Antibody Drug Conjugates for Cancer Therapy
Antibody Drug Conjugates for Cancer Therapy
Antibody Drug Conjugates for Cancer Therapy
- No tags were found...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
<strong>Antibody</strong> <strong>Drug</strong> <strong>Conjugates</strong> <strong>for</strong> <strong>Cancer</strong> <strong>Therapy</strong>2010 AAPSNational Biotechnology ConