Cell lysates were collected, and gel electrophoresis occurred as described above. CK2 phosphorylation was detected by rabbit EIF2S2-pS2\\u003csup\\u003e51\\u003c/sup\\u003e (gifted by Dr. David Litchfield, University of Western Ontario, London, ON, Canada) and normalized with mouse β-actin (A2228). Secondary antibodies and imaging occurred as described above.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBioluminescence\\u003c/h2\\u003e \\u003cp\\u003eTHP-1 NF-kB-Luc2 cells were seeded at 6x10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well of an opaque flat 96-well plate (Corning) in 100 \\u0026micro;l of RMPI complete media. Selected plates were differentiated with 100 nM PMA for 48 h, washed once with HBSS, then rested in serum free RPMI complete selection media for 24 h. Next, all the media was removed, replaced with 100 \\u0026micro;l of RMPI complete media containing 150 \\u0026micro;g/ml D-luciferin (Syd Labs, location), and treated with various conditions. The undifferentiated plates were immediately treated with 150 \\u0026micro;g/ml D-luciferin and various conditions. Plates were incubated in a Cytation 5 imager (Agilent Technologies) at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e for 24 h and bioluminescence was read every hour for 24 h.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eStatistical analyses were performed using GibStat or GraphPad Prism, Version 9. All data were presented as the mean plus/minus the standard error of the mean. Statistical significance was detected at p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05. Biological replicates were denoted by (N) whereas technical replicates were denoted by (n). One-way and two-way ANOVAs were performed with appropriate post hoc tests for multiple comparisons as noted in the figure legends.\\u003c/p\\u003e \\u003c/div\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003e\\u003cstrong\\u003eCompeting Interests\\u003c/strong\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eThere are two patents awarded and one patent pending. Japanese Awarded Patent Number: 7328762Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly United States Awarded Patent Number: US 11,873,340 B2Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly Canadian Pending Patent Number: Patentees: The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada; Inventors: Gregory Dekaban, Arthur Brown, Lynne Weaver. Patentees: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. Inventors: Barrett Allan, Kristine Kikly Specific Aspect of the manuscript covered by the two awarded and one pending patent(s): All three patents address the design of the antibodies used in the manuscript. Eli Lilly and Company did not provide any direct funding to any of the authors of this manuscript. Eli Lilly supplied the antibodies that the authors report on in this manuscript, as an in-kind contribution to the development and functional characterization of the humanized anti-CD11d antibodies. None of the authors of this manuscript have any financial interests of any kind with Eli Lilly. Nor do any of the authors have personal or professional relations with Eli Lilly and Company or members of said company. Thus, the authors declare no competing interests.\\u003c/p\\u003e\\n\\u003ch2\\u003eAdditional Information\\u003c/h2\\u003e\\n\\u003cp\\u003eThere are two patents awarded and one patent pending.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cu\\u003eJapanese Awarded Patent Number: \\u0026nbsp;7328762\\u003c/u\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePatentees:\\u003c/strong\\u003e The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInventors:\\u003c/strong\\u003e\\u0026nbsp; Gregory Dekaban, Arthur Brown, Lynne Weaver. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePatentees:\\u003c/strong\\u003e\\u0026nbsp; Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInventors:\\u003c/strong\\u003e\\u0026nbsp; Barrett Allan, Kristine Kikly\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cu\\u003eUnited States Awarded Patent Number: US 11,873,340 B2\\u003c/u\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePatentees:\\u003c/strong\\u003e The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInventors:\\u003c/strong\\u003e\\u0026nbsp; Gregory Dekaban, Arthur Brown, Lynne Weaver. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePatentees:\\u003c/strong\\u003e\\u0026nbsp; Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInventors:\\u003c/strong\\u003e\\u0026nbsp; Barrett Allan, Kristine Kikly\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCanadian Pending Patent Number:\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePatentees:\\u003c/strong\\u003e The University of Western Ontario, 100 Colip Circle, Suite 105, Ontario N6G 4X8 Canada;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInventors:\\u003c/strong\\u003e\\u0026nbsp; Gregory Dekaban, Arthur Brown, Lynne Weaver. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePatentees:\\u003c/strong\\u003e\\u0026nbsp; Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 U.S.A. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInventors:\\u003c/strong\\u003e\\u0026nbsp; Barrett Allan, Kristine Kikly\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSpecific Aspect of the manuscript covered by the two awarded and one pending patent(s)\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u003c/strong\\u003e\\u0026nbsp; All three patents address the design of the antibodies used in the manuscript. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eEli Lilly and Company did not provide any direct funding to any of the authors of this manuscript. Eli Lilly supplied the antibodies that the authors report on in this manuscript, as an in-kind contribution to the development and functional characterization of the humanized anti-CD11d antibodies. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eNone of the authors of this manuscript have any financial interests of any kind with Eli Lilly. Nor do any of the authors have personal or professional relations with Eli Lilly and Company or members of said company. Thus, the authors declare no competing interests. \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\n\\u003cp\\u003eThe research reported here was supported in part by the U.S. Department of Defense CDRMP SCIRP grant SC090328P, CIHR OPG 363209 and the National Hockey League Players Associate Concussion Challenge Fund.\\u003c/p\\u003e\\n\\u003ch1\\u003eAuthor Contribution\\u003c/h1\\u003e\\n\\u003cp\\u003eThe concept for this paper was that of Gregory Dekaban with contributions from Lynne Weaver and Arthur Brown. The manuscript was written by Eoin N. Blythe and edited by Drs. Gregory Dekaban, Lynne Weaver and Arthur Brown. Christy Barreira also contributed to the editing of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 1.\\u003c/strong\\u003e The experimental design was devised by Gregory Dekaban and Christy Barreira. Data were acquired and analyzed by Christy Barreira.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 2.\\u003c/strong\\u003e The experimental design was devised by Lynne Weaver, Arthur Brown and Gregory Dekaban. Figure 2B conducted by Lynne Weaver\\u0026rsquo;s staff. Figure 2C conducted by Gregory Dekaban, Lynne Weaver and Dr. Weaver and Dr. Brown\\u0026rsquo;s staff. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 3.\\u003c/strong\\u003e The experimental design was devised by Eoin N. Blythe and Gregory Dekaban. Data were acquired and analyzed by Eoin N. Blythe.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 4.\\u003c/strong\\u003e The experimental design was devised by Eoin N. Blythe and Gregory Dekaban. Data were acquired and analyzed by Eoin N. Blythe.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 5.\\u003c/strong\\u003e The experimental design was devised by Eoin N. Blythe, Gregory Dekaban and Corby Fink. Data acquisition and analysis were conducted by Corby Fink.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSupplemental Figure 1.\\u003c/strong\\u003e The experimental design was devised by Eoin N. Blythe and Gregory Dekaban. Data were acquired and analyzed by Eoin N. Blythe.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSupplemental Figure 2.\\u003c/strong\\u003e The experimental design was devised by Gregory Dekaban and Christy Barreira. Data were acquired and analyzed by Christy Barreira.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSupplemental Figure 3.\\u003c/strong\\u003e The experimental design was devised by Eoin N. Blythe and Gregory Dekaban. Data were acquired and analyzed by Eoin N. Blythe.\\u003c/p\\u003e\\n\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\n\\u003cp\\u003eWe thank Feng Bao, Todd Hryciw, Nicole Geremia and Kevin Braden from the Robarts Research Institute laboratories of Drs. Lynne C. Weaver, Arthur Brown and Gregory Dekaban, respectively, for conducting important experiments in this report.\\u003c/p\\u003e\\n\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\n\\u003cp\\u003eUpon reasonable request, all data can be acquired by contacting the corresponding author.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eBlythe, E. N., Weaver, L. C., Brown, A. \\u0026amp; Dekaban, G. 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I. \\u003cem\\u003eet al.\\u003c/em\\u003e Development of a potent and selective chemical probe for the pleiotropic kinase CK2. \\u003cem\\u003eCell Chem Biol\\u003c/em\\u003e \\u003cstrong\\u003e28\\u003c/strong\\u003e, 546-558.e10 (2021).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4764783/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4764783/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eImmunomodulatory agents targeting the CD11d/CD18 integrin are in development for the treatment of several pathophysiologies including neurotrauma, sepsis, and atherosclerosis. Previous rodent models have successfully improved neurotrauma recovery using murine anti-CD11d therapeutic antibodies. Here, we present the progression of anti-CD11d therapy with the development of humanized anti-CD11d monoclonal antibodies. Flow cytometric analysis demonstrated that the humanized anti-CD11d-2 clone binds both human monocytes and neutrophils. Using a THP-1 model, the humanized anti-CD11d-2 clone was then determined to bind both active and inactive CD11d/CD18 conformations without inducing inflammatory cell signaling. Finally, an investigation into the impact of CK2 phosphorylation on CD11d/CD18 expression found that CK2 inhibition downregulated all β2 integrins. By developing humanized anti-CD11d monoclonal antibodies, new tools are now available to study CD11d/CD18 physiology. The subsequent characterization of these humanized anti-CD11d antibodies makes their use in therapeutic interventions possible.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Humanized anti-CD11d monoclonal antibodies suitable for basic research and therapeutic applications\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-08-20 09:59:39\",\"doi\":\"10.21203/rs.3.rs-4764783/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"f4b542f3-e43e-489a-8a12-e2b1b55439a2\",\"owner\":[],\"postedDate\":\"August 20th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":36184102,\"name\":\"Biological sciences/Immunology/Immunotherapy\"},{\"id\":36184103,\"name\":\"Biological sciences/Immunology/Neuroimmunology\"},{\"id\":36184104,\"name\":\"Biological sciences/Immunology\"},{\"id\":36184105,\"name\":\"Biological sciences/Immunology/Inflammation\"}],\"tags\":[],\"updatedAt\":\"2024-08-28T03:52:30+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-08-20 09:59:39\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4764783\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4764783\",\"identity\":\"rs-4764783\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}