AntiIdiotype Antibodies: Powerful Tools for Antibody Drug DevelopmentM - PDF document

AntiIdiotype Antibodies: Powerful Tools for Antibody Drug DevelopmentMichelle Parker, Ph.D.Michelle.parker@genscript.com Make Research Easy What is an AntiIdiotype ntibody?Table of ontents��2 &#x/MCI; 15;&#x 000;&#x/MCI; 15;&#x 000;Obstacles & Solutions to the Generation of AntiIdiotype AbsFeatures of GenScript’s AntiIdiotype Antibody ServicesAntiIdiotype Antibody ApplicationsDownstream Assay DevelopmentGenScript AntiIdiotype Antibody Packages 1 2 3 4 5 6 Make Research Easy Overall structure:2 identical light chains (blue)2 identical heavy chains (green/purple)Variable regions constant regions5 classes of Abs:IgG, IgA, IgM, IgD, IgEAll contain either or light chainsBiological effector functions are mediated by the C domainChemical structure explains 3 functions of Abs:Binding versatilityBinding specificityBiological activityAntibody: Structure and Function��3 &#x/MCI; 78;&#x 000;&#x/MCI; 78;&#x 000;Antibody(Ab)Recognitionproteinsfoundtheserumotherbodilyfluidsvertebratesthatreactspecificallywiththeantigensthatinducedtheirformation Make Research Easy Antibody Binding Regions Idiotopethe antigenic determinants in or close to the variable portion of an antibody (Ab) Paratopethe part of an Ab that recognizes an antigen, the antigenbinding site of an Ab or complementarity determining region CDR Epitopethe part of the antigen to which the paratope binds Make Research Easy Antiidiotype antibodies (AntiIDs) Abs directed against the paratope (or CDR region) of another AntiIdiotype Antibodies Hypervariable regions (or the idiotype of an Ab) are the antigenic determinants Make Research Easy Different types of antiIDs��6 &#x/MCI; 9 ;&#x/MCI; 9 ;Anti ParatopespecificInhibitoryNeutralizingDetects free drugAntigenblockingAntibody drugAnti Drug TargetNot paratopespecificNot inhibitoryDetects total drug (free, partially bound, fully bound)Nonblocking AntiAntibody drugDrugtarget complex specificNot inhibitoryDetects bound drug onlyComplexspecific Make Research Easy Why use antiIDs?Ideal for preclinical research and antibody drug development studiesAllow monitoring of therapeutic Abs in samples Allow detection of Ab biotherapeutics that closely resemble circulating human IgAntiIdiotype Antibodies AntiIDs, specific for the unique variable region of the therapeutic Ab, are ideal for this purpose Make Research Easy AntiIDs can be powerful reagents for the following applications:Pharmacokinetic (PK) studiesUsed to measure the drug level in patient samplesImmunogenicity (antidrug antibody) assaysUsed as a positive control or reference standardAntiID Applications Make Research Easy Vaccine DevelopmentAntiID Applications General flow chart of antiidiotype antibody network Make Research Easy ��10 &#x/MCI; 6 ;&#x/MCI; 6 ;Antibody Production Overview Make Research Easy ��11 &#x/MCI; 6 ;&#x/MCI; 6 ;How are Monoclonal Antibodies Made? Make Research Easy 12 How are Polyclonal Antibodies Made? Make Research Easy CDR region of Ab may not be very immunogenic Antigen immunogenicity:The ability of a particular molecule to elicit an immune response determined by whether the immune system can recognize the antigen Percentage in antiserum very lowMost Abs raised target the Fc regionAntiID clone may be missed if conventional screening methods are usedMust optimize clone selection from numerous antiIDsChallenges During AntiID Production Make Research Easy Overcoming the Obstacles to AntiID Production Make Research Easy Solution Use antibody drugKLH conjugate as immunogenUse F(ab)(fragment, antigen binding) as immunogenConstant heavy chain determinants are absentAdjust immunization scheduleConsider different animal host1. CDR Region Not Immunogenic Make Research Easy Solution Use proper secondary Ab (antimouse Fc to prevent crossreaction with human Ig)HTP binding screeningCapture ELISA to select natural epitope recognized by Ab. Low Percentage of AntiIDs in Serum Make Research Easy Solution Antibody pairing (for PK study)Affinity ranking offranking)Epitope binning (ELISA or SPRbased) Antigen blocking 3. Optimize Clone Selection from Numerous AntiIDs��17 &#x/MCI; 28;&#x 000;&#x/MCI; 28;&#x 000;Antigen blocking = signal Mouse antiID & antigen mixture Target human Ab coated plateAntimouse Ab mix Antigen blocking assay Make Research Easy Optimizing clone selection: Epitope binning��18 &#x/MCI; 17; 00;&#x/MCI; 17; 00;&#x/MCI; 10;&#x 000;&#x/MCI; 10;&#x 000;Abs tested in a pairwise