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Review ArticleUnraveling the Effect of Immunogenicity on the PKPDEfficacy and Safety of Therapeutic Proteins

Alison Smith1 Hugh Manoli1 Stacey Jaw1 Kimberley Frutoz1 Alan L Epstein1

Leslie A Khawli1 and Frank-Peter Theil2

1Department of Pathology Keck School of Medicine of University of Southern California Los Angeles CA 90089 USA2Nonclinical Development UCB Biopharma SPRL 1420 Braine-lrsquoAlleud Belgium

Correspondence should be addressed to Leslie A Khawli lkhawliuscedu and Frank-Peter Theil petertheilucbcom

Received 7 March 2016 Accepted 12 July 2016

Academic Editor Douglas C Hooper

Copyright copy 2016 Alison Smith et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Biologics have emerged as a powerful and diverse class of molecular and cell-based therapies that are capable of replacing enzymesediting genomes targeting tumors and more As this complex array of tools arises a distinct set of challenges is rarely encounteredin the development of small molecule therapies Biotherapeutics tend to be big bulky polar molecules comprised of protein andornucleic acids Compared to their small molecule counterparts they are fragile labile and heterogeneous Their biodistribution isoften limited by hydrophobic barriers which often restrict their administration to either intravenous or subcutaneous entry routesAdditionally their potential for immunogenicity has proven to be a challenge to developing safe and reliably efficacious drugs Ourdiscussion will emphasize immunogenicity in the context of therapeutic proteins a well-known class of biologics We set out todescribe what is known and unknown about the mechanisms underlying the interplay between antigenicity and immune responseand their effect on the safety efficacy pharmacokinetics and pharmacodynamics of these therapeutic agents

1 Introduction

Biologics are some of the most promising innovationsin modern drug therapy and represent a diverse arrayof molecular tools ranging in complexity and size Thesebiologically derived therapeutic agents are engineered byexploitingmolecular and cellularmachinery already found innature They are capable of performing complex and precisefunctions theirmedicinal applications are seemingly endlessThey greatly differ from the more traditional concept of adrug the small molecule Small molecules are typically orallyadministered lipophilic organic compounds that are low inmolecular weight (lt900Da) and a nanometer in size [1]These characteristics allow them to diffuse throughout thebody binding promiscuously to both target and off-targetsites On the other hand biologics encompass a wide range ofbiomolecular products ranging in size from short peptides toorgans for transplantation [2ndash5] As these products increasein size and complexity so do their molecular and functionalprofiles

Biotherapeutics are a subclass of biologics that are syn-thesized using recombinant DNA andor hybridoma tech-nologies They include monoclonal antibodies cytokinesgrowth factors hormones and other regulatory peptides [6]They differ from small molecule drugs in that they are oftenhydrophilic and interact with a far more discrete set of targetsmaking them an attractive alternative to traditional smallmolecule therapies associated with severe toxicities such aschemotherapeutics and immunosuppressants For instanceantitumor antibodies can be conjugated to small moleculepayloads markedly decreasing the effective dose and toxiceffects of the moiety Once considered science fiction theadvancement of biologic technology has made treatmentslike enzyme replacement therapies gene therapy and tissueengineering a reality

Biologics have some clear advantages over smallmoleculedrugs but they are not a panacea Due to their high antigenicpotential biologics frequently stimulate an immunogenicresponse [7 8] Some clinical investigations of a biothera-peutic have reported antidrug antibody (ADA) formation

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2016 Article ID 2342187 9 pageshttpdxdoiorg10115520162342187

2 Journal of Immunology Research

incidence rates in gt90 of patients [9ndash12] These immuno-genic responses sometimes result in altered efficacy andimmunotoxicity profiles that can vary frompatient to patientImmunogenicity-related adverse events (AEs) may be asbenign as amild itchy rash or as serious as infusion reactionsanaphylaxis and cytokine release syndrome (CRS) [13 14] Asan example tumor necrosis factor (TNF) inhibitors can acti-vate autoantibodies leading to Drug-Induced Autoimmunity(DIA) [15] Luckily the majority of immunogenic responsesare not toxic and do not lead to sustained autoimmunepathologies Rather encounters with ADAs affect efficacyby either accelerating or impeding clearance [16 17] Itis in these instances that immunogenicity complicates theinterplay of pharmacokinetics (PK) and pharmacodynamics(PD) which further obfuscates the development of a reliablepreclinical model correlating with clinical events Withoutthese data research and development (RampD) are severelyimpaired further potentiating the already abysmal attritionrates (852) of biologics once they reach phase 1 clinicaltrials [18] As the library of biologics continues to grow sodoes our need to understand how immunogenicity affectsefficacy and safety Here we set out to describe the biologyof immunogenic response and its impact on the PKPDof biologics We do this in an effort to shed light on thechallenges and pitfalls facing the successful development ofsafe and efficacious biologics

2 Immunogenic Risk Factors and the Need forPredictive Tools

Attributes of both the biologic and the patient contributeto the risk of immunogenic response The antigenicity of atherapy along with patient immune status can be used topredict the occurrence and severity of a potential immuno-genic response Patients with hyperreactive immune diseaseautoimmunity or history of biotherapeutic administrationare all at an increased risk of reactions against biologicsConversely immunosuppressed patients are less likely tomount a response [19 20] Hypervigilant immune systemscan be especially problematic when treating autoimmunediseases with biotherapeutics For instance an observationalstudy monitored patients being treated for Crohnrsquos diseasewith infliximab (IFX) a chimeric anti-TNF-alpha therapy[21] It was found that patients with high anti-IFX antibodiesin serum and low trough IFX levels correlated with loweredefficacy or poor IFX persistence These clinical observationsand immunoassays generated a PK profile of IFX that allowedclinicians to monitor Crohnrsquos patients for bioanalytical mark-ers that predicted treatment outcomes in the context of ADANonetheless this kind of data is often not applicable tobiosimilars or other biotherapeutics and is difficult to validateacross institutions administering identical therapies due tovariances in sample collection in vitro immunoassays dataanalysis and records In order to standardize the clinicalassessment of immunogenic response procedures and stan-dards must be applied and interpreted by predeterminedcriteria The US Food and Drug Administration (FDA) andthe European Medicine Agency (EMA) provide regulatorydocuments detailing such guidelines [22ndash24]

Genetically derived markers predicting immunogenicitycould potentially be applied across all treatment groups andeven across biotherapies For instance the Human LeukocyteAntigen (HLA) genes that encode for Major Histocompati-bility Complex (MHC) class II haplotypes have been shownto play a role in the immunogenic response [25 26] Aclinical study monitored patients diagnosed with MultipleSclerosis (MS) treatedwith Interferon-Beta (INF-120573) An asso-ciation between a commonMHC class II allele (DRB1lowast0701)present in 23 of the patients studied and an increasedlikelihood of producing an ADA response against INF-120573was discovered Screening for this allele could guide futuretreatment plan decisions of MS patients Establishing reliableand clinically relevant PK profiles of biotherapeutics andidentifying genetic markers predictive of immunogenicitywill allow clinicians to more effectively monitor and guidebiotherapeutic administration

