Name: in vitro methods for comparing target binding and cdc induction between therapeutic antibodies: applications in biosimilarity analysis
Uploaded: 2017-05-04T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis at JoVE.com

Gli autori non hanno riconoscimenti.

1. Valutazione del legame di target mediante citometria a flusso <ol><li> Preparazione di materiali biologici e reagenti <ol><li> Realizzare 500 ml di terreno di coltura RPMI integrato con 10% di siero di bovini fetali inattivati ​​termicamente (H-IFBS). </li><li> Cultura Le cellule del linfoma di Daudi Burkitt (Daudi) e le cellule di Daudi GFP + utilizzando RPMI e colaschi di coltura da 75 cm. Mantenere le colture a 37 ° C in un&#39;atmosfera umidificata al 5% di CO 2 fino ...

<ol>
	<li>Schimizzi, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosimilars+from+a+practicing+rheumatologist+perspective:+An+overview.">Biosimilars from a practicing rheumatologist perspective: An overview.</a> Autoimmun Rev. 15 (9), 911-916 (2016).</li><li>Cuello, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparability+of+Antibody-Mediated+Cell+Killing+Activity+Between+a+Proposed+Biosimilar+RTXM83+and+the+Originator+Rituximab.">Comparability of Antibody-Mediated Cell Killing Activity Between a Proposed Biosimilar RTXM83 and the Originator Rituximab.</a> Bio Drugs. 30 (3), 225-231 (2016).</li><li>Iwamoto, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validated+LC/MS+Bioanalysis+of+Rituximab+CDR+Peptides+Using+Nano-surface+and+Molecular-Orientation+Limited+(nSMOL)+Proteolysis.">Validated LC/MS Bioanalysis of Rituximab CDR Peptides Using Nano-surface and Molecular-Orientation Limited (nSMOL) Proteolysis.</a> Biol Pharm Bull. 39 (7), 1187-1194 (2016).</li><li>Chapman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waiving+in+vivo+studies+for+monoclonal+antibody+biosimilar+development:+National+and+global+challenges.">Waiving in vivo studies for monoclonal antibody biosimilar development: National and global challenges.</a> MAbs. 8 (3), 427-435 (2016).</li><li>Zembruski, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=7-Aminoactinomycin+D+for+apoptosis+staining+in+flow+cytometry.">7-Aminoactinomycin D for apoptosis staining in flow cytometry.</a> Anal Biochem. 429 (1), 79-81 (2012).</li><li>Salinas-Jazmin, N., Hisaki-Itaya, E., Velasco-Velazquez, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometry-based+assay+for+the+evaluation+of+antibody-dependent+cell-mediated+cytotoxicity+(ADCC)+in+cancer+cells.">A flow cytometry-based assay for the evaluation of antibody-dependent cell-mediated cytotoxicity (ADCC) in cancer cells.</a> Methods Mol Biol. 1165, 241-252 (2014).</li><li>Teeling, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biological+Activity+of+Human+CD20+Monoclonal+Antibodies+Is+Linked+to+Unique+Epitopes+on+CD20.">The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique Epitopes on CD20.</a> J Immunol. 177 (1), 362-371 (2006).</li><li>Miranda-Hernandez, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+physicochemical+properties+of+rituximab+related+to+its+immunomodulatory+activity.">Assessment of physicochemical properties of rituximab related to its immunomodulatory activity.</a> J Immunol Res. 2015, 910763 (2015).</li><li>Visser, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+and+functional+comparability+between+the+proposed+biosimilar+rituximab+GP2013+and+originator+rituximab.">Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab.</a> BioDrugs. 27 (5), 495-507 (2013).</li><li>Ylera, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-rate+screening+for+selection+of+high-affinity+anti-drug+antibodies.">Off-rate screening for selection of high-affinity anti-drug antibodies.</a> Anal Biochem. 441 (2), 208-213 (2013).</li><li>Broyer, L., Goetsch, L., Broussas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+complement-dependent+cytotoxicity+using+ATP+measurement+and+C1q/C4b+binding.">Evaluation of complement-dependent cytotoxicity using ATP measurement and C1q/C4b binding.</a> Methods Mol Biol. 988, 319-329 (2013).</li><li>Herbst, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+depletion+in+vitro+and+in+vivo+with+an+afucosylated+anti-CD19+antibody.">B-cell depletion in vitro and in vivo with an afucosylated anti-CD19 antibody.</a> J Pharm Exp Ther. 335 (1), 213-222 (2010).</li><li>Lazar, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+antibody+Fc+variants+with+enhanced+effector+function.">Engineered antibody Fc variants with enhanced effector function.</a> Proc Natl Acad Sci U S A. 103 (11), 4005-4010 (2006).