combinatorial mannerAbs that compete for binding to the same binding region are grouped into the same epitope bin. EpitopeAssess relative affinity rankEpitope Epitope Epitope Epitope 4F55B113E519C123H93B35G7Mouse antiHRP (against epitope 1)Target human Ab coated plate Mouse antiID (against epitope 2)Same epitope results in signal Epitope binning by Octet RED 96Case Study: Make Research Easy GenScript AntiID Development Protocol Animals are immunized with antibody drug candidates Titer tested for reactivity with speciesspecific antibody Hybridoma screening for specific reactivity to immunogen antibody by ELISA or Fab expressing cells by FACS; multiple closely related counter proteins Optimal clone selection: epitope binning/antibody pairing, antigen blocking and affinity ranking Antibody candidates subcloning and antibody production Immunogenecity and PK assay development and sample test Make Research Easy Downstream Assay Development Make Research Easy Due to different binding modes and levels of affinityUsing a combination of antiIDs one can obtain:PK profileDegree of target saturation by the antibody drugPossible binding modes of antiIDs:ComplexspecificantiIDs detect boundAb drugs directlyAntigenblocking(neutralizing) antiIDs detect freedrugNonblocking(noninhibitory) antiIDs detect free and bound drug AntiIDs Offer Flexibility for Specialized Assay Development��21 &#x/MCI; 55;&#x 000;&#x/MCI; 55;&#x 000;Anti Antibody drugAnti Drug TargetAntigenblockingblocking AntiAntibody drugComplexspecific Antibody drug Make Research Easy Guide for how to use concentration data Concentration dataIntended usefreetotalTo characterize in vivo PK behavior and to project human PKTo assess PK and PD responserelationships in animal modelsTo calculate the safety margin and determine the safe starting dose or efficacious dosefreeTo assess theinhibition of freefollowing drug treatment; understand the dynamics of freetotalTo assess the redistribution/modulation of totalfollowing drugtreatment; understand the dynamics of totalfreetotalratioTo compare in vivo and in vitro binding affinitiesmAb and L dynamic relationshipTo understandthe underlying dynamic relationship between mAb and L to facilitate dose and dosing schedule selection= target ligand in circulationmAb = monoclonal antibody therapeuticTable adapted from The AAPS Journal, Vol.13 (1), 99110, March 2011. Make Research Easy Downstream applications of antiIDs Pharmacokinetic Assays Immune Response Assays 1 2 Make Research Easy Purpose:Detect and quantitate human Ab drugs in serum Human serum contains 9.5 mg/ml IgG1Required sensitivity for Ab drug is in the ng/ml range Thus, assays must accurately detect Ab drug despite a millionfold excess of similar moleculesPossible agents for detection include:Antigen (i.e. drug target)AntiID antibodiesPharmacokinetic (PK) Assays Make Research Easy Typical PK Assay Formats AntiBridging AntiAntigenBridging AntiAntibody drug (in human serum)AntiID (labeled) AntiAntibody drug (in human serum)Antigen (labeled) 2 4 Antigen Capture AntiFc (labeled)AntigenAntibody drug (in human serum) 1 AntiAntibody drug (in human serum)AntiHu IgG Fc (labeled) AntiID Capture Sandwich 3 Make Research Easy Overview of Design Elements FDA recommends a multitiered approachTier 1: rapid, sensitive screening assay Tier 2:Positive should then be subjected to a confirmatory assay, such as ligand or antigen competition assayTier 3:Further characterization: neutralization/class/isotype/titerAspects of Assay Development: Highly sensitiveAble to detect all isotypes (IgM, IgE and IgG subtypes); carefully consider the avidity of control used to evaluate the assay; Should conduct assay performance test in the same concentration of matrix as that used to assess patient samples.Immune Response (IR) Assays Make Research Easy Direct and Bridging ELISAs Antibody drug AntiHu IgG Fc (labeled) Antidrug antibodyDirect Binding ELISA Antibody drug Antibody drug (labeled) Antidrug antibodyBridging ELISAAntiID(mAb or pAb) serve as the best positive controls for IR assay development Make Research Easy Direct and Bridging ELISA assaysAdvantages:Sensitive, inexpensive, equipment readily availableDisadvantages: May not detect early immune response and may be influenced by high levels of circulating drugRadioimmuno precipitation assay (RIPA)Advantages:sensitive, inexpensive, equipment readily availableDisadvantages:May not detect early immune response and may be influenced by high levels of circulating drugSurface Plasmon Resonance (SPR)Advantages: Method of choice for detecting early immune response and has Ab characterization capabilitiesDisadvantages:Expensive equipment, generally less sensitive than RIP or ELISA/ECL Electrochemiluminescence (ECL) assayAdvantages: Sensitive, can be modified to respond in the presence of high levels of circulating drugDisadvantages:Equipment can be expensive, may not easily detect rapidly dissociating AbsTypical IR Assay Formats Make Research Easy Successful GenScript Case Studies��29 &#x/MCI; 12;&#x 000;&#x/MCI; 12;&#x 000;Figure 1: Final subclones with high specificity to humanized when screened against different counter screenings.Figure 2: Final subclones with high specificity to humanized when screened against isotypecontrol, normal human IgGand human serum.Case study 1: AntiID mAb binds specifically to idiotype Ab, but not other negative controls ClonesAntibody drug HumanIgG1 Normal IgG Human serum No.12.6320.0860.2850.124No.22.6910.190.1860.285No.31.5760.0940.0840.253No.42.1330.210.0850.111No.52.4690.0930.1160.483No.62.5460.1220.0910.176No.71.8030.0840.1530.311No.82.1490.090.0810.133No.92.4980.0940.1820.082No.102.3990.1950.0840.195 0.51.52.5Antibody drugHuman IgG1Normal IgGHuman serum No.1 No.2 No.3 No.4 No.5 No.6 No.7 No.8 No.9 Make Research Easy Successful GenScript Case Studies��30 &#x/MCI; 12;&#x 000;&#x/MCI; 12;&#x 000;Figure 3: Affinity Ranking of antibodies by using BIAcore; clones with subaffinity 1E-092E-093E-094E-09Clone 1Clone 2Clone 3Clone 4KD (M) Figure 4: Affinity determination of Clone 2 ntibodies by using BIAcore Clone 2Case study 2 : GenScript can generates high affinity antiidiotype antibody with subnanomolarlevel Make Research Easy Successful GenScript Case Studies��31 &#x/MCI; 8 ;&#x/MCI; 8 ;Case study 3 :Figure 2: Specific inhibition of antiIdiotype mAbs in an antigen ligand blocking assay. 10%20%30%40%50%60%70%80%90%100%1:11:101:1001:500% antigen blocking Complete blocking clone Partial blocking clone Non-bloking clone Non - blocking clone Make Research Easy Features of GenScript AntiID Platform:High specificity and high affinityHigh speed production: 3 monthsAntigen ligand blocking, epitope binning, and antibody pairingAntiID polyclonal antibody packages (rabbit & goat)AntiID monoclonal antibody packages (mouse & rat)Proven track record: 100% success rateIR assay developmentGenScript Full AntiID Development Services GenScript has developed a panel of high affinity, high specificity antiIDs for use in PK and IR assays Make Research Easy GenScript AntiID Packages AntiIdiotype Antibody PackagesServicesAntiidiotype mAb (SC1184Antiidiotype pAb (SC1185Starting materialTarget antibody drug 23 mgTarget antibody drug 20 mg or moreCrossreactivity with control IgG 0%Crossreactivity with control IgG 0%DeliverablesHybridoma cell lines, supernatants and purified antiID antibody (optional)0.53 mg purified antiID antibody/rabbit Ordering is easy:http://www.genscript.com/antiidiotypeantibody.html Make Research Easy Sequence to purified antibody service with no need to provide an antigenOptimized immunization using our OptimumAntigen™ design tool and intelligent Antigen Strategy increasing specificity and affinity of antibodies Guaranteed results: quantity of antibodies or hybridoma, ELISA titer, and WB guarantee (varies with specific package)Fast turnaround time: delivery of purified pAb or development of specific hybridoma in 45 days.Certified facility: AAALAC International accreditation and OLAW certification, demonstrating our commitment to responsible animal care and use. Advantages of GenScript Custom Abs Make Research Easy Sequence to purified antibody service with no need to provide an antigenOptimized immunization using our OptimumAntigen™ design tool and intelligent Antigen Strategy increasing specificity and affinity of antibodies Guaranteed results: quantity of antibodies or hybridoma, ELISA titer, and WB guarantee (varies with specific package)Fast turnaround time: delivery of purified pAb or development of specific hybridoma in 45 days.Certified facility: AAALAC International accreditation and OLAW certification, demonstrating our commitment to responsible animal care and use. Advantages of GenScript Custom Abs Make Research Easy Sequence to purified antibody service with no need to provide an antigenOptimized immunization using our OptimumAntigen™ design tool and intelligent Antigen Strategy increasing specificity and affinity of antibodies Guaranteed