21 Predicting Antigenicity From discovery to clinicimmunogenicity must constantly be monitored yet ourcurrent preclinical models have poor predictive powerfor clinically relevant immunogenicity [27] For examplehumanized therapies are designed to evade the immunesystem of patients yet they are inherently antigenic inimmunocompetent animal models and often exaggerate theantigenicity of the therapeutic agent Nevertheless the effortto combine preclinical data from in silico in vitro and in vivomethods remains worthwhile and may one day yield validcriteria for immunogenicity risk assessment

In silico computational tools have broad applications inthe identification and assessment of antigenicity Some screenfor leads such as cancer neoantigens and peptides for vaccinetargets [28ndash30] Others troubleshoot antigenicity by optimiz-ing the design of peptide nanoparticles or by determiningdeimmunization methods for antileukemic L-asparaginase[31 32] As this technology matures and provides morereliable predictions we will improve our ability to navigatethe effects of immunogenicity

In vitro modelling of antigenicity utilizes T cell assays Bcell assays and mimotopes to identify potentially immuno-genic epitopes [33 34] whereas in vitro characterization ofimmunogenic response from serum samples includes qual-itative and quasiquantitative immunoassays such as enzymelinked immunosorbent assays (ELISAs) radioimmunoassays(RIAs) and immunoradiometric assays (IRMAs) [35] Cell-based assays remain a popular choice formodeling antigenic-ity via the immunophenotyping proliferation tracking andcytokine secretion profiling of immune cells [36 37] How-ever these techniques remain costly since they are labor andtime intensive In vitromodeling and in vitro characterizationwork together to describe mechanisms of immune activationas well as immunogenic markersphenotypes belonging toboth disease and nondisease states Ideally this informationcan be used to predict the magnitude and frequency ofimmunogenic response and to discern which patients aremost at risk of developing this response The limitations ofthe clinical utility of in vitro assays for immunogenic markerdiscovery were discussed in a previous section Howeverpredictive preclinical in vitro assays would help protect both

Journal of Immunology Research 3

patients and companies from the expense and risk associatedwith failed clinical trials The development of such assayscan be informed by selecting clinically tested therapeuticproteins and then validated if the clinically observed antibodyresponse can be reconstructed in the preclinical setting [38]Expanding the repertoire of valid yet simplified assays willpush forward our preclinical evaluation of biologic designhopefully improving biologic design at its earliest stages

Preclinical in vivo methods monitoring the antigenicityof a therapy have failed to correlate with clinical events andare considered to have low predictive value for immuno-genic response in humans according to the InternationalConference on Harmonisation (ICH) S6 guidance How-ever they still hold value in the preclinical investigationof immunogenicity [39] Nonhuman primates have provedsuccessful in the modeling of toxicology but an analysisof their use in nonclinical trials revealed that they are notparticularly good for evaluating efficacy or immunogenicitysince they both under-overpredicted immunogenic response[40] Consequently researchers are still searching for ways tomodel human immunogenic response in vivo One strategy isto engineer transgenic mice to express human antigens thattolerize them to humanized biotherapeutics In these miceresearchers were able to model immune complex aggregationand extended-dose related ADA formation which closelymirror the phenomena observed in humans [41] Vaccines areoften evaluated in transgenic mice with a humanized haplo-type which has proved to be an effective tool for predictingCD4+ T cell activation [42 43] Savvy strategies like theseare gradually improving our preclinical in vivo investigationshowbeit animal modeling remains an unreliable predictor ofclinically relevant immunogenicity

Over the past few decades investigators have devel-oped sophisticated assessment strategies and significantlyexpanded our understanding of immunogenicity While reg-ulatory agencies have agreed upon and established inter-national guidelines for certain preclinical standards thenonclinical evaluation of allergy and pseudoallergy remainsunclear [44]

3 Immunotoxicity

Immunotoxicity is not unique to biologics Penicillin is a clas-sic example of a potentially lethal hypersensitivity reaction toa drug therapy and DIA can be induced via small moleculetherapies as well As early as the 1950s drug-induced lupuswas documented in response to hydralazine [45] Sincebiologics have greater immunogenic potential they are morelikely to elicit immunotoxic effects Here we will briefly dis-cuss immunogenic response emphasizing ADA formationand how these can affect the toxicity profile of the therapy

Interestingly many biotherapeutic agents are immuno-modulators capable of stimulating or suppressing immunefunctionThis immunogenicity is engineered to accomplish atherapeutic goal whereas unwanted immunogenic responsemay impair therapeutic outcome by affecting safety andorefficacy Immunotoxicology is associated with four derange-ments of immune function immunosuppression immunos-timulation hypersensitivity and autoimmunity [46 47]

These derangements can result in an unintentional blockadeof endogenous function CRS infusion reactions and ana-phylaxis While these AEs can have serious consequencesthe majority of immunogenic response elicits mild AEs orremains clinically silent Prior to the start of a clinical trialit is necessary that researchers evaluate the therapy in orderto establish appropriate criteria for monitoring immuno-genicity and to define unacceptable immunotoxicity thatwould haltend the study

Immunologically based AEs are often ADA-mediatedThe formation ofADA is triggeredwhen the classical pathwayrecognizes a ldquoforeignrdquo antigen via antigen presentation cellsor by BT cell mediated breaking of tolerance to humanizedrecombinant therapies [17 48] The latter functions via twopathways the T cell dependent (TD) and the T cell indepen-dent (TI) pathway TD accounts for 90 of ADA formationand relies on antigen stimulation of CD4+ T cell whichmounts a full adaptive immune response TD activatingtherapeutic agents have an epitope that engages CD4+ Tcells thus generating high affinity IgGs and memory B cellsleading to a more persistent and robust response In contrastTI-ADA are thought to arise from the direct stimulation ofB cell receptors (BCRs) by molecular therapies containingpatterned structural motifs that is polymeric repeats orcarbohydrate molecules capable of crosslinking BCRs TIactivation generates pentavalent IgM or low-affinity IgGantibodies that bypass T cellmediated affinitymaturation andisotype switching This immature antibody response fails toinduce memory phenotypes T cell epitope screening shouldbe considered along with the construct design of the therapyin order to mitigate ADA response by either selecting forimmunosuppressive phenotypes like Tregs or avoiding stableepitopes all together

Each biologic must be evaluated case by case for potentialimmunotoxicity Careful attention is requiredwhen consider-ing bispecific antibodies antibody drug conjugates and otherproducts lacking an endogenous analog since they are morelikely to elicit ADA formation [49] For instance HemophiliaA is a life-threatening genetic disease in which patients lackan essential clotting factor and is treated with Factor VIIIreplacement therapy [50] Unfortunately this therapy triggersADA response in up to a third of patients and often worsensthe condition in patients with high ADA titers Even thoughsupplementary recombinant proteins are often constructedfrom the native protein sequences already expressed inpatients they can elicit ADAwhich also neutralized the nativeproteins resulting in thrombocytopenia and red-cell aplasia[51 52] Additionally tissue deposition of immune complexescan precipitate disease such as nephrotic syndrome and drug-induced vasculitis [53 54] Preclinical models and clinicalprecedence can provide clues pointing to possible immuno-genic outcomes of a biologic in the clinic however immuno-toxicity remains an unpredictable threat to patient safety