</li><li>Winiarska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statins+impair+antitumor+effects+of+rituximab+by+inducing+conformational+changes+of+CD20.">Statins impair antitumor effects of rituximab by inducing conformational changes of CD20.</a> PLoS medicine. 5 (3), e64 (2008).</li><li>Zhou, X., Hu, W., Qin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+complement+in+the+mechanism+of+action+of+rituximab+for+B-cell+lymphoma:+implications+for+therapy.">The role of complement in the mechanism of action of rituximab for B-cell lymphoma: implications for therapy.</a> Oncologist. 13 (9), 954-966 (2008).</li><li>Hayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gemcitabine+enhances+rituximab-mediated+complement-dependent+cytotoxicity+to+B+cell+lymphoma+by+CD20+upregulation.">Gemcitabine enhances rituximab-mediated complement-dependent cytotoxicity to B cell lymphoma by CD20 upregulation.</a> Cancer Sci. 107 (5), 682-689 (2016).</li><li>Mossner, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+efficacy+of+CD20+antibody+therapy+through+the+engineering+of+a+new+type+II+anti-CD20+antibody+with+enhanced+direct+and+immune+effector+cell-mediated+B-cell+cytotoxicity.">Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity.</a> Blood. 115 (22), 4393-4402 (2010).</li><li>Lapalombella, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Raji-Burkitt's+lymphoma+model+for+preclinical+and+mechanistic+evaluation+of+CD52-targeted+immunotherapeutic+agents.">A novel Raji-Burkitt's lymphoma model for preclinical and mechanistic evaluation of CD52-targeted immunotherapeutic agents.</a> Clin Cancer Res. 14 (2), 569-578 (2008).</li><li>Mitoma, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+cytotoxic+effects+of+anti-tumor+necrosis+factor+agents+on+transmembrane+tumor+necrosis+factor+alpha-expressing+cells:+comparison+among+infliximab,+etanercept,+and+adalimumab.">Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor alpha-expressing cells: comparison among infliximab, etanercept, and adalimumab.</a> Arthritis Rheum. 58 (5), 1248-1257 (2008).</li><li>Kaymakcalan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparisons+of+affinities,+avidities,+and+complement+activation+of+adalimumab,+infliximab,+and+etanercept+in+binding+to+soluble+and+membrane+tumor+necrosis+factor.">Comparisons of affinities, avidities, and complement activation of adalimumab, infliximab, and etanercept in binding to soluble and membrane tumor necrosis factor.</a> Clin Immunol. 131 (2), 308-316 (2009).</li><li>Zent, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+complement+dependent+cytotoxicity+in+CLL+cells+from+patients+with+high-risk+early-intermediate+stage+chronic+lymphocytic+leukemia+(CLL)+treated+with+alemtuzumab+and+rituximab.">Direct and complement dependent cytotoxicity in CLL cells from patients with high-risk early-intermediate stage chronic lymphocytic leukemia (CLL) treated with alemtuzumab and rituximab.</a> Leuk Res. 32 (12), 1849-1856 (2008).</li><li>Goswami, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+complement-dependent+cytotoxicity+by+TGF-beta-induced+epithelial-mesenchymal+transition.">Regulation of complement-dependent cytotoxicity by TGF-beta-induced epithelial-mesenchymal transition.</a> Oncogene. 35 (15), 1888-1898 (2016).</li><li>Wang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+anti-EGFR+immune+response+with+mimotopes+identified+from+a+phage+display+peptide+library+by+panitumumab.">Induction of anti-EGFR immune response with mimotopes identified from a phage display peptide library by panitumumab.</a> Oncotarget. , (2016).</li><li>Ueda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytotoxic+effects+of+certolizumab+pegol+and+golimumab+mediated+by+transmembrane+tumor+necrosis+factor+alpha.">The cytotoxic effects of certolizumab pegol and golimumab mediated by transmembrane tumor necrosis factor alpha.</a> Inflamm Bowel Dis. 19 (6), 1224-1231 (2013).</li><li>Nesbitt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+action+of+certolizumab+pegol+(CDP870):+in+vitro+comparison+with+other+anti-tumor+necrosis+factor+alpha+agents.">Mechanism of action of certolizumab pegol (CDP870): in vitro comparison with other anti-tumor necrosis factor alpha agents.</a> Inflamm Bowel Dis. 13 (11), 1323-1332 (2007).</li><li>Teeling, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+new+human+CD20+monoclonal+antibodies+with+potent+cytolytic+activity+against+non-Hodgkin+lymphomas.">Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas.</a> Blood. 104 (6), 1793-1800 (2004).</li></ol>

