Antibody engineering and therapeutics, the annual meeting of the antibody society December 7-10

37 Pages·2016·0.96 MB·English

by  Matthias Pauthner

mAbs
ISSN: 1942-0862 (Print) 1942-0870 (Online) Journal homepage: http://www.tandfonline.com/loi/kmab20
Antibody engineering & therapeutics, the annual
meeting of the antibody society December 7–10,
2015, San Diego, CA, USA
Matthias Pauthner, Jenny Yeung, Chris Ullman, Joost Bakker, Thierry Wurch,
Janice M. Reichert, Fridtjof Lund-Johansen, Andrew R.M. Bradbury, Paul J.
Carter & Joost P.M. Melis
To cite this article: Matthias Pauthner, Jenny Yeung, Chris Ullman, Joost Bakker, Thierry
Wurch, Janice M. Reichert, Fridtjof Lund-Johansen, Andrew R.M. Bradbury, Paul J. Carter
& Joost P.M. Melis (2016) Antibody engineering & therapeutics, the annual meeting of
the antibody society December 7–10, 2015, San Diego, CA, USA, mAbs, 8:3, 617-652, DOI:
10.1080/19420862.2016.1153211
To link to this article: http://dx.doi.org/10.1080/19420862.2016.1153211
© 2016 The Author(s). Published with Accepted author version posted online: 24
license by Taylor & Francis Group, LLC© Feb 2016.
Matthias Pauthner, Jenny Yeung, Chris Published online: 24 Feb 2016.
Ullman, Joost Bakker, Thierry Wurch, Janice
MSu.b Rmeiict hyeorut,r Farritdictjloef t Lou tnhdis- Jjoohuarnnsael n, Andrew Article views: 1104
R.M. Bradbury, Paul J. Carter, and Joost P.M.
Melis
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Download by: [University of London] Date: 24 June 2016, At: 07:33
MABS
2016,VOL.8,NO.3,617–652
http://dx.doi.org/10.1080/19420862.2016.1153211
MEETINGREPORT
Antibody engineering & therapeutics, the annual meeting of the antibody society
December 7–10, 2015, San Diego, CA, USA
MatthiasPauthnera,JennyYeungb,ChrisUllmanc,JoostBakkerd,ThierryWurche,JaniceM.Reichertf,
FridtjofLund-Johanseng,AndrewR.M.Bradburyh,PaulJ.Carteri,andJoostP.M.Melisj
aDepartmentofImmunologyandMicrobiology,TheScrippsResearchInstitute,LaJolla,USA;bUniversityCollegeLondon,London,UK;cParatopixLtd,
Cambridge,UK;dScicomvisuals,Amsterdam,TheNetherlands;eCenterdeRechercheServier,France;fReichertBiotechnologyConsultingLLC,
Framingham,USA;gDept.ofImmunology,OsloUniversityHospitalRikshospitalet,Oslo,Norway;hLosAlamosNationalLaboratory,LosAlamos,USA;
iAntibodyEngineeringDepartment,Genentech,SouthSanFrancisco,USA;jGenmab,Utrecht,TheNetherlands
ABSTRACT ARTICLEHISTORY
The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society Received5February2016
united over800participants fromallovertheworldinSanDiegofrom6–10December2015.Thelatest Accepted8February2016
16 innovations and advances in antibody research and development were discussed, covering a myriad of KEYWORDS
20 antibody-relatedtopicsbymorethan100speakers,whowerecarefullyselectedbyTheAntibodySociety. Antibodyengineering;
e As a prelude, attendees could join the pre-conference training course focusing, among others, on the
n antibodytherapeutics;
u engineeringandenhancementofantibodiesandantibody-likescaffolds,bispecificantibodyengineering
J antibodyeffectorfunc-
4 andadaptationtogeneratechimericantigenreceptorconstructs.Themaineventcovered4dofscientific
2 tions;antibody-drugcon-
3 sessions that includedantibodyeffector functions,reproducibility ofresearchanddiagnosticantibodies, jugates;bispecific
3 new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new
don] at 07: tirrneeedcpsihiescntraatotoniloiocrneegssiet,otshaarcnonltiunidbgicoahadplipdeilimsisccmaottoviuoennhroisantrhfgnoe,errcsabospinsytpfi.hreTemchiifienccegAlalnuantnltaiibdbroocdidmayitemaSslo,ougacnniieenttgiybs’yonssdetsywepmethgc,eiearbrlamupsileleidnsusientiiogcIngsVcffoHoormcgunpesorennede-hcseo,annnasnci“evdArenoatvnIigbedVrocHodor-impegsheinnatgnoe annpnooretsloictbilgcionydai;cniieamtslib;mcouldinnieiocsta;hli;emdramiapguy--;
on watch”in2016.Anotherspecialsessionputthespotlightonthelimitationsofthenewdefinitionsforthe
L
f assignmentofantibodyinternationalnonproprietarynamesintroducedbytheWorldHealthOrganization.
o The convention concluded with workshops on computational antibody design and on the promise and
y
sit challenges of using next-generation sequencing for antibody discovery and engineering from synthetic
r andinvivolibraries.
e
v
ni
U Abbreviations:ADCC,antibody-dependentcell-mediatedcytotoxicity;ADC,antibody-drugconjugate;AE,adverse
y [ events;AML,acutemyeloidleukemia;BiTE,bi-specificTcellengagers;bsAb,bispecificantibody;CR,complete
b response;CDR,complementarity-determiningregion;DAR,drug-to-antibodyratio;Fc,fragmentcrystallizable;HIV,
ed humanimmunodeficiencyvirus;IgG,immunoglobulinG;IgVH,immunoglobulinvariableregionheavychain;IHC,
d
a immunohistochemistry;mAb,monoclonalantibody;MDR,multidrugresistance;NSCLC,non-smallcelllungcancer;
o
nl PDX,patient-derivedxenograft;PK,pharmacokinetics;RA,rheumatoidarthritis;scFv,single-chainvariablefragment;
w SCLC,smallcelllungcancer
o
D
MondayDecember7,2015,keynotepresentations glycoproteins.Thesedifferentstructurescanpresenthurdlesto
effectively create antibody therapeutics, since target sites are
JoostMelis
sometimeslostorhiddenorserveaspotentialdecoys.Research
has only partially elucidated how viruses can best be targeted.
The human anti-Ebola monoclonal antibody (mAb) KZ52
AntibodiesagainstEbolavirus:Aglobalcollaboration
from a 1995 survivor has been shown to neutralize the virus,
Erica Ollmann Saphire (The Scripps Research Institute) and is able to protect mice and Guinea pigs, but not non-
opened the conference with an overview of the work of the human primates. In 2011, cocktails combining several anti-
Viral Hemorrhagic Fever Immunotherapeutic Consortium Ebola mAbs protected against infection in non-human pri-
(VIC). The VIC is a global, field-wide open collaboration for mates, but at least one of these cocktails contained antibodies
antibodytherapeuticsagainstEbolaandrelatedviruses. thatwerenon-orweaklyneutralizing.In2012,manyquestions
Filoviruses,suchastheEbolaVirusandtheMarburgVirus, regarding cocktails needed answers: which type of cocktails is
have different structural manifestations of their envelope most suitable, which mAbs are possibly synergistic, which
CONTACT JoostP.M.Melis [email protected]
PublishedwithlicensebyTaylor&FrancisGroup,LLC©MatthiasPauthner,JennyYeung,ChrisUllman,JoostBakker,ThierryWurch,JaniceM.Reichert,FridtjofLund-Johansen,AndrewR.M.