4 The Impact of Immunogenicity onPKPD and Efficacy

In order for a therapy to be successful once it reaches marketclinicians must have confidence in its therapeutic potential


and be able to prescribeadminister the drug with a reliablyeffective dosing schedule What the body does to the drug(PK) and what the drug does to the body (PD) need to befully characterized and understoodThis requires the accrualof data pertaining to treatment outcomes and clinical eventsthat can be generalized to the biologicrsquos target populationThis has proven to be a challenging task for biotherapeuticsin part because immunogenicity unpredictably perturbsPKPD and efficacy Regardless the success of biologicswould be improved by defining the impact of unwantedimmunogenicity on PK profiles especially since the primaryreason for drug failure in phase II clinical trials is lack ofclinical efficacy [55]

Small molecules and therapeutic proteins are comprisedof entirely distinct molecular classes which are not onlydistinguished by their chemical profiles but their immuno-genicity and PK profiles as well [17 56] Small molecule PKprofiles can be interpreted from direct measurements of theactive drug and its metabolites in serum their ADME is wellcharacterized and employs simplified compartment modelsTheir PD can be interpolated from relationships such as dose-exposure-response curves and does not require a nuancedunderstanding of the drugrsquosmechanism of actionThePKPDof biologics are driven by target-mediated drug disposition(TMDD) meaning that the high affinity of the therapy for itstarget (PD) strongly influences its PK Theoretically TMDDshould yield a large volume of distribution with a highaccumulation of therapy in its target tissue However tissueuptake can be slowed by a variety of mechanisms thusconfining the therapy to its administration site Currently wedo not understand ADME well enough to fully describe andcharacterize this interplay

Serum measurements of biologics must consider theunbound partially bound and bound forms [57ndash59] Distin-guishing between the free and bound forms of the biologicand its metabolites is necessary in order to identify the effec-tive fraction of bioactive drug in circulation The terminalhalf-lives of these forms tend to vary with the free formoften displaying the shortest half-life [60] Many of the well-established small molecule models employed by RampD fail todescribe the PKPD and ADME of biotherapeutics Addi-tionally unwanted and unanticipated immunogenic responseperturbs PKPD thereby diminishing the predictive power ofpreclinical data Our inability to describe and anticipate thesephenomena hinders our ability to develop biologics that willprove efficacious and reliable once they reach clinical trialHere we discuss what is known about immunogenicityrsquos rolein affecting PKPD and howwe hope to apply this knowledgein order to improve the predictive power of biologic models

41 The Influence and Modes of Action of Immunogenicity onPKPD Assessment The immunogenic potential of biologicscan be affected at any stage of their journey startingwith theirroute of administration or even after the biologic is clearedby leaving a lasting impression on the immunomemoryof the patient Most biotherapeutics are large hydrophobicmolecules that cannot survive the harsh conditions of theGI tract therefore they have poor bioavailability by oraladministration Traditionally therapeutic proteins have been

given intravenously (IV)maximizing bioavailability andmin-imizing immunogenic risk However this method is bothinconvenient and expensive Newer formulations allow forsubcutaneous (SC) intraperitoneal or inhalation routes [61ndash63] SC administration is more cost effective and convenientthan IV yet it is often slow and incomplete In particularfor large molecules (gt20 kDa) that tend to migrate moreslowly through the extracellular matrix (ECM) SC poses anincreased risk of ADA formation by dendritic cells processingwhich drain directly to the lymphatics Depot formulationscan aid absorption by including ECM degrading enzymeslike hyaluronidase and by stabilizing proteins preventingdegradation and aggregation [64] Currentlywe are in need offormulations that can deliver an efficacious dose in affordableand convenient ways

Aswas previously describedmyriad patient and product-related factors can influence immunogenicityrsquos effect onPKPD and efficacy ADAs are described by their mode ofaction classifying themas either neutralizingADAs (nADAs)or nonneutralizing ADAs (non-nADAs) [65] The nADAsinteract directly with biologics andor bind to pharmaco-logically relevant sites obscuring interactions between thetherapy and its target thereby decreasing the overall efficacyAnother classification categorizes ADA as sustaining or clear-ing which indicates their influence on protein drug clearance[66] Both nADA and non-nADA can either prolong orshorten the half-life of biologics Most of the time immunecomplex formation comprised of drug-ADA aggregateswill increase the clearance of the biotherapeutic This phe-nomenon is most notable in patients with high affinity hightiter ADA In some cases isotype interactions with Fc recep-tors (FcR) also influence serumdrug concentration For IgGsbinding to the neonatal FcR (FcRn) increases bioavailabilitypossibly due to FcRn-mediated protection from catabolismat the injection site andor draining lymphatics [67 68]Coupling immunoprotective technologies such as Fc fusionproteins with improved delivery formulations could stabilizecirculating concentrations of biotherapeutics and their over-all bioavailability This effect will vary from patient to patientdepending on factors like theirMHChaplotypes andwhetherTD-ADA versus TI-ADA are triggered Since we do not fullyunderstand the patient and product-related factors governingthis response it is difficult to establish general guidelinesdefining an efficacious and reliable dosing regimen

In order to characterize the effect of ADA on the PKprofile of a biologic an immunogenicity profile must beestablished ADAs are a temporally evolving heterogeneouspopulation circulating in serum alongside a multitude ofendogenous proteins and antibodies [69 70] In most ani-mals ADAs become detectable within 2ndash4 weeks after thefirst dose administration and around 10 days in mice Theyare usually polyclonal antibodies against multiple epitopescomprised of more than one isotype circulating at varyingconcentrations amongst a multitude of endogenous proteinsTo overcome this daunting task of identifying and classi-fying ADA response a standard bioanalytical schematic isemployed (1) sensitive screen (2) confirmatory assay and (3)functional characterization The detection of these ADA islimited to capture antibody concentrations gt20ndash1000 ngmL


with Kd of K-9 to K-8 respectively [71ndash73] Occasionally thetherapeutic is administered at such a low dose that it is rapidlyconsumed by the target site In this absence of detectablecirculating drug PD measurements of target binding cancorrelate drug exposure This method of analysis can also beuseful in instances where a therapeutic protein has bioactivemetabolites not measured by PK assays [74]

Studies looking at therapeutic antibodies such as natal-izumab and IFX observed an increase in clearance anda reduction in efficacy when ADAs were persistent ratherthan transient throughout the first 12 weeks of therapy Theyalso found that clearing ADA formation was detectable 8ndash16weeks after treatment initiation [8 75 76]These labile kinet-ics suggest that when a measurement is taken significantlyit impacts the interpretation of the bioanalytics thereforedosing and sampling schedules should be well-defined andinclude peak and troughmeasurements as well as time pointssampled long after the circulating drug has been clearedHigher concentrations of ADAs are more likely to impactPK profiles and interfere with bioanalytical measurementsmaking simultaneous quantification of both the biologic andADA difficult This hampers the ability to track the timecourse of ADA response As a result the impact of immuneresponse on biologics cannot be fully investigated and ourunderstanding remains incomplete

Ultimately the development of a physiologically basedpharmacokinetic (PBPK) model akin to the small moleculecompartment model that also accounts for immunogenicitywould be ideal Both ldquotop downrdquo and ldquobottomuprdquo approachesare being used to derive mathematical models of ADME Forexample biodistribution coefficients (BC) can estimate tissuespecific distribution of protein fragments based onmolecularweight and plasma concentrations [77] The BC50 values formost tissues were found to be sim35 kDa Clinical derivationof these models could be achieved with a meta-analysisof clinical PK bioanalytical data but inconsistent reportingof bioanalytical parameters regarding ADA response makesstudies incomparable between institutions [78] Fortunatelyby collecting sparse or dense samples from many clinicalstudies Population PK (PopPK) allows for the inclusion ofdata from a variety of un-balanced designs Most advan-tageously PopPK looks at the target population of interestreceiving clinically relevant doses rather than the healthysubjects in traditional PK studies These studies enable theidentification and measurement of variances and can pointto potential explanations if identifying factors (demographicenvironment and pathophysiology) are found to correlatewith altered PK Currently groups are working to validateparameters and refinemodels relating PK and immunogenic-ity they have yet to design a reliable model generalizable to adiverse array of biotherapeutics

ADAs affect both the clearance and efficacy of biologicsBuilding off of this basic tenant we have been able to partiallyclassify the interplay between immunogenicity and PKPDyet we are unable to fully describe these phenomena Unlikethe PKPD of small molecules which began with bioanalyti-cal measurements that later yielded theories to describe thosemeasurements the PKPD of biologics is mostly understoodthrough its apparent differences from small molecule ADME

We cannot rely on our current bioanalytical techniques topredict or describe PKPD or clinical efficacy in the contextimmunogenic response

5 Strategies for Overcomingthe Pitfalls of Immunogenicity

As we have discussed the PKPD and the ADME of biologicscan be dramatically affected by immunogenic response Sohow then can we mitigate the consequences of those effectsWe can approach this challenge from both RampD and clinicalperspectives

51 Biotherapeutic Production and Design Design and man-ufacturing processes can mitigate or exacerbate the inherentantigenicity of biotherapeutics Good manufacturing pro-cesses and quality assurance recognize the inherent hetero-geneity of biologic batches Antigenic impurities such asendotoxin contamination and detergents aggregate forma-tion and variation in biologic activity must be kept to amini-mum in order to ensure a safe and reliably efficacious product[79] Careful handling of the protein product throughoutthe entire manufacturing process is critical in preventingaggregate formation Not only are aggregated and misfoldedproteins less efficacious but also they precipitate immunecomplex formation and immunogenic response Data suggestthat proteins exposed to stress freeze-thawing and pHshifts with surface active agents or hydrophobic surfacesare more likely to form aggregates that can crosslink BCRsthereby inducing TI-ADA formation [80 81] Additionallysome biotherapeutics do not tolerate high concentrationformulations well and may require more frequent dosing[82] By improving production processes and conservativelyadministering these therapies we can prevent the acquisitionof these unnecessary antigenic properties

Construct design is at the heart of biologic engineeringNew and exciting therapies such as bispecific antibodiesand antibody drug conjugates are expanding the utility andfunction of biotherapeutics even so they may introduceantigenicity with their inadvertently novel structures Infact it was the drive to decrease antigenicity that evolvedantibody therapies Initial antibody therapies were murine-derived and found to be highly immunogenic Eventuallychimeric antibody technologies evolved until we achievedfully humanized antibodies In theory a humanized antibodyfree of antigenic properties should not be immunogenic Inreality the immense variance of CD4+ T cell epitopes andMHC haplotypes allows for fully humanized antibodies toremain immunogenic in some patients [83 84] To mitigatethis deimmunization strategies can be used when designingprotein therapeutics This method utilizes in silico predic-tions of antigenic peptide sequences to identify potentiallyantigenic epitopes in a therapeutic protein These peptidesare then synthesized and their antigenicity is evaluated within vitro binding assays to TCRs and MHC II receptorsImmunogenic epitopes are then mutated to a more toleratedsequence and subsequent constructs are iteratively screenedTolerization is an ingenious twist on deimmunization thatselects for and engineers biologics with Treg activating


epitopes that can induce tolerance of the biologic [85] Com-bining both deimmunization with tolerization has provedan effective method for reducing immunogenic responsePosttranslational modifications of biologics also appear toinfluence immunogenicity For instance glycosylation canshield antigens from ADA binding and retard processing Inlinewith this finding a synthetic kind of glycosylation knownas PEGylation can prevent protein aggregation and maskantigenic epitopes thereby reducing immune response [86]However sometimes this process introduces an immuno-genic linker Additionally some forms of xeno-glycation mayhave immunostimulatory effects The risk is especially highwhen eukaryotic organisms like yeast are used to expressproteins since they may introduce their own microorganismspecific glycosylation patterns For instance yeast posttrans-lational processing adds mannose glycan which can bind tomannose receptors on human immune cells [87] Thoughthis is not an exhaustive list of all the possible considerationsthat should be taken into account when developing biologicsthese examples illustrate the usefulness of optimizing abiologicrsquos design to minimize immunogenicity

52 The Clinical Approach Clinicians already manageimmunogenicity associated with a variety of pathologies andtreatments such as rheumatoid arthritis and hematopoieticstem cell transplantation Clinical strategies to mitigateimmunogenic response include dose manipulation andimmunosuppressive agents such as methrotrexate andprednisone Some patients diagnosed with Infantile Pompedisease formed ADAs when treated with the enzymereplacement therapy recombinant human acid 120572-glucosidase(rhGAA) [88 89] A subgroup of patients form nADAs butadditional work showed that this subgroup can be tolerizedagainst rhGAA by complementing the replacement therapywith combinatorial immunosuppressive therapy comprisedof rituximab and methotrexate Fortunately this tolerancepersisted even after termination of B cell recovery andimmunosuppression Dosing-through is another methodthat has also proven to be a successful strategy in overcomingthe deleterious effects of ADA on efficacy [90] For MSpatients receiving long term IFN-s therapy nADAs havealso proven to be problematic Interestingly recent studieshave shown that high doses of IFN-sv32 result in lowernADA titers due to the saturation of these nADAs and therestoration of IFN-s binding to its receptor High doses ofIFN-s can also induce long term high dose tolerance of theimmune system

6 Summary and Conclusions

The advancement of biologics has revolutionized targetedtherapy By utilizing biologically derived tools we can admin-ister drugs with complex and precise functions which oftenmimic the bodyrsquos natural processes However it is this keycharacteristic of molecular mimicry that potentiates the anti-genicity of biologics and leads to undesirable ADA-mediatedoutcomes such as immunotoxicity and decreased efficacyADAs against biologics can alter ADME thereby greatlyconfounding the interpretation of PKPD assessments Thus

it is vital that we develop bioanalytical tools that can reliablypredict and model the complex interplay between immuno-genicity and PKPD Outside of patient immune status andexposure history we do not currently possess adequatetools or knowledge of immunogenic markers to identifywhich patient-product interactions carry an increased riskof immunogenic response Though we described variouspreclinical in silico in vitro and in vivo tools used forimmunogenic risk assessment these methods are not recog-nized as predictive We must rely on clinical investigationsAs novel biotherapeutic proteins continue to shape the courseof modern medicine we must work to fill these knowledgegaps in order to ensure that patients receive safe and effectivetreatments

Competing Interests

The authors declared no conflict of interests

Acknowledgments

The authors would like to express their gratitude toWolfgangF Richter PhD degree holder Michael Diaz Ishani Synghaland Michelle Khawli for their valuable and constructivesuggestions and comments Their willingness to give theirtime so generously has been very much appreciated

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[4] N Kubicki C Laird L Burdorf R N Pierson III and A MAzimzadeh ldquoCurrent status of pig lung xenotransplantationrdquoInternational Journal of Surgery vol 23 pp 247ndash254 2015

[5] A M Bailey J Arcidiacono K A Benton Z Taraporewalaand S Winitsky ldquoUnited states food and drug administrationregulation of gene and cell therapiesrdquoAdvances in ExperimentalMedicine and Biology vol 871 pp 1ndash29 2015

[6] M Wadhwa I Knezevic H-N Kang and R ThorpeldquoImmunogenicity assessment of biotherapeutic products anoverview of assays and their utilityrdquoBiologicals vol 43 no 5 pp298ndash306 2015

[7] C A Weber P J Mehta M Ardito L Moise B Martin and AS De Groot ldquoT cell epitope friend or foe Immunogenicity ofbiologics in contextrdquo Advanced Drug Delivery Reviews vol 61no 11 pp 965ndash976 2009

[8] R Ponce L Abad L Amaravadi et al ldquoImmunogenicity ofbiologically-derived therapeutics assessment and interpreta-tion of nonclinical safety studiesrdquo Regulatory Toxicology andPharmacology vol 54 no 2 pp 164ndash182 2009

[9] K Kuus-Reichel L S Grauer L M Karavodin C Knott MKrusemeier and N E Kay ldquoWill immunogenicity limit theuse efficacy and future development of therapeuticmonoclonal


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[10] E Koren L A Zuckerman and A R Mire-Sluis ldquoImmuneresponses to therapeutic proteins in humansmdashclinical signifi-cance assessment and predictionrdquo Current Pharmaceutical Bio-technology vol 3 no 4 pp 349ndash360 2002

[11] H Schellekens and N Casadevall ldquoImmunogenicity of recom-binant human proteins causes and consequencesrdquo Journal ofNeurology vol 251 supplement 2 pp II4ndashII9 2004

[12] H Schellekens ldquoImmunogenicity of therapeutic proteins clini-cal implications and future prospectsrdquoClinicalTherapeutics vol24 no 11 pp 1720ndash1740 2002

[13] T Corona C Leon and L Ostrosky-Zeichner ldquoSevere anaphy-laxis with recombinant interferon betardquo Neurology vol 52 no2 p 425 1999

[14] R Stebbings L Findlay C Edwards et al ldquolsquoCytokine stormrsquoin the Phase I trial of monoclonal antibody TGN1412 betterunderstanding the causes to improve preclinical testing ofimmunotherapeuticsrdquo Journal of Immunology vol 179 no 5 pp3325ndash3331 2007

[15] M De Bandt J Sibilia X Le Loet et al ldquoSystemic lupus erythe-matosus induced by anti-tumour necrosis factor alpha therapya French national surveyrdquo Arthritis Research amp Therapy vol 7no 3 pp R545ndashR551 2005

[16] V Jawa L P Cousens M Awwad E Wakshull H Kropshoferand A S De Groot ldquoT-cell dependent immunogenicity ofprotein therapeutics preclinical assessment and mitigationrdquoClinical Immunology vol 149 no 3 pp 534ndash555 2013

[17] H Kropshofer and W F Richter ldquoImmunogenicity its impacton ADME of therapeutic biologicsrdquo Pharmaceutical SciencesEncyclopedia vol 11 pp 1ndash2 2015

[18] M Hay D W Thomas J L Craighead C Economides andJ Rosenthal ldquoClinical development success rates for investiga-tional drugsrdquo Nature Biotechnology vol 32 no 1 pp 40ndash512014

[19] Y Vugmeyster J Harrold and X Xu ldquoAbsorption DistributionMetabolism and Excretion (ADME) studies of biotherapeuticsfor autoimmune and inflammatory conditionsrdquo AAPS Journalvol 14 no 4 pp 714ndash727 2012

[20] P Ragnhammar H J Friesen J E Frodin et al ldquoInductionof antirecombinant human granulocyte-macrophage colony-stimulating factor (Escherichia coli-derived) antibodies andclinical effects in nonimmunocompromised patientsrdquo Bloodvol 84 no 12 pp 4078ndash4087 1994

[21] S Ben-HorinM Yavzori L Katz et al ldquoThe immunogenic partof infliximab is the F(ab1015840)2 but measuring antibodies to theintact infliximab molecule is more clinically usefulrdquo Gut vol60 no 1 pp 41ndash48 2011

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[23] EMA ldquoGuideline on immunogenicity assessment of biotech-nology-derived therapeutic proteinsrdquo Tech Rep EMACHMPBMWP143272006 EMA London UK 2007

[24] EMA ldquoGuideline on immunogenicity assessment of mono-clonal antibodies intended for in vivo clinical userdquo Tech RepEMACHMPBMWP862892010 EMA London UK 2012

[25] M D F S Barbosa J Vielmetter S Chu D D Smith andJ Jacinto ldquoClinical link between MHC class II haplotype andinterferon-beta (IFN-120573) immunogenicityrdquo Clinical Immunol-ogy vol 118 no 1 pp 42ndash50 2006

[26] V Zota A Nemirovsky R Baron et al ldquoHLA-DR alleles inamyloid 120573-peptide autoimmunity a highly immunogenic rolefor the DRB1lowast1501 allelerdquo Journal of Immunology vol 183 no 5pp 3522ndash3530 2009

[27] V Brinks D Weinbuch M Baker et al ldquoPreclinical modelsused for immunogenicity prediction of therapeutic proteinsrdquoPharmaceutical Research vol 30 no 7 pp 1719ndash1728 2013

[28] A Desrichard A Snyder and T A Chan ldquoCancer neoantigensand applications for immunotherapyrdquo Clinical Cancer Researchvol 22 no 4 pp 807ndash812 2016

[29] R Zaheer C L Klima and T A McAllister ldquoExpeditiousscreening of candidate proteins for microbial vaccinesrdquo Journalof Microbiological Methods vol 116 pp 53ndash59 2015

[30] P Carlos V Roupie S Holbert et al ldquoIn silico epitope analysisof unique and membrane associated proteins from Mycobac-terium avium subsp paratuberculosis for immunogenicity andvaccine evaluationrdquo Journal of Theoretical Biology vol 384 pp1ndash9 2015

[31] T A P F Doll R Dey and P Burkhard ldquoDesign and optimiza-tion of peptide nanoparticlesrdquo Journal of Nanobiotechnologyvol 13 no 1 article 73 2015

[32] L N Ramya and K K Pulicherla ldquoStudies on deimmuniza-tion of antileukaemic L-asparaginase to have reduced clinicalimmunogenicitymdashan in silico Approachrdquo Pathology and Oncol-ogy Research vol 21 no 4 pp 909ndash920 2015

[33] E M Moussa J Kotarek J S Blum E Marszal and E MTopp ldquoPhysical characterization and innate immunogenicity ofaggregated intravenous immunoglobulin (IGIV) in an in vitrocell-based modelrdquo Pharmaceutical Research vol 33 no 7 pp1736ndash1751 2016

[34] V Dhir M Fort A Mahmood et al ldquoA predictive biomimeticmodel of cytokine release induced by TGN1412 and other ther-apeutic monoclonal antibodiesrdquo Journal of Immunotoxicologyvol 9 no 1 pp 34ndash42 2012

[35] L Yin X Chen P Vicini B Rup and T P Hickling ldquoThera-peutic outcomes assessments risk factors andmitigation effortsof immunogenicity of therapeutic protein productsrdquo CellularImmunology vol 295 no 2 pp 118ndash126 2015

[36] V Rombach-Riegraf A C Karle B Wolf et al ldquoAggregationof human recombinant monoclonal antibodies influences thecapacity of dendritic cells to stimulate adaptive T-cell responsesin vitrordquo PLoS ONE vol 9 no 1 Article ID e86322 2014

[37] H Kropshofer and T Singer ldquoOverview of cell-based tools forpre-clinical assessment of immunogenicity of biotherapeuticsrdquoJournal of Immunotoxicology vol 3 no 3 pp 131ndash136 2006

[38] P Gaitonde and S V Balu-Iyer ldquoIn vitro immunogenicity riskassessment of therapeutic proteins in preclinical settingrdquoMeth-ods in Molecular Biology vol 716 pp 267ndash280 2011

[39] V Brinks W Jiskoot and H Schellekens ldquoImmunogenicity oftherapeutic proteins the use of animal modelsrdquo PharmaceuticalResearch vol 28 no 10 pp 2379ndash2385 2011

[40] P J K vanMeer M Kooijman V Brinks et al ldquoImmunogenic-ity of mAbs in non-human primates during nonclinical safetyassessmentrdquomAbs vol 5 no 5 pp 810ndash816 2013

[41] M Haji Abdolvahab A Fazeli A Halim A S Sediq M RFazeli and H Schellekens ldquoImmunogenicity of recombinanthuman interferon beta-1b in immune-tolerant transgenic micecorresponds with the biophysical characteristics of aggregatesrdquoJournal of InterferonampCytokine Research vol 36 no 4 pp 247ndash257 2016


[42] P Riese S Trittel K Schulze and C A Guzman ldquoRodentsas pre-clinical models for predicting vaccine performance inhumansrdquo Expert Review of Vaccines vol 14 no 9 pp 1213ndash12252015

[43] Y Zeng T Gao G Zhao et al ldquoGeneration of humanMHC (HLA-A11DR1) transgenic mice for vaccine evaluationrdquoHuman Vaccines amp Immunotherapeutics vol 12 no 3 pp 829ndash836 2015

[44] J-Y Han Y Yi A-H Liang et al ldquoReview on requirements ofdrug allergy or pseudoallergic reactions in pre-clinical evalu-ationrdquo Zhongguo Zhong Yao Za Zhi vol 40 no 14 pp 2685ndash2689 2015

[45] A T Borchers C L Keen andM E Gershwin ldquoDrug-inducedlupusrdquo Annals of the New York Academy of Sciences vol 1108pp 166ndash182 2007

[46] J Descotes and A Gouraud ldquoClinical immunotoxicity oftherapeutic proteinsrdquo Expert Opinion on Drug Metabolism andToxicology vol 4 no 12 pp 1537ndash1549 2008

[47] F Dedeoglu ldquoDrug-induced autoimmunityrdquo Current Opinionin Rheumatology vol 21 no 5 pp 547ndash551 Sep 2009

[48] A S De Groot and D W Scott ldquoImmunogenicity of proteintherapeuticsrdquo Trends in Immunology vol 28 no 11 pp 482ndash490 2007

[49] M B Hock K E Thudium M Carrasco-Triguero and N FSchwabe ldquoImmunogenicity of antibody drug conjugates bio-analytical methods and monitoring strategy for a novel ther-apeutic modalityrdquo The AAPS Journal vol 17 no 1 pp 35ndash432015

[50] M Ing N Gupta M Teyssandier et al ldquoImmunogenicityof long-lasting recombinant factor VIII productsrdquo CellularImmunology vol 301 pp 40ndash48 2016

[51] C Pollock D W Johnson W H Horl et al ldquoPure red cellaplasia induced by erythropoiesis-stimulating agentsrdquo ClinicalJournal of the American Society of Nephrology vol 3 no 1 pp193ndash199 2008

[52] M de Serres B Ellis J E Dillberger et al ldquoImmunogenicity ofthrombopoietin mimetic peptide GW395058 in BALBc miceand New Zealand white rabbits evaluation of the potentialfor thrombopoietin neutralizing antibody production in manrdquoSTEM CELLS vol 17 no 4 pp 203ndash209 1999

[53] T E Hunley D Corzo M Dudek et al ldquoNephrotic syndromecomplicating 120572-glucosidase replacement therapy for pompediseaserdquo Pediatrics vol 114 no 4 pp e532ndashe535 2004

[54] R G Grau ldquoDrug-induced vasculitis new insights and achanging lineup of suspectsrdquo Current Rheumatology Reportsvol 17 no 12 article 71 2015

[55] P van der Graaf ldquoInfluencing early portfolio decision makingusing preclinical MampS how early is early and when is it toolaterdquo in Proceedings of the AAPS National Biotechnology Con-ference San Francisco Calif USA 2010

[56] S Shi ldquoBiologics an update and challenge of their pharmacoki-neticsrdquo Current Drug Metabolism vol 15 no 3 pp 271ndash2902014

[57] H Salimi-Moosavi J Lee B Desilva and G Doellgast ldquoNovelapproaches using alkaline or acidguanidine treatment to elim-inate therapeutic antibody interference in the measurement oftotal target ligandrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 51 no 5 pp 1128ndash1133 2010

[58] A C Bautista D Wullner M Moxness S J Swanson N Chir-mule and V Jawa ldquoImpact of matrix-associated soluble factorson the specificity of the immunogenicity assessmentrdquo Bio-analysis vol 2 no 4 pp 721ndash731 2010

[59] J W Lee M Kelley L E King et al ldquoBioanalytical approachesto quantify lsquototalrsquo and lsquofreersquo therapeutic antibodies and theirtargets technical challenges and PKPD applications over thecourse of drug developmentrdquo AAPS Journal vol 13 no 1 pp99ndash110 2011

[60] M P Hall C Gegg K Walker et al ldquoLigand-binding massspectrometry to study biotransformation of fusion proteindrugs and guide immunoassay development strategic approachand application to peptibodies targeting the thrombopoietinreceptorrdquo AAPS Journal vol 12 no 4 pp 576ndash585 2010

[61] W F Richter and B Jacobsen ldquoSubcutaneous absorption of bio-therapeutics knowns and unknownsrdquo Drug Metabolism andDisposition vol 42 no 11 pp 1881ndash1889 2014

[62] S Tamilvanan N L Raja B Sa and S K Basu ldquoClinicalconcerns of immunogenicity produced at cellular levels by bio-pharmaceuticals following their parenteral administration intohuman bodyrdquo Journal of Drug Targeting vol 18 no 7 pp 489ndash498 2010

[63] R U Agu M I Ugwoke M Armand R Kinget and N Ver-beke ldquoThe lung as a route for systemic delivery of therapeuticproteins and peptidesrdquo Respiratory Research vol 2 no 4 pp198ndash209 2001

[64] S Rosengren S S Dychter M A Printz et al ldquoClinicalimmunogenicity of rHuPH20 a hyaluronidase enabling subcu-taneous drug administrationrdquo The AAPS Journal vol 17 no 5pp 1144ndash1156 2015

[65] M Krishna and S G Nadler ldquoImmunogenicity to bio-therapeuticsmdashthe role of anti-drug immune complexesrdquo Fron-tiers in Immunology vol 7 article 21 2016

[66] N Chirmule V Jawa and B Meibohm ldquoImmunogenicity totherapeutic proteins impact on PKPD and efficacyrdquo AAPSJournal vol 14 no 2 pp 296ndash302 2012

[67] S AMousaviM Sporstoslashl C Fladeby R Kjeken N Barois andT Berg ldquoReceptor-mediated endocytosis of immune complexesin rat liver sinusoidal endothelial cells is mediated by Fc120574RIIb2rdquoHepatology vol 46 no 3 pp 871ndash884 2007

[68] E S Ward S C Devanaboyina and R J Ober ldquoTargetingFcRn for the modulation of antibody dynamicsrdquo MolecularImmunology vol 67 no 2 pp 131ndash141 2015

[69] G Shankar S Arkin L Cocea et al ldquoAssessment and reportingof the clinical immunogenicity of therapeutic proteins andpeptides-harmonized terminology and tactical recommenda-tionsrdquo AAPS Journal vol 16 no 4 pp 658ndash673 2014

[70] J Wang J Lozier G Johnson et al ldquoNeutralizing antibodiesto therapeutic enzymes considerations for testing preventionand treatmentrdquoNature Biotechnology vol 26 no 8 pp 901ndash9082008

[71] A R Mire-Sluis Y C Barrett V Devanarayan et al ldquoRecom-mendations for the design and optimization of immunoassaysused in the detection of host antibodies against biotechnologyproductsrdquo Journal of Immunological Methods vol 289 no 1-2pp 1ndash16 2004

[72] G Shankar V Devanarayan L Amaravadi et al ldquoRecommen-dations for the validation of immunoassays used for detectionof host antibodies against biotechnology productsrdquo Journal ofPharmaceutical and Biomedical Analysis vol 48 no 5 pp 1267ndash1281 2008

[73] M Liang S L Klakamp C Funelas et al ldquoDetection ofhigh- and low-affinity antibodies against a human monoclonalantibody using various technology platformsrdquo Assay and DrugDevelopment Technologies vol 5 no 5 pp 655ndash662 2007


[74] J A Lofgren S Dhandapani J J Pennucci et al ldquoComparingELISA and surface plasmon resonance for assessing clinicalimmunogenicity of panitumumabrdquo Journal of Immunology vol178 no 11 pp 7467ndash7472 2007

[75] M K de Vries G J Wolbink S O Stapel et al ldquoInefficacy ofinfliximab in ankylosing spondylitis is correlated with antibodyformationrdquo Annals of the Rheumatic Diseases vol 66 no 1 pp133ndash134 2007

[76] M G Tovey and C Lallemand ldquoImmunogenicity and otherproblems associated with the use of biopharmaceuticalsrdquoTher-apeutic Advances in Drug Safety vol 2 no 3 pp 113ndash128 2011

[77] Z Li B Krippendorff S Sharma A C Walz T Lave and DK Shah ldquoInfluence of molecular size on tissue distribution ofantibody fragmentsrdquomAbs vol 8 no 1 pp 113ndash119 2016

[78] L Aarons ldquoPopulation pharmacokinetics theory and practicerdquoBritish Journal of Clinical Pharmacology vol 32 no 6 pp 669ndash670 1991

[79] S K Singh ldquoImpact of product-related factors on immuno-genicity of biotherapeuticsrdquo Journal of Pharmaceutical Sciencesvol 100 no 2 pp 354ndash387 2011

[80] K D Ratanji J P Derrick R J Dearman and I KimberldquoImmunogenicity of therapeutic proteins influence of aggre-gationrdquo Journal of Immunotoxicology vol 11 no 2 pp 99ndash1092014

[81] A S Rosenberg ldquoEffects of protein aggregates an immunologicperspectiverdquo AAPS Journal vol 8 no 3 pp E501ndashE507 2006

[82] M J Treuheit A A Kosky and D N Brems ldquoInverse relation-ship of protein concentration and aggregationrdquo PharmaceuticalResearch vol 19 no 4 pp 511ndash516 2002

[83] D R Jandrlic G M Lazic N S Mitic and M D PavlovicldquoSoftware tools for simultaneous data visualization and Tcell epitopes and disorder prediction in proteinsrdquo Journal ofBiomedical Informatics vol 60 pp 120ndash131 2016

[84] R R Mettu T Charles and S J Landry ldquoCD4+ T-cell epitopeprediction using antigen processing constraintsrdquo Journal ofImmunological Methods vol 432 pp 72ndash81 2016

[85] A S De Groot F Terry L Cousens and W Martin ldquoBeyondhumanization and de-immunization tolerization as a methodfor reducing the immunogenicity of biologicsrdquo Expert Reviewof Clinical Pharmacology vol 6 no 6 pp 651ndash662 2013