results: quantity of antibodies or hybridoma, ELISA titer, and WB guarantee (varies with specific package)Fast turnaround time: delivery of purified pAb or development of specific hybridoma in 45 days.Certified facility: AAALAC International accreditation and OLAW certification, demonstrating our commitment to responsible animal care and use. Advantages of GenScript Custom Abs Make Research Easy Sequence to purified antibody service with no need to provide an antigenOptimized immunization using our OptimumAntigen™ design tool and intelligent Antigen Strategy increasing specificity and affinity of antibodies Guaranteed results: quantity of antibodies or hybridoma, ELISA titer, and WB guarantee (varies with specific package)Fast turnaround time: delivery of purified pAb or development of specific hybridoma in 45 days.Certified facility: AAALAC International accreditation and OLAW certification, demonstrating our commitment to responsible animal care and use. Advantages of GenScript Custom Abs Make Research Easy Variety of GenScript Antibody Services Make Research Easy Over 500 Publications Citing Our Ab Services Methylation protects microRNAs from an AGO1associated activity that uridylates 5’ RNA fragments generated by AGO1 cleavage. Yu B, Chen X, Vinovskis C, etc.PNAS, (Apr 2014)HYPERSENSITIVE TO HIGH LIGHT1 Interacts with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 and Functions in Protection of Photosystem II from Photodamage in Arabidopsis. Wang HB, Wang J, Qi K, etc.Plant Cell, (Mar 2014)Tousledlike kinases phosphorylate Asf1 to promote histone supply during DNA replication. Groth A, Jensen ON, Nielsen ML, etc.Nature Communications, (Mar 2014)Dirigent domaincontaining protein is part of the machinery required for formation of the ligninbased Casparian strip in the root. Hosmani PS, Kamiya T, Danku J, etc.PNAS, (August 2013)PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum Jiang L, Mu J, Zhang Q, Ni T, etc.Nature, (July 2013)Wheat Mds1 encodes a heatshock protein and governs susceptibility towards the Hessian fly gall midge. Liu X, Khajuria C, Li J, Trick HN, Huang L,etcNature Communications, (Jun 2013) Make Research Easy About GenScript Gene Peptide Protein Antibody Discovery Biology Cell Line http://www.genscript.com Make Research Easy Thank you for your participationWe wish you all success in your researchEmail me: Michelle.Parker@GenScript.com��41 &#x/MCI; 11;&#x 000;&#x/MCI; 11;&#x 000;Register for other webinars in the GenScript Webinar Series @ http://www.genscript.com/webinars.html November 25, 2014/8:00 am and 2:00 pm EST Peptide design strategy: basics, optimization, and application Tiffany GuptonCampolongo, PH.D. December 3, 2014/8:00 am and 2:00 pm EST Fusion partner for recombinant soluble protein production in E. Coli Keshav Vasanthavada Please complete the surveyQ & A Make Research Easy Karl Erik Hellstrom, Dale E. Yelton, H. Perry Fell, Donna Beaton, Margit Gayle, Michael Maclean, Maria Kahn, and IngegerdHellstrom. (1990) “Epitope Mapping and use of AntiIdiotypicAntibodies to the L6 Monoclonal AnticarcinomaCancer Research50, 24492454.James A. Lofgren, SripriyaDhandapani, Jason J. Pennucci, Christina M. Abbott, Daniel T. MytychArunanKaliyaperumal, Steven J. Swanson, and Michael C. Mullenix. (2007). “Comparing ELISA and Surface Plasmon Resonance for Assessing Clinical Immunogenicity of PanitumumabThe Journal of Immunology178: 74677472.Karen Weiss, M.D., FDA. News along the Pike, December 3, 2003Anthony R. Mire, Yu Chen Barrett, ViswanathDevanarayan, Eugen Koren, Hank Liu, Mauricio Maia, Thomas Parish, George Scott, GopiShankar, Elizabeth Shores, Steven J, Swanson, Gary Taniguchi, Daniel Wierda, Linda A. Zuckerman (2004). Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology.”Journal of Immunological Methods289: 116.Guidance for Industry Assay Development for Immunogenicity Testing of Therapeutic Protein , December 2009.Jean W. Lee, Marian Kelley, Lindsay E. King, JihongYang, HosseinSalimiMoosaviMeinaT. Tang, JianFeng Lu, John Kamerud, Ago Ahene, Heather Myler, Cindy Rogers. (2011) . Bioanalyticalapproaches to quantify “total” and “free” therapeutic antibodies and their targets: technical challenges and PK/PD applications over the course of drug development.”The AAPS Journal, 13(1): 99110.Shankar, G., Pendley, C., Stein, K. (2007). A riskbased bioanalyticalstrategy for the assessment of antibody immune responses against biological drugs.”Nature Biotechnology25: 555561.References