La scadenza brevettuale di un mAb terapeutico sta promuovendo lo sviluppo di biosimilari. Pertanto, vi è la necessità di metodi semplici che possono identificare le differenze nelle attività clinicamente rilevanti di questi prodotti. Le cellule colture CD20 + sono state impiegate per la valutazione di due funzioni funzionali chiave di rituximab: binding target e induzione CDC. L&#39;attività precedente richiede il riconoscimento di CD20 dalla regione Fab della mAb, mentre quest&#39;ultima dipende principa...

Anticorpi terapeutici sono anticorpi monoclonali ricombinanti (mAbs) sviluppati per il trattamento di diverse patologie, incluso il cancro, autoimmuni e malattie croniche, disturbi neurologici, e altri 1. Attualmente, la FDA ha concesso l&#39;approvazione a più di 40 anticorpi monoclonali terapeutici, e più si prevede di raggiungere il mercato nei prossimi anni. Rituximab è un anticorpo monoclonale chimerico IgG1 ad alta affinità approvato per il trattamento del linfoma CD20 + cellule B non-Hodgkin (NHL), CD20 + NHL follicolare, leucemia linfatica cronica, e l&#39;artrite reumatoide 2, 3. Il riconoscimento del CD20, che è sovraespressa in cellule B, da Rituximab induce apoptosi; attivazione del complemento; e cellule anticorpo-dipendente citotossicità mediata (ADCC) 3. I brevetti di questo farmaco sono scaduti in Europa e negli Stati Uniti nel 2013 e 2016, Rispettivamente. Così, le aziende farmaceutiche di tutto il mondo stanno sviluppando biosimilari rituximab. Come in qualsiasi altro farmaco per il consumo umano, biosimilari richiedono l&#39;approvazione da agenzie di regolamentazione. Le linee guida internazionali indicano che per anticorpi monoclonali, biosimilarity deve essere dimostrata confrontando le caratteristiche fisico-chimiche, la farmacocinetica, l&#39;efficacia e la sicurezza dei nuovi prodotti e di riferimento 4. Di conseguenza, le metodologie utilizzate per le comparazioni devono valutare le caratteristiche strutturali e funzionali dei mAbs, in particolare quelli con rilevanza clinica. A tal fine, saggi in vitro mostrano diversi vantaggi rispetto esperimenti in vivo (recensiti Chapman et al.) 5: i) in vitro sono più sensibili alle differenze tra il prodotto biosimilare e il riferimento proposto; ii) studi in vivo devono essere eseguite in specie, le quali sono per vari mAbsprimati non umani; e iii) poiché il meccanismo d&#39;azione, la tossicologia preclinica, e gli effetti clinici del prodotto di riferimento sono ben noti, in studi in vivo con biosimilari non possono fornire ulteriori informazioni utili. Di conseguenza, guida dell&#39;Unione europea per biosimilari consente ai candidati di entrare in studi clinici basati su robusti dati in vitro solo 6. Qui vi presentiamo due saggi veloci, economiche e semplici che valutano l&#39;attività biologica di rituximab utilizzando cellule in coltura CD20 +. Questi test possono essere inclusi come parte dell&#39;esercizio di comparabilità per i candidati biosimilari rituximab.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>ATCC</td>
			<td>30-2001</td>
			<td>Modify the culture depending on the cell line</td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>0.4%, liquid, sterile-filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Daudi Burkitt&#39;s Lymphoma Cells</td>
			<td>ATCC</td>
			<td>CCL-213</td>
			<td>You can modify the cell line depending on the antibody of interest</td>
		</tr>
		<tr>
			<td>Fetal bovine serum(FBS)</td>
			<td>GIBCO</td>
			<td>16000-044</td>
			<td>You can modify the source of serum depending of requirements of the cell line</td>
		</tr>
		<tr>
			<td>Normal Human Serum Complement</td>
			<td>Quidel</td>
			<td>A113</td>
			<td>It is therefore appropriate for use in biocompatibility experiments including drug development, biomaterials testing and other applications</td>
		</tr>
		<tr>
			<td>7AA-D</td>
			<td>BDPharmigen</td>
			<td>559925</td>
			<td>You can use broad range of color options, compatible with most instrument configurations for to analyze viability.</td>
		</tr>
		<tr>
			<td>PECy5 Mouse Anti-human IgG</td>
			<td>BDPharmigen</td>
			<td>551497</td>
			<td>Change fluorochrome depending on the filter and laser of your flow cytometer.</td>
		</tr>
		<tr>
			<td>Human IgG Isotype Control</td>
			<td>ThermoFisher Scientific</td>
			<td>07-7102</td>
			<td>Change depending to mAb</td>
		</tr>
		<tr>
			<td>BDCytofix</td>
			<td>BDPharmigen</td>
			<td>554655</td>
			<td>Flow Cytometry Fixation Buffer (1-4% formaldehyde or paraformaldehyde )</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 10X (Phosphate buffer saline)</td>
			<td>GIBCO</td>
			<td>70011-044</td>
			<td>Phosphatebuffer without Ca2+/Mg2+ [137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4] and endotoxin free.</td>
		</tr>
		<tr>
			<td>Cell culture plates 96 well, V-bottom</td>
			<td>Corning</td>
			<td>29442-068</td>
			<td>12 x 75 mm round bottom test tubes or 96-well V- or U- bottom microtiter plates</td>
		</tr>
		<tr>
			<td>MabThera (Rituximab)</td>
			<td>Roche</td>
			<td>&#8212;</td>
			<td>Reference product</td>
		</tr>
		<tr>
			<td>Rituximab</td>
			<td>Indian</td>
			<td>&#8212;</td>
			<td>Biosimilar product</td>
		</tr>
		<tr>
			<td>15- or 50-mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430290 or 430052</td>
			<td>&#8212;</td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td>Eppendorf</td>
			<td>&#8212;</td>
			<td>Multiple volume configurations are necessary</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf</td>
			<td>&#8212;</td>
			<td>Adjustable-volume pipettes are necessary</td>
		</tr>
		<tr>
			<td>Centrifuge 5430/ 5430R model</td>
			<td>Eppendorf</td>
			<td>&#8212;</td>
			<td>Refrigerated variable-speed centrifuge (4 to 25 &#176; C) with speeds ranging from 10 to 30,130 &#215; g</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Dickinson</td>
			<td>&#8212;</td>
			<td>BD FACSAria III or other flow cytometer</td>
		</tr>
		<tr>
			<td>Olympus optical and light microscope</td>
			<td>Olympus</td>
			<td>&#8212;</td>
			<td>To quantify and evaluate cell growth</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>SANYO</td>
			<td>&#8212;</td>
			<td>Incubatorfor temperature andCO2 control to culture cells</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet</td>
			<td>CHC BIOLUS</td>
			<td>&#8212;</td>
			<td>Biological safety cabinet that is used to protect the researcher, product and environment.</td>
		</tr>
	</tbody>
</table>

in vitro methods for comparing target binding and cdc induction between therapeutic antibodies: applications in biosimilarity analysis