[86] R Jefferis ldquoGlycosylation as a strategy to improve antibody-based therapeuticsrdquo Nature Reviews Drug Discovery vol 8 no3 pp 226ndash234 2009

[87] A VonDelwig DM Altmann J D Isaacs et al ldquoThe impact ofglycosylation on HLA-DR1-restricted T cell recognition of typeII collagen in a mouse modelrdquo Arthritis and Rheumatism vol54 no 2 pp 482ndash491 2006

[88] Y H Messinger N J Mendelsohn W Rhead et al ldquoSuccessfulimmune tolerance induction to enzyme replacement therapy inCRIM-negative infantile Pompe diseaserdquo Genetics in Medicinevol 14 no 1 pp 135ndash142 2012

[89] K P Pratt ldquoEngineering less immunogenic and antigenic FVIIIproteinsrdquo Cellular Immunology vol 301 pp 12ndash17 2016

[90] F Deisenhammer ldquoInterferon-beta neutralizing antibodiesbinding antibodies pharmacokinetics and pharmacodynam-ics and clinical outcomesrdquo Journal of Interferon amp CytokineResearch vol 34 no 12 pp 938ndash945 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


incidence rates in gt90 of patients [9ndash12] These immuno-genic responses sometimes result in altered efficacy andimmunotoxicity profiles that can vary frompatient to patientImmunogenicity-related adverse events (AEs) may be asbenign as amild itchy rash or as serious as infusion reactionsanaphylaxis and cytokine release syndrome (CRS) [13 14] Asan example tumor necrosis factor (TNF) inhibitors can acti-vate autoantibodies leading to Drug-Induced Autoimmunity(DIA) [15] Luckily the majority of immunogenic responsesare not toxic and do not lead to sustained autoimmunepathologies Rather encounters with ADAs affect efficacyby either accelerating or impeding clearance [16 17] Itis in these instances that immunogenicity complicates theinterplay of pharmacokinetics (PK) and pharmacodynamics(PD) which further obfuscates the development of a reliablepreclinical model correlating with clinical events Withoutthese data research and development (RampD) are severelyimpaired further potentiating the already abysmal attritionrates (852) of biologics once they reach phase 1 clinicaltrials [18] As the library of biologics continues to grow sodoes our need to understand how immunogenicity affectsefficacy and safety Here we set out to describe the biologyof immunogenic response and its impact on the PKPDof biologics We do this in an effort to shed light on thechallenges and pitfalls facing the successful development ofsafe and efficacious biologics

2 Immunogenic Risk Factors and the Need forPredictive Tools

Attributes of both the biologic and the patient contributeto the risk of immunogenic response The antigenicity of atherapy along with patient immune status can be used topredict the occurrence and severity of a potential immuno-genic response Patients with hyperreactive immune diseaseautoimmunity or history of biotherapeutic administrationare all at an increased risk of reactions against biologicsConversely immunosuppressed patients are less likely tomount a response [19 20] Hypervigilant immune systemscan be especially problematic when treating autoimmunediseases with biotherapeutics For instance an observationalstudy monitored patients being treated for Crohnrsquos diseasewith infliximab (IFX) a chimeric anti-TNF-alpha therapy[21] It was found that patients with high anti-IFX antibodiesin serum and low trough IFX levels correlated with loweredefficacy or poor IFX persistence These clinical observationsand immunoassays generated a PK profile of IFX that allowedclinicians to monitor Crohnrsquos patients for bioanalytical mark-ers that predicted treatment outcomes in the context of ADANonetheless this kind of data is often not applicable tobiosimilars or other biotherapeutics and is difficult to validateacross institutions administering identical therapies due tovariances in sample collection in vitro immunoassays dataanalysis and records In order to standardize the clinicalassessment of immunogenic response procedures and stan-dards must be applied and interpreted by predeterminedcriteria The US Food and Drug Administration (FDA) andthe European Medicine Agency (EMA) provide regulatorydocuments detailing such guidelines [22ndash24]

Genetically derived markers predicting immunogenicitycould potentially be applied across all treatment groups andeven across biotherapies For instance the Human LeukocyteAntigen (HLA) genes that encode for Major Histocompati-bility Complex (MHC) class II haplotypes have been shownto play a role in the immunogenic response [25 26] Aclinical study monitored patients diagnosed with MultipleSclerosis (MS) treatedwith Interferon-Beta (INF-120573) An asso-ciation between a commonMHC class II allele (DRB1lowast0701)present in 23 of the patients studied and an increasedlikelihood of producing an ADA response against INF-120573was discovered Screening for this allele could guide futuretreatment plan decisions of MS patients Establishing reliableand clinically relevant PK profiles of biotherapeutics andidentifying genetic markers predictive of immunogenicitywill allow clinicians to more effectively monitor and guidebiotherapeutic administration

21 Predicting Antigenicity From discovery to clinicimmunogenicity must constantly be monitored yet ourcurrent preclinical models have poor predictive powerfor clinically relevant immunogenicity [27] For examplehumanized therapies are designed to evade the immunesystem of patients yet they are inherently antigenic inimmunocompetent animal models and often exaggerate theantigenicity of the therapeutic agent Nevertheless the effortto combine preclinical data from in silico in vitro and in vivomethods remains worthwhile and may one day yield validcriteria for immunogenicity risk assessment

In silico computational tools have broad applications inthe identification and assessment of antigenicity Some screenfor leads such as cancer neoantigens and peptides for vaccinetargets [28ndash30] Others troubleshoot antigenicity by optimiz-ing the design of peptide nanoparticles or by determiningdeimmunization methods for antileukemic L-asparaginase[31 32] As this technology matures and provides morereliable predictions we will improve our ability to navigatethe effects of immunogenicity

In vitro modelling of antigenicity utilizes T cell assays Bcell assays and mimotopes to identify potentially immuno-genic epitopes [33 34] whereas in vitro characterization ofimmunogenic response from serum samples includes qual-itative and quasiquantitative immunoassays such as enzymelinked immunosorbent assays (ELISAs) radioimmunoassays(RIAs) and immunoradiometric assays (IRMAs) [35] Cell-based assays remain a popular choice formodeling antigenic-ity via the immunophenotyping proliferation tracking andcytokine secretion profiling of immune cells [36 37] How-ever these techniques remain costly since they are labor andtime intensive In vitromodeling and in vitro characterizationwork together to describe mechanisms of immune activationas well as immunogenic markersphenotypes belonging toboth disease and nondisease states Ideally this informationcan be used to predict the magnitude and frequency ofimmunogenic response and to discern which patients aremost at risk of developing this response The limitations ofthe clinical utility of in vitro assays for immunogenic markerdiscovery were discussed in a previous section Howeverpredictive preclinical in vitro assays would help protect both





3 Immunotoxicity
































Competing Interests


Acknowledgments


References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Immunotoxicity
































Competing Interests


Acknowledgments


References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































Competing Interests


Acknowledgments


References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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