Bradbury,PaulJ.Carter,andJoostP.M.Melis
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttribution-Non-CommercialLicense(http://creativecommons.org/licenses/by-nc/3.0/),whichpermitsunre-
strictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Themoralrightsofthenamedauthor(s)havebeenasserted.
618 M.PAUTHNERETAL.
mAbs are best to be included or which mAbs could possibly polymorphism and micro-neutralization titers to H5N1. Fur-
competewithoneanother,andwhichinvitroassaysshouldbe ther analyses indicated correlations between IGHV1-69 clonal
usedtoselectmAbs.Theseunknownscreatedacomplexprob- signature frequencies,copynumberandutilizationoftheanti-
lem that needed a significant sample size and input to solve. bodiesasBnAbs.Also,immunoglobulinlocuswideassociations
ThisinitiatedtheformationoftheVIC,whichisnowaimingto wereassessedandindicatedthatIGHV1-69F/Lpolymorphism,
mapepitopes,findpredictorsofwhatapproachworksbestand copynumber,andgeneduplicationaredifferentamongethnic
howtocreatethebestcocktails. groups. Genetic background should therefore be taken into
So far, the VIC has mapped the epitopes of 160 anti-Ebola account during vaccine development. Currently, several
mAbs and investigated which of these mAbs are able to neu- approaches toward structured-based universal stem-directed
tralize the virus and which are able to protect in vivo. Results influenza vaccines are ongoing. For these current and future
showedthatsomeepitopesorlocationsaremorelikelytocause developments, not only serological studies should be used, but
neutralization than others, however the potency to neutralize also genotyping and phenotyping signatures. Moreover, estab-
doesnotpredictthelevelofprotection. lishing an IGHV haplotype map could prove to be beneficial
Additionally,immuneeffectorfunctionsofmAbsmightalso fordevelopmentofBnAbs.
be of importance for efficacy. The VIC analyzed the effector
functionsrelatedtothe160availablemAbs.Theinitialanalyses
Aspatialsystemsbiologicalviewofcancer
demonstratedadiscrepancybetweenhumanandmouseresults,
andindicateddifferentassaysormodelswerenecessarytobet- JoeGray(OregonHealth&ScienceUniversity)thendiscussed
6 ter address the importance of effector functions. The Marburg a spatial systems biological view of cancer. To generate robust
01 virus is the next big challenge for the VIC, which is currently solutions to battle cancer, cancer should be managed as a het-
2
e mappingepitopesofavailablemAbsforthisvirus. erogeneous, adaptive system that includes extrinsic proximal
un influences, distal influences and intrinsic cell biology. This
J
24 Thebasisandbiasofhumananti-influenza- wholesystemtogetherdictatescellresponsessuchasprolifera-
3 tion or apoptosis and the eventual clinical outcome. Cancer
3 neutralizingantibodyresponses
7: heterogeneityexistsatmanyscales,fromtissuelevelalltheway
0
at The second keynote lecture was given by Wayne Marasco tomolecularstructures.Nowadays,imagingtechnologiesallow
] (Harvard Medical School). Certain scientific barriers interfere us to study and integrate biological systems at multiple scales.
n
do with developing a “universal” influenza vaccine, such as the Electron microscopy, conventional and super-resolution light
n
o antigenic drift that can abolish the binding properties of microscopy,plusanatomicalimagingsuchaspositronemission
L
f broadly neutralizing antibodies (BnAbs). However, very little tomography (PET), magnetic resonance imaging (MRI) and
o
y changes in the stem domain of the influenza virus occur in computerized tomography (CT) together with bioengineering
rsit respecttotheglobularheaddomain.Theinfluenzahemaggluti- orcomputationalbiologycanbeintegratedtoassessandman-
e
v nin (HA) stem is able to elicit a population of antibodies with age tumor heterogeneity. Intrinsic tumor heterogeneity can
ni
U broadneutralizingactivities,whicharealsorecalcitranttoneu- now be visualized in 3D by using technologies such as light
[
y tralization escape. BnAb F10 is able to bind all subtypes of sheet microscopy and tissue clearing; an example of this was
b
d group1HAforexample.F10blockspH-dependentconforma- shownfortriple-negativebreastcancer.
e
ad tional change and escape from F10 coincides with loss of viral Toaddressthequestionifintrinsictumorheterogeneityisof
nlo fitness. functionalimportance,acellsystemusing~80breastcancercell
w
Structural analyses of 38 stem-directed IGHV1-69-sBnAbs lines was used to model many aspects of tumor heterogeneity
o
D
showedseveralhighlyconserved aminoacids thatareessential such as genomic aberrations, transcriptional subtypes and dif-
forhighaffinitybinding,suchasphenylalanine52andtyrosine ferentiation state. Using MEK and PI3 K inhibitors, it was
97–99.Theseconservedaminoacidscreatepromisingopportu- shown that the differentiation state of cancer cells changes in
nities for development of BnAbs. IGHV3-30 germline gene- different directions. This is also reflected by dose-dependent
derived BnAbs contain a complementarity-determining region changes measured by several “Omics” techniques. Monitoring
(CDR) 3 HC hydrophobic pocket-stabilized epitope and are and directing these changes creates opportunities to possibly
effective against group 1 and 2 influenza A strains, which is steer cancer cells into a more homogeneous state that is more
broaderthanIGHV1-69BnAbs. easily treated. This was initially attempted by driving cancer
Part two of the presentation focused on the question if it is cells into a state of deep CK19C quiescence. However, once
possible to vaccinate all individuals of a population, since it is releasedfromselectivepressure,thecellsstartedgrowingagain.
known that there is quite some variation within populations, Another option is to prevent cells from undergoing epigenetic
bothpre-andpost-vaccination.Theresearchhereinfocusedon changes, and early results indicate that chromatin modifiers
the role of genetic V segment polymorphisms in anti-influ- appeartocounterresistance-associatedepigenomicevolution.
H
enzaantibodyresponses.Inaclinicalcohortof85H5N1vacci- To manage tumor extrinsic heterogeneity, microenviron-
nated individuals, genotype/phenotype BnAb responses were mental signals that influence tumor cell behavior should be
correlated.Forthis,theexpressedantibodyrepertoire,theeffect identified.Microenvironmentmicroarraysanalysiswasusedto
of IGHV1-69 genotype on clonal distribution, precursor fre- unravel which signals are important and which ones are not.
quencyandcopynumberwereanalyzed.Indifferentgenotypic Both soluble and insoluble proteins were printed on arrays on
groups, HA stem-directed Abs were monitored over time and which each spot hosts space for growth of »100 cells. Several
this analysis uncovered a correlation between IGHV1-69 aspectsoftumorbiologyweremeasuredonthesespots,suchas
MABS 619
morphology,metabolism,cellcycle,nuclearactivityandlineage products. ImmunoPET can provide info on tumor presence,
status. Some microenvironment proteins (MEPs) profoundly cell surface phenotype, target expression for therapy selection,
affected differentiation and proliferation of cancer cells. target occupancy, internalization/catabolism, response to ther-
Selected MEPs can convert gene targeted therapies into stimu- apy,andmechanisticorquantificationpurposes.Dataofa124I
latory agents. For example, NRG1 and HGF make neratinib PSCA-minibodyforquantitativeimmunoPETimagingofpros-
stimulatoryinHER2luminalcells,whichisprobablycausedby tate cancer and of an 89Zr-DFO-H2 anti-MET minibody mea-
trimerization of human epidermal growth factor receptors suring c-MET expression as a marker for resistance to anti-
(HERs).Thiscreatesopportunitiesofreceptormultimerforma- EGFRtherapywaspresented.
tiontomanageextrinsicmicroenvironment. Antibody-based imaging of both cancer cells and immune
Results of immuno-targeted siRNA delivery indicated cells can shed more light on immune responses, immune cells
siRNA can be used to overcome resistance of cancer cells to subsets, expansion, trafficking and activation. These imaging
HER2-targeting therapeutics. Anti-HER2-siHER2-NP demon- techniques can be applied in the field of inflammatory and
stratedtumorgrowthinhibitioninmicethatweretumorresis- autoimmune disease and cancer immunotherapy for instance.
tanttoTM1andpaclitaxel,butbecamesensitivetothesedrugs Since CD8 cytotoxicTcells are very potent immunecells, sev-
againbyadditionofthesiRNAtherapeutic. eralexamplesofCD8Tcellquantitationandbiodistributionin
Multispectral super resolution fluorescence microscopy can tissues were shown: 1) imaging using 89Zr-radiolabeled anti-
be used for direct assessment of receptor complexes on cancer CD8-169 cys-diabody successfully showed T cell repopulation
cells. 3D imaging of cancer cells and the interaction with the followingHSCtransplant,2)imagingofCD8Tcellinfiltration
6 microenvironment can be visualized and indicates where the in anti-PD-L1 tumor immunotherapy in syngeneic BALB/c
01 receptors are localized on cancer cells. Many of the receptors mouse model for CT26 colon carcinoma, and 3) imaging the
2
e arepresentonlongprotrusions.Functionalassessmentofpro- massiverecruitmentofTcellsintotumorandlymphoidtissues
un trusionsdemonstratedaclearroleinreceptortransport.Recep- upon 4–1BB antibody treatment. This work is now being
J
4 tors were shown to be moving along these protrusions and extendedtoCD4andCD20imaging.
2
3 receptors were visualized being transported between cells.
3
7: These results initiated the search for protrusion-targeted
0
at therapies. TrackA:Antibodyeffectorfunctions
]
on JennyYeung
nd Beyondbiopsies–enablingprecisionmedicine The afternoon session on antibody effector functions was
o
L throughantibody-basedimaging chaired by Paul Parren (Genmab) and Dennis Burton (The
f
o ScrippsResearchInstitute).
y ThefinalkeynotelecturewasgivenbyAnnaWu(DavidGeffen
rsit School of Medicine at University of California Los Angeles).
e
v Molecular diagnostics in oncology are mainly still performed
ni Suppressionofantibodyeffectoractivityduring
U bymaking use of in vitro-basedtechnologies, such as biopsies,
[ persistentvirusinfection
y blood tests and more novel techniques like circulating tumor
b
d cell detection or next-generation sequencing. Molecular imag- Persistentviralinfectionsareaglobalhealthconcern,withover
e
ad ing using PET can be used to assess the in vivo situation, ana- 500 million people infected with HIV and hepatitis B and C
o
nl lyzing living tissue in context and performing spatial and viruses. As discussed by David Brooks (Princess Margaret
w
temporal assessments. Since antibody-based therapeutics have Cancer Center and University of Toronto), what they all have
o
D improved significantly over the last decade, the use of anti- in common is a mechanism that evades or circumvents the
body-based products in molecular imaging can be meaningful immune response, creating an immunosuppressive environ-
for in vivo diagnostics. Several aspects of immune-conjugates ment. Understanding what governs immunosuppression may
forimagingwerediscussed,such astargeting strategies, conju- help to develop ways in which to restore immune function in
gation/linker strategies and signal considerations. Improve- persistentinfection.
ments in positron-emitting radionuclides have been made for The comparison of the humoral responses elicited by mice
theiruseinImmunoPET,e.g.,availabilityofisotopeswithlon- infectedwith2variantstrainsoflymphocyticchoriomeningitis
ger half-lives. Also, optimized antibodies for imaging are now virus (LCMV), which induced either an acute infection (Arm-
available; human or humanized antibodies show reduced strong) or a persistent infection (clone 13), led to the elucida-
immunogenicity,antibodyengineeringcaneliminateunwanted tion of a mechanism of immunosuppression mediated by
effectorfunctions andcreatesoptions forsite-specificconjuga- immune complexes. During persistent infection, high total
tion. Engineering can also influence the pharmacokinetics and antibody levels and high levels of immune complexes exist in
targeting capabilities of the antibody-based imaging product. tandem with decreased virus-specific antibody levels, which
TheuseofeitherintactIgGversusdiabodyorminibodymole- resultinsuppressionofantibodyfunctions.Usingexogenously
cules can greatly affect exposure and clearance, and conse- administered cell-depleting anti-CD4 or anti-CD20antibodies,
quentlythelevelofbackgroundactivity.Forinstance,anintact it was observed that depletion of target cells was significantly
antibodytakesaweektoclearfromthebodywhileadiabodyis suppressedinpersistentlyLMCVinfectedwildtypemicewhere
eliminatedwithin4–7hours,whicheffectivelymeansdiabodies antibodylevelswerehigh.Depletionoftargetcellswasrestored
can be used for same day imaging. Cysteine modification of in mice with compromised B cell responses and therefore low
diabodies allows site-specific conjugation and more uniform antibodylevels.
620 M.PAUTHNERETAL.
Invitroandinvivostudiesshowedthatthehighamountsof (conatumumab and tigatuzumab). In seven of 9 murine xeno-
(cid:1)
immunecomplexesgeneratedinpersistentlyinfectedwildtype graftmodels,theHexabody combinationshowedconsistently
mice led to compromised FcgR-mediated effector functions, better potency than conatumumab. The maximal therapeutic
such as macrophage phagocytosis and dendritic cell mediated effectwasachievedat0.5mg/kg(lowestdosetested)or2.0mg/
cross-presentation. Immune complex formation blocked acti- kg. These data demonstrate the therapeutic potential of the
vating FcgRs and resulted in suppression of IgG-mediated Hexabody(cid:1) platform for potentiating therapeutic antibodies
effector functions. This suppression of the immune system forthetreatmentofcancer.
functionisreversible,asexvivocellsfrompersistentlyinfected Afterthenetworkingbreak,DavidDiLillo(RockefellerUni-
mice had decreased levels of antibody on the cell surface after versity) discussed the long-term effects of immunotherapy.
resting, which subsequently restored phagocytosis. These data Recent insights showed that next to direct Fc-mediated cyto-
illustrate a new mechanism through which persistent virus toxiceffects,therapeuticantibodiesmayalsoinducepotentand
infectionscansuppresstheimmuneresponseandhaveimpor- long long-lasting vaccine effects. The potential of improving
tantimplicationforthedesignofantibodytherapiestocontrol this aspect, which is mediated via an interaction between anti-
persistentinfections.1 body Fc and the IgG receptor FcgRIIa, provides new opportu-
GeorgeGeorgiou(UniversityofTexas)nextpresentedaset nitiesformarryingvaccineandimmunotherapyapproaches.
of novel aglycosylated Fc domains with an exquisite ability to
specifically activate individual Fc-mediated effector mecha-
Neutrophilskillantibody-opsonizedcancercellsby
nisms. Notably, he demonstrated the importance of the long-
trogoptosis
6 disputed significance of in vivo complement activation in the
01 anti-tumoractivityofCD20antibodies. TimoK.vandenBerg(SanquinResearchandAcademicMed-
2
e ical Center) discussed the process of trogoptosis. Antibody-
n
u dependent cell-mediated cytotoxicity (ADCC), mediated by
J Functionalaspectsandtherapeuticapplicationof
4 natural killer (NK) cells and phagocytic cells such as macro-
2 antigen-dependentformationofIgGhexamersatthe
3 phages andneutrophils, contributeto theanti-tumoreffectsof
3 cellsurface
7: therapeuticantibodiesdevelopedforcancertreatment.Eachof
at 0 Esther Breij (Genmab) presented the Genmab Hexabody(cid:1) these effector cell types express Fcg receptors (FcgRs) and
] platform, which developed from their earlier elegant work form cell-to-cell synapses with their antibody-coated target
n
o
d showing how monomeric IgGs assemble into an ordered hex- cells. NK cells induce target cell apoptosis by release of gran-
n
o americstructurepromotestoactivationofthecomplementcas- zyme B and perforin, and macrophages phagocytose tumor
L
f cade.2,3 The understanding of this mechanism can aid or cellsanddestroythemthroughintracellularlysosomaldegrada-
o
y enhance the efficacy of therapeutic antibodies.4 Formation of tion. How neutrophils killantibody-opsonized tumor cells was
rsit the hexamers is determined by Fc:Fc interactions mediated by however not fully understood. As has been previously shown
e
v critical residues. Indeed enhanced formation of hexamers as a for macrophages, inhibition of the Signal Regulatory Protein
ni
U resultofmutationsinkeyresiduescanenhancebindingtoC1q Alpha (SIRPa)/CD47 signaling pathway potentiated cell death
[
y and enhanced complement-dependent cytotoxicity, which cre- ofopsonizedcancercellsbyneutrophils.
b
d ates potential for developing more effective therapeutics. The Formation of a cytotoxic synapse between the neutrophil
e
ad Hexabody(cid:1)platformcanbeappliedtoalargerangeofantibod- and opsonized target was a necessary pre-requisite for neutro-
o
nl iesandtodifferenttargets. phil-induced tumor cell death. Blocking SIRPa/CD47 interac-
w
Death receptor (DR5) is a receptor for TRAIL, which upon tion increased synapse formation, a process dependent on
o
D bindingtotheligand,initiatesan‘outside-in’signalingpathway CD11b/CD18 integrin. Live imaging studies showed that neu-
leading to apoptotic cell death of TRAIL-sensitive cancer cells. trophilsusedatrogocytosis-relatedprocess,termedtrogoptosis
While pre-clinical findings showed compelling anti-tumor (trogo is Greek for gnaw) to kill antibody-coated tumor cells.
activity,anti-DeathReceptorantibodieshavenotperformedas This novel cell death process did not involve anti-microbial
well in clinical trials. Cross-linking of DR5 is a key factor for properties of neutrophils, i.e., NAPDH oxidase or cytotoxic
the anti-tumor activity. Two anti-DR5 antibodies that do not granules release. Neutrophils from these patients with genetic
(cid:1)
competeforDR5bindingweredevelopedusingtheHexabody defectsinthesepathwayswerestillcapableofkillingantibody-
platformandstudiedinvitroandinvivo:Hx-DR5-03andHx- coatedtumorcells.
DR5-07. The potency of the Hx-DR5-03 and Hx-DR5-07 was During trogoptosis, neutrophils disrupt the plasma mem-
enhanced compared to the wild type IgG1 antibodies IgG1- braneofopsonizedtumorcellsbytearingoffpiecesatthecyto-
DR5-03 and IgG1-DR5-07. When used as a combination, the toxic synapse, leading to leakage of cellular contents and
(cid:1)
potency of the HexaBody molecules was enhanced even subsequent tumor cell death. Use of pharmacological com-
further, in a number of different DR5-positive cell lines. pounds showed that several signaling pathways were involved
The potency was relatively ratio independent, whereby a in the triggering of trogoptosis, including the calmodulin, Syk,
Hx-DR5-03:Hx-DR5-07 ratio of 9:1 or 1:9 was always more PI3K and MLCK pathways. There was evidence to show that
potent than the single antibodies, although a 1:3 or 3:1 ratio trogoptosisisapotentiallyrelevantprocessinvivoaswellasin
appearedtobeoptimal.Thekeyelementwasthatitwasamix- vitro. Using a murine melanoma model, GFPC neutrophils
ture of antibodies. To potently induce cytotoxicity, the Hx- could be correlated with death of anti-gp75-opsonized B16F10
DR5-03/07combinationdoesnotneedasecondarycross-link- melanoma cells in the liver of mice. Furthermore, in biopsies
ing agent, which is necessary for other anti-DR5 antibodies taken from HER2C breast cancer patients after trastuzumab
MABS 621
treatment, significant infiltration of neutrophils was apparent manufacturers may not solve the problem, because even good
andtheseneutrophilscontainedHER2Cintracellularmaterial. antibodies that are well characterized are not defined or
Insummary,trogoptosisisanovelformofneutrophil-medi- archivedforeverinawaythatallowsscientiststounequivocally
ated cell death of antibody-coated cancer cells, which can be usethesameantibodyasthatusedinapreviousstudy.
potentiated by disruption of the CD47/SIRPa pathway. In considering solutions, a key point is that research anti-
Together this could potentially improve the activity of thera- bodies are sold on the basis of what they (purportedly) recog-
peuticantibodiesagainstcancer. nize,nottheirphysicalidentity.Dr.Bradburymadeitclearthat
Marie Kosko-Vilbois (NovImmune) continued on this antibodies should be well characterized and validated; this is
theme by presenting a bispecific antibody approach. Bispecific the solution to poorly functioning antibodies. However, in
antibodiescomprisingaCD47bindingarmoptimizedforaffin- order to resolve the problem of irreproducibility, antibodies
ity,atumorantigenbindingarmandanactiveIgG1Fcdomain need to be expressed recombinantly and identified by unique
provide a novel means for specific and enhanced tumor cell “bar codes,” i.e., their publicly available sequences. This would
killing. address reproducibility issues, as researchers can then repeat/
reproduceexperimentswiththesamereagentsasthoseusedin
previouspublications.Goodbinderswillbecomeimmortaland
TrackB:GettingtoreproducibilityinresearchandDx
neverlost,andcompletecharacterizationisrequiredonlyonce.
antibodies
In fact, some non-therapeutic antibody companies are selling
JaniceReichert,AndrewBradbury,FridtjofLund-Johansen recombinants,butnotrevealingsequences.TheseincludeAbD-
6 Asresearchreagents,commerciallyavailableantibodieshave Serotech and Creative Diagnostics (that use in vitro methods),
01 been widely criticized for being poorly characterized, and for Biosite, REAfinity and ABfinity (that generate recombinant
2
e yielding irreproducible experimental results because they are antibodies from immunized animals), Absolute Antibodies
un either not of the correct specificity or not suitable for the type (makerecombinantantibodiesbysynthesisinggenesfrompat-
J
4 of experiment.5 In this session chaired by Andrew Bradbury entsequences)andAbCam(»5,000recombinantrabbitmono-
2
3 (Los Alamos National Laboratory), speakers from the aca- clonals,originallyderivedbyEpitomics).
3
7: demic, non-profit and commercial sectors reiterated the prob- Dr. Bradbury stated that the problem needs to be solved by
0
at lem,discussedtheapproachescurrentlyusedtoamelioratethe many different constituencies, including the funding agencies,
] situation,andproposedsolutions. journalsandcommercialantibodyproviders.Fundingagencies
n
o
d can contribute by insisting that all antibodies used in research
n
o aresequencedandrecombinantlyexpressed,withtheirsequen-
L Atthecrossroads:Gettingtoreproducibleresearch
f cespublishedinpublicallyaccessibledatabases.Asthisisavery
o antibodies
y ambitiousgoal,itwillrequirescientiststobeinformedofthese
rsit Andrew Bradbury provided an overview of the nature and requirements with a long lead time (e.g., 5–10 years). Because
e
v scaleoftheproblemswithresearchantibodies,andsuggesteda ofthecommercialconsiderations,itislikelythatthiswouldbe
ni
U solution.Theresearchantibodybusinessincludesover300ven- best implemented if funding agencies established specialized
[
y dors(aslistedonantibodyresource.com) and>2,000,000 anti- centers for binder selection, sequencing and characterization
b
d bodies (as listed on CiteAb). Many companies set up their that would be able to provide well characterized sequenced
e
ad developmentormanufacturingsitesoverseastotakeadvantage antibodies to scientists, much like NCI, or Neuromab.
o
nl of cost advantages, and most producers sell their antibodies to Publishers can contributebyInsisting that thesequences of all
w
multiple vendors, who add their own labels. The vendors also antibodies used in published papers are disclosed (e.g., within
o
D buyandsellfromeachother,andthenaddtheirownlabels.It 5–10 years), and that methods descriptions include antibody
isthuspossibletogetthesameantibodywhenpurchasingfrom concentrationsandthebuffersused.Antibodysequenceswould
different vendors. Characterization is expensive (>$200 for a bebest disclosedusing anaccession numbersystemthatcould
western blot; >$60 per IHC slide), so data from vendors is becitedinpublications.Ideallythiswouldbeinaseparatefield,
oftenminimalandhistorical(i.e.,itoftendoesnotapplytothe whichwouldallowautomaticarchiving.Heacknowledgedthat
specificlotpurchased).Thelargenumberofantibodiesagainst some companies are not keen on the idea of publishing anti-
popular targets (e.g., receptor-type tyrosine-protein phospha- body sequences as they are considered intellectual property,
tase C is targeted by 3255 antibodies; 42 proteins have over and there are no cheap ways to protect antibody sequences,
1,000 antibodies; 3483 proteins have 100 or more antibodies) since antibodies used in research do not make enough money
makes the selection of effective and reproducible antibodies to invest in patenting (see the description of Dr. Polakiewicz’s
extremely challenging. Dr. Bradbury noted that substantial seminarbelow).Onewayto address company concerns would
time and money (estimated at >$1.1 billion annually world- be the publically funded, specialized centers for binder selec-
wide)iswastedonbadantibodies.Thisrepresentsonlythecost tion, sequencing and characterization described above. In this
of the defective reagents themselves, and takes no account of scenario, once antibodies are derived and characterized, compa-
the money and time wasted on additional secondary reagents, nies could compete on the production, cost or quality of the
or trying to reproduce irreproducible experiments. In fact, publicly available sequence validated antibodies. Dr. Bradbury
poorlycharacterized,irreproducibleantibodieshavebeencited alsoraisedthepossibilitythattechnologymaybedevelopedthat
asbeingoneofthecommonestcausesofpublicationsthatcan- renders the generation, characterization and production of anti-
notberepeated.6-8Unfortunately,betterantibodycharacteriza- bodies so cheapthatpublicationofsequence maynotbecomea
tion or ordering from only the best, most reliable concern.However,hethoughtthiscouldtakesometime.
622 M.PAUTHNERETAL.
In conclusion, Dr. Bradbury described progress made by a long-standing problems with these reagents. He discussed the
number of organizations. The Global Biological Standards fact that flawed assumptions and experiments lead to the
Institute (GBSI) has established a Research Antibodies and reportingofflaweddataandconclusions,andacascadeofneg-
Standards Task Force that will focus on the application of ativeeffectscanoccurwhenresearchersthenattempttofollow
standards thatcanadvancethequality,efficacy,andreproduc- up on the flawed work. As an example of this, Dr. Elliott
ibility of antibodies used in basic and preclinical research and reviewed flawed data that suggested erythropoietin stimulating
development. The objectives of the Task Force are to identify agents promoted tumor cell growth through activation of
specific areas where standards for research antibodies are EpoR.Dr.Elliottrelatedthatheandcolleaguesinvestigatedthe
urgently needed, what types of standards could be designed specificity of the anti-EpoR polyclonal antibodies used for the
andimplementedtoaddresstheseneeds,andhowtheserecom- immunoblottingandimmunostaining,andshowedthatinreal-
mendationscanbeeffectivelyimplementedbycommercialpro- ityonlyone was suitable for usein immunoblotting,andnone
ducers and vendors. With The Antibody Society, GBSI is were suitable for immunohistochemistry.11 Use of these anti-
organizing a meeting at Asilomar in the fall of 2016 for all bodies,however,hascontinuedandledtowidespreaddissemi-
stakeholdersontheuseofantibodiesinrigorousandreproduc- nation of misinformation about the receptor. Dr. Elliott then
ibleresearch.TheHumanProteomeOrganization(HUPO)has related a more detailed examination of the problem. Of 219
establishedanAntibodyStandardsCommitteewiththegoalto studies of the “EpoR cancer” hypothesis, only 10% were prop-
define and propose common standards for affinity reagents, erly controlled, i.e., used positive and negative control cells or
and to engage the broader scientific community in dialog tissue,usedvalidatedreagentsandreporteddatathatsupported
6 around this issue. Ultimately the goal would be to publish theconclusions.Atotalof140ofthe219studiesincludedanti-
01 guidelines to be endorsed by publishers that offer guidance to EpoR antibody data, and only 8 of these were properly con-
2
e bothantibodyusersandproviders.TheFederationofAmerican trolled,i.e.,hadEpoRpositiveandnegativetissuesorcelllines,
n
u Societies for Experimental Biology (FASEB), is organizing and, for westerns, size markers were used and band matched
J
4 workshops on the use of antibodies in rigorous reproducible the size of EpoR. Notably, none of the studies that reported
2
3 research, and is also planning to publish guidelines on the use resultssupportiveofthehypothesiswerecontrolled.Dr.Elliott
3
7: ofantibodiestobeadoptedbyresearchers. highlighted the specific problems with many of the studies,
at 0 includinguseofnon-specificanti-EpoRantibodies,inappropri-
] ate or insufficient use of controls and the reporting of conclu-
on Useandabuseofantibodiesinresearchandtheclinic
d sions that were not supported by the data. He then elaborated
n
o DavidRimm(YaleUniversity)notedthattheproblemsassoci- on the difficulty of validating antibodies. False-positive results
L
f ated with antibody reagents sold by some vendors pose a bar- arecommon,evenwithwesterns,andmaybedifficulttodetect.
o
y riertosuccessfultranslationalresearch.Forexample,inastudy For example, a band on a gel may be the correct size, but the
rsit reportedin2008,9thesuccessratesforvalidationof5436anti- bandmayresultfromoff-targetbinding.Inaddition,false-pos-
e
v bodiesfrom51providersforuseinWesternblottingandtissue itive data may be tissue specific. Thus, investigators must be
ni
U microarrays was only 49% on average, and ranged from 0 to extremely cautious, always use appropriate positive and nega-
[
y 100%. Prof. Rimm indicated that this unpredictability as to tive control samples and include additional confirmatory data,
b
d whetherantibodyreagentsaresuitableforspecificapplications e.g., duplicate experiments with a second validated antibody.
e
ad means that researchers must validate the reagents to ensure Dr.Elliottconcludedbystatinghisviewonthetopic:Inscien-
nlo that their experiments yield scientifically rigorous and repro- tific endeavors it is rewarding to be right. But it takes courage
w ducible data. He then discussed the Rimm Lab’s algorithm,10 toadmityouarewrongandthencorrecttherecord.Failureto
o
D
which is focused on validation of antibodies for immunohis- dosomaybefarmoredamagingthanyourealize.
tochemistry(ICH)orquantitativeimmunofluorescenceonpar-
affin-embedded tissues, but could be equally valid or modified
Improvingantibodyqualitybycombiningnovel
for other antibody-based assays. Although antibody validation
technologyandclassicconceptsforresearchantibody
by the end-user is time-consuming, Prof. Rimm emphasized
discoveryanddevelopment
theneedforquantitativeanalysis,assessmentofsensitivityand
specificity, as well as reproducibility across lots, platforms, RobertoPolakiewicz(CellSignalingTechnology)providedthe
operatorsandlabs.ForbothICHandscientificresearchappli- perspective of Cell Signaling Technology (CST), a commercial
cations, use of monoclonal and preferably recombinant anti- supplier of research antibodies. He first acknowledged that
bodies was recommended. In concluding Prof. Rimm noted commercial antibodies are getting bad press. One issue is the
that, although companies should in theory validate antibodies, economicsofthemarketforresearchantibodies,whichissmall
the end users are ultimately responsible for the accuracy of compared to the markets for diagnostic and therapeutic anti-
their experimental data, and therefore the quality of the anti- bodies(»$2.5bnvs$15bnand$55bn,respectively)andcom-
bodiestheyuse. posedofover300providersthatofferover2.5millionantibody
reagents. Suppliers may be aggregators or re-sellers of the
Nonspecificantibodies,whatcouldpossiblygo reagents, and many do not validate their products or provide
validationdata.Theproblemsareexacerbatedbypoorornon-
wrong?theEpoRstory
existent quality-control (QC) systems or technical support. He
Steve Elliott (Amgen) who has extensive knowledge of anti- then differentiated CST’s approach by noting that their mAbs
bodies targeting the erythropoietin receptor (EpoR), discussed are primarily recombinant mAbs and that CST emphasizes
MABS 623
application validation, QC and technical support. CST does concluding, he discussed steps that could yield substantial
95% of antibody development and all product-manufacturing improvements in preclinical reproducibility rates, including
in-house. encouragingvendorstoofferonlyvalidatedreagents(e.g.,anti-
Dr.Polakiewiczthendiscussedtheprocessofmakingagood bodies, cell lines) and broad utilization of these reagents by
mAb,includingproductdesign,antibodycapture/isolation,val- principle investigators; ensuring that research funder policies
idation, formulation, and the use of appropriate screening require documented use of validated and non-contaminated
assays at each step. He emphasized that appropriate controls reagents and adequate funding to cover these additional costs;
and testing on multiple applications to verify specificity are ensuring that procedures to document reagent validation are
important,andthateachlotmustbeassessedandcomparedas requiredbypublishers.12
part of the QC process. Such a process requires investment of
scientific expertise, time and money. Sequenced antibodies
have been proposed as a solution to the problems associated Reproducibilitycrisis:Don’tblametheantibodies,
with research antibodies, but Dr. Polakiewicz noted that plac- arraythem
ing the sequences in the public domain jeopardizes the invest-
Fridtjof Lund-Johansen (Oslo University Hospital) argued
mentinvalidationandsupportbecausetherevenuefromeach
that researchers may be ignoring a large number of excellent
productistoosmalltojustifycostsofpatenting(upto$300k)
antibodies. Currently, sales are largely limited to a small num-
and potential litigation. In concluding, Dr. Polakiewicz pro-
berofreagentsthathavebeenusedinmanypublications.How-
vided recommendations that, if followed, could alleviate the
ever, there is no evidence that top-cited antibodies are top
6 issuesassociatedwithresearchantibodies.Inparticular:1)anti- performers. To find good antibodies with few or no citations,
01 body suppliers should validate properly, share data (excluding
2 Prof. Lund-Johansen and co-workers have developed array
ne sequences) and provide customer support; 2) antibody users technology thatallows parallel validation ofthousands of anti-
Ju shouldtrustcompaniesthatvalidateandsharedata,followpro- bodies. In Microsphere Affinity Proteomics (MAP), antibodies
4 tocols suggested by the vendor, use antibodies for recom-
2 bound to color-coded microspheres are used to capture bioti-
3 mended applications, seek technical support, avoid purchasing
3 nylated proteins from cell lysates. In a multiplexed version of
7: from large aggregators offering antibodies of unknown origin,
0 the protein gel blot, the labeled proteins are denatured, sepa-
at andprefermonoclonals(recombinants,ifavailable);3)journals rated by gel electrophoresis and eluted into liquid fractions.
] shouldrequireprecisecitationofantibodysources(e.g.,catalog
on The fractions are next incubated with microsphere-based anti-
d and lot numbers), validation data, and detailed protocols for
n body arrays, and proteins captured onto the surface of the
o experiments involving use of antibodies; 4) independent inter-
L microspheres are labeled with fluorescent streptavidin and
of net sources should help disseminate data, citations and user detected by flow cytometry. In a separate assay referred to as
y feedback; and 5) academic and funding institutions should
rsit train young investigators on how to find and use good Native-MAP, sample proteins are first separated according to
e subcellular localization and then by size-exclusion chromatog-
v antibodies.
ni raphy to resolve monomeric proteins and protein complexes.
U
[ Numerical data from MAP are visualized as line plots that
y
b Closingthereproducibilitygapwithstandardsand showantibodycaptureacrossthefractions.Differenttargetsof
d
e bestpracticesforantibodies thesamecaptureantibodyareseenasdiscretepeaksacrossthe
d
a
o fractions. To discriminate those that correspond to intended
wnl GlobalBiologicalStandardsInstitute antibodytargetsandcross-reactivity,analiquotofeachfraction
Leonard Freedman (Global Biological Standard Institute)
Do isanalyzedbymassspectrometry.Thusfar,theMAPtechnique
madethecasethatstandardsfacilitatethealignmentofconsen-
has been used with more than 3000 commercially available
sus-based best practices, reduce variance, and improve repro-
antibodies, and the results show that »10% of antibodies with
ducibility in biomedical research. He discussed the progress
no or few citations outperform top-cited products. Attendees
made with establishing standards for authentication of human
asked whether MAP can predict performance of antibodies in
cell lines using short tandem repeat DNA profiling, which is a
standardapplicationssuchaswesternblottingandimmunohis-
well-established technique that unambiguously characterizes a
tochemistry (IHC). Prof. Lund-Johansen replied that there is
number of different loci in the human genome, but noted that
good correspondence between MAP and protein gel blotting,
there are no universally-accepted guidelines or standardized
andthatstudiesonIHCareinprogress.Fromatheoreticalper-
methods for validation of research antibodies. Dr. Freedman
spective,antibodiesthatperformwellincapturearelikelytobe
notedthattheGlobalBiologicalStandardsInstitute(GBSI)has
high affinity reagents, and since the assay is used both with
launched a research antibody validation initiative. The
nativeanddenaturedproteins,onewouldexpectthatmostrel-
“Research Antibodies: Solutions for Today and Tomorrow”
evantepitopesshouldbecovered.
workshop GBSI is organizing with The Antibody Society, has
the goals to: 1) identify a set of standards to validate research
antibodies,includingrecommendationsforadoptionbyacade-
TrackA:Newaspectsofprodrugtargeting
mia, industry, funders, and journals; 2) develop recommenda-
tions for an independent proficiency certification system or ChrisUllman
open access user ratings service; and 3) develop recommenda- The second day of the conference opened with a session
tions, timeline, and follow-up plan for the introduction of chaired by Andreas Plu€ckthun (University of Zu€rich) that
sequenced recombinant antibodies as research reagents. In focused on Prodrug targeting, addressing the challenge of how
624 M.PAUTHNERETAL.
antibodies and antibody drug conjugates can be engineered to growtharrestofpathway-addictedcells.Goodaffinitiesagainst
bemoreselectivefordiseasetissue. bothmoleculeshavebeenachieved.
Theseexamplesservedtodemonstrateinterestingfunctional
formats for bispecifics, but the toxicity associated with some
Proteasemediatedactivationoftissue/tumor-
targets can be problematic. A proposed solution is to enable
targetingantibodiesandprodrugapproaches
local activation of function through protease cleavage, particu-
Ulrich Brinkmann (Roche) opened this session. The basis of larly relevant when binding to death receptors or targets that
the approach was an adaption of the use of bi/multi-specific are also expressed on non-diseased tissue.14 Overexpression of
antibodies,usedtomodulateproliferativeorinflammatorypro- proteases in tumors can be exploited by activating the binding
cesses. There are over 50 different bispecific formats described capacity of half a bispecific. Thus, half of the bispecific tethers
inthe literature, which allows the researcher to adjust the size, the molecule to the diseased cell, thereby bringing a cleavable
valency, flexibility, half-life and biodistribution offering an linker in proximity to the protease to allow activation of the
overwhelming choice.13 Brinkmann stated that he was not a second binding modality. This cleavable disulfide-stabilized Fv
believer in one size and one format fits all diseases and all tar- formatwasdescribedforabispecifictargetingHER3andcMet,
gets, thus rather than to ask which format is best, it is best to inwhichthebindingofcMetisdrivenbyaV andV domain
H L
consider which format is optimal for which disease. Modifica- at the C-terminus of the heavy chain that is activated through
tionstothestructuresofthebispecificcanaddadditionalselec- cleavage by an enzyme (such as MMP2 or 9, urokinase-type-
tivitysothattheybecomeactiveonlyatthedesiredlocation. plasminogen activator or furin). The two anti-cMet variable
6 Ingeneral,thereare2groupsofbsAbs,thosethathaveanFc domains are linked by a disulfide bond (VH 44- VL 100) that
01 andthosethatdonot.Thenon-Fc-containingbispecificsfunc- maintains pairing following the cleavage of the protease sensi-
2
e tionbyformingabridgebetweenTcellsandcancerouscelltar- tive linker ((G4S2)2-protease site (G4S2)2) connecting VL to
un gets. Examples of these formats are Amgen’s bi-specific T cell CH3. The cloaking of the binding site affects on-rate of bind-
J
4 engagers (BiTEs) or Macrogenic’s Dual-Affinity Re-Targeting ing,butnottheoff-ratetocMet,andreducesaffinityforrecep-
2
3 (DART) fragments. A disadvantage of the format is the short torswherethereisanabsenceofprotease.
3
7: half-life, which may require products to be administered via
0
at continuous intravenous infusion. Inclusion of an Fc confers Probodytherapeuticsredefinethelandscapeof
] advantages of solubility, stability, half-life extension and effec-
don tor function, each of which can be modified by genetic engi- antibodymodalitiesinoncology
n
o neering.PurificationisalsoeasierforFc-containingmolecules. The theme was continued by Luc Desnoyers’ (CytomX) pre-
L
f The production of such multichain bsAb can be improved by sentation “ProbodyTM Therapeutics Redefine the Landscape of
o
y ‘knobs-into-holes’ technology, or variations of this theme, to Antibody Modalities in Oncology.” CytomX’s Probody tech-
rsit ensurecorrectheavychainassembly. nology differs by the fact that the antigen binding site of the
e
v Dr. Brinkmann offered examples from the Roche Group. antibodyismaskedwithapeptidelinkedtotheN-terminusof
ni
U RG7716 (in Phase 1for wetage-related macular degeneration) thelightchainthroughaproteasecleavablelinkerandisappli-
[
y andRG7221(inPhase2forcolorectalcancer)werebuiltusing cabletotherapeuticsandimaging.15,16Inthecaseofanti-EGFR
b
d CrossMabtechnology,whichallowscorrectheavy-lightpairing Probody PB1, the 21–amino acid masking peptide is recombi-
e
ad drivenbyswappingtheCH1ofoneheavychainwiththecorre- nantly fused tothelight chain through a26–amino acidlinker
nlo sponding CL region of the light chain and vice versa. These carrying the 8-residue protease cleavable substrate, flanked by
w antibodiessimultaneouslyinactivate2ligandsthataremodula- Gly-Ser–rich peptide linkers. Protease activation of the Pro-
o
D
tors for angiogenesis - angiopoietin2 and VEGF-A. The bsAb body antibody is tightly regulated, producing binding when
binds each ligand with each arm of an IgG-like molecule con- cleaved by tumor-associated proteases, such as urokinase-type
taining functional Fc region and prevents activation of their plasminogenactivator(uPA),membrane-typeserineprotease1
respectivereceptors,Tie-2andVEGFreceptorkinases.Thebis- (MT-SP1/matriptase), and legumain, a lysosomal protease
pecific format offers advantages as 2 factors are depleted, foundtobereleasedandactiveintheacidicextracellulartumor
thereby allowing treatment of tumors that escape angiogenic microenvironment.15 This potentially provides a better safety
singleanti-VEGFtreatment. profileandspecificexamplesweredescribedincludingCX-072,
RG6013(developedbyChugai),usingacommonlightchain a protease-cleavable anti-PD-L1 Probody therapeutic. CX-072
approach to simplify production, targets FIXa and FX for has been shown to provide equivalent anti-tumor activity in
hemophilia in a full IgG format. The bispecific binding brings micetothatofitsparentalantibody,whileminimizingbinding
together2proteinsofthesamecascadeintothesamecomplex to peripheral cells through local activation within the tumor,
that is necessary for sufferers of hemophilia A who lack factor thereby reducing induction of autoimmune diabetes in NOD
VIIIa, which naturally complexes FIXa and FX. The antibody mice caused by the parental antibody. Further examples
hasadvantagesoversupplementationofFVIIIbyhavingalon- included anti-human CD166 Probody drug conjugate (PDC).
ger half-life in vivo and is less immunogenic in patients. In CD166isnotanobvioustargetforanantibody-drugconjugate
addition, a dual action Fab (DAF) antibody directed at HER1 (ADC) because it is expressed on normal tissues, but is highly
andHER3,isinPhase2clinicaltrialforheadandneckcancer. expressed in many cancers. The anti-CD166 Probody-SPDB-
ThisantibodysimultaneouslyblocksHER1andHER3through DM4therapeuticwasselectedbecauseitbindshumanandcyn-
bindingthesamevariableregions,potentiallyimprovingcancer omolgus CD166, but doesn’t bind to the rodent equivalent. It
treatment by inhibition of MAPK and AKT signaling and was well tolerated at 5 mg/kg, with no evidence of on-target
MABS 625
toxicity, and off-target toxicity typical of SPDB-DM4 noted at developed as a prodrug and both linear and branched PEG
15 mg/kg. This study demonstrates the potential use of Pro- moieties were added at specific sites using a linker designed
body drug conjugates for promising and previously inaccessi- to be susceptible to tumor-specific protease cleavage,
ble cancer antigens through reducing the collateral damage to unmasking its full activity through loss of PEG. The native
normal tissues. A Probody T cell-binding bispecific (TCB) REDLK sequence of the toxin was replaced with the canonical
platform has also been developed by creating an anti-CD3 eukaryotic ER-retention signal KDEL. Two key positions, one
Probody bispecific. T cell engagers have shown efficacy in within the catalytic domain and one close to the C-terminal
00 00
treating hematologic malignancies, but can have toxic side- KDEL sequence of Ec1-ETA (Ec1-ETA 486Aha-AhaKDEL-
effects due to binding healthy tissue. An anti-EGFR/anti-CD3 3C-PEG) were identified that lowered cytotoxicity 1000-fold
C
Probody TCB eliminated established EGFR HT-29 colorectal in EpCAM-positive tumor cells when both were PEGylated
tumors in human T cell engrafted NSG mice, having potency with 20 kDa linear PEG. However, following proteolytic cleav-
within 2- to 3-fold of an antibody-TCB, but gaining a >30- age of the linker-PEG moiety, the molecule was fully potent.
00
fold increase in the maximum tolerated dose in cynomolgus The PEGylated Ec1-ETA was much better tolerated than
00
monkeys relative to the Ab-TCB. Again this demonstrates the Ec1-ETA , providing a longer circulation half-life (82 min
potential benefit to safety as CytomX have noted limited leak- compared to 7.5 min for the unPEGylated format) and an
age of the Probody therapeutic from the sites of cleavage almost 10-fold increased area under the curve (AUC), follow-
withintumors. ingsystemicintravenous delivery.17
Dr. Desnoyers also commented that the masking of each Intracellular delivery was also investigated using fusions
6 paratoperequiredaspecificpeptidemaskthatcanreduceaffin- of EpCAM-targeting DARPins with the full-length pore-
01 itybyupto1000-fold,although,forexample,acetuximabPro- forming protein of anthrax toxin, “protective antigen”
e 2 body therapeutic demonstrates a »30-fold reduction. (N682A/D683A mutant), or the translocation domain of
un Immunogenicity hasn’t been studied widely, but in proof of ETA (252–412) and cargo DARPins. Interestingly, for the
J
4 conceptstudieswithaProbodyvariantofcetuximab,therewas anthrax toxin, the stability of the cargo had to be less than
2
3 noincreaseintheanti-Igresponse. the threshold thermodynamic stability for anthrax pores
3
7: due to the requirement for unfolding in retrograde translo-
0
at Probodytherapeuticsredefinethelandscapeof cation. Therefore, a destabilized DARPin mutant carrying a
] point mutation was necessary. Assays monitoring delivery
don antibodymodalitiesinoncology were developed that required biotinylation of an AviTagTM
n
o Andreas Plu€ckthun (University of Zu€rich) addressed the sequence (carried on the cargo) by prokaryotic biotin ligase
L
f theme from an alternative angle in his lecture, presenting data (BirA) expressed in a FlpIn 293 EpCAM/BirA cell line or
o
y for designed ankyrin repeat protein (DARPin)-toxin fusions MCF7 cells that transiently overexpress BirA. Studies using
rsit whoseselectivitywasengineeredbybioorthogonalveiling. the ETA domain and phosphorylated ERK-binding N2C
e
v DARPins are a non-antibody class of small protein affinity DARPin demonstrated efficient uptake and high nM con-
ni
U reagents engineered from natural ankyrin repeat proteins, one centrations of delivered cargo, even saturating the pathway.
[
y of the most common binding proteins in nature and responsi- Importantly, the modular systems described in the presenta-
b
d ble for diverse functions such as cell signaling and receptor tion have the potential to overcome critical barriers for the
e
ad binding. The DARPins have favorable properties of high therapeutic use of toxins to modulate targets beyond the
nlo potency, high stability, high affinity, flexible architecture and reach of current biological drugs in a safe manner.18
w
ease of production that can be used singularly or in combina-
o
D
tionin2ormoretobindmultipletargetsorepitopes.Examples
Paretooptimalbiotherapeuticdeimmunization:P99
of clinical phase molecules are Abicipar, an anti-VEGF-A
beta-lactamaseasacasestudyforADEPTfusion
DARPin forwetAMD entering Phase 3andMP0250,an anti-
partners
HGFandVEGFbispecificDARPininPhase1clinicalstudyfor
solid tumor cancers and hematological malignancies (devel- Antibody-targeted toxins and prodrug converting enzymes
opedbyMolecularPartnersAG). can be powerful anticancer treatments, but therapeutic pro-
The DARPins are free from Cys and Met and can be tein payloads present a risk of undesirable immunogenicity.
made Lys-free, thereby offering choices for coupling drugs, Karl Griswold (Thayer School of Engineering) described
such as monomethylauristatin F toxin and Pseudomonas experimental validation of integrated protein deimmuniza-
aeruginosa exotoxin A (ETA), through compatible biorthog- tion algorithms that deplete immunogenic T cell epitopes
onal click chemistry. However, reducing the toxicity caused while maintaining protein activity. The Pareto optimal
by target-independent uptake of such molecules fused to methods efficiently and accurately mapped the protein
biological drugs remains a challenge. Therefore, Plu€ckthun design space, enabling aggressive molecular engineering that
and colleagues have developed methods to cloak DARPin- balances the tradeoffs between immunogenic potential and
ETA fusions to improve pharmacological properties using therapeutic function.
site-specific addition of polyethylene glycol (PEG) moieties
using azide alkyne cycloaddition. PEG serves as a veil to
CRISPRlibrariesforfunctionalgenomics
reduce immunogenicity, improve selectivity and improve
the half-life of the molecule. An anti-EpCAM DARPin David E. Root (Broad Institute of Harvard and MIT) dis-
00
(Ec1) and a domain I-deleted variant of ETA (ETA ) was cussed CRISPR, which has emerged as a powerful tool for