Gli anticorpi monoclonali terapeutici (mAb) sono rilevanti per il trattamento di diverse patologie, inclusi i tumori. Lo sviluppo di mAb biosimilar da parte delle aziende farmaceutiche è un&#39;opportunità di mercato, ma è anche una strategia per aumentare l&#39;accessibilità dei farmaci e ridurre i costi associati alla terapia. I protocolli qui descritti descrivono la valutazione della binding target e l&#39;induzione del CDC da rituximab nelle cellule Daudi. Queste due funzioni richiedono diverse regioni strutturali dell&#39;anticorpo e sono rilevanti per l&#39;effetto clinico indotto da rituximab. I protocolli permettono il confronto side-by-side di un rituximab di riferimento e un biosimilar di rituximab commercializzato. I prodotti valutati hanno mostrato differenze sia in associazione target e induzione CDC, suggerendo che esistano differenze fisico-chimiche che sottendono e evidenziano la necessità di analizzare l&#39;impatto di tali differenze nell&#39;ambiente clinico. I metodi qui riportati sono semplici e poco costosi in vitro/ Em&gt; modelli per la valutazione dell&#39;attività dei biosimilari rituximab. Così, essi possono essere utili durante lo sviluppo biosimilare, nonché per il controllo della qualità nella produzione biosimilare. Inoltre, i metodi presentati possono essere estrapolati ad altri anticorpi monoclonali terapeutici.

Usando i protocolli sopra descritti, il binding obiettivo e l&#39;induzione CDC di rituximab di riferimento sono stati confrontati in parallelo con quelli di un rituximab biosimilar prodotto e commercialmente disponibile in Asia. Nelle cellule di Daudi, entrambi i mAb hanno legato CD20 in modo dipendente dalla concentrazione ( Figura 1D ). Le regressioni non lineari dei dati di legame hanno mostrato una r <sup...

Watch this Scientific Journal Video about In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis at JoVE.com

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Questo protocollo descrive il confronto in vitro di due funzionalità funzionali chiave di rituximab: indotta da bersaglio e da induzione a dipendenza della citotossicità (CDC). I metodi sono stati impiegati per un confronto tra rituximab di riferimento e biosimilar di rituximab. Questi test possono essere impiegati durante lo sviluppo biosimile o come controllo di qualità nella loro produzione.

Metodi in vitro per il confronto obiettivo vincolante e CDC induzione Tra terapeutiche Anticorpi: Applicazioni nel Analisi Biosimilarity

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Research

JoVE Journal

Immunology and Infection

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Ricerca

Immunologia e infezioni

Introducing Shear Stress in the Study of Bacterial Adhesion

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Creazione di un liquido coperto Cultura polarizzata Airway epiteliali umane Calu-3 celle per studio cellulare risposta dell&#39;ospite agli agenti patogeni delle vie respiratorie<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Totale Estrazione di proteine ​​e 2-D elettroforesi su gel Metodi per<em&gt; Burkholderia</em&gt; Specie

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

A Novel<em&gt; In vitro</em&gt; Modello per lo studio delle interazioni tra l&#39;uomo di sangue intero e l&#39;endotelio

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Un<em&gt; In vitro</em&gt; Test di rilascio di Caseum che prevede la penetrazione di farmaci nelle lesioni della tubercolosi

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

Induzione di paralisi e di lesioni del sistema visivo in topi da specifiche cellule T per neuromielite ottica Autoantigen acquaporina-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Metodi alternativi In Vitro per la determinazione dell'integrità strutturale del capside virale

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Monostrati Colonoid umana per studiare le interazioni tra gli agenti patogeni, Commensals ed epitelio intestinale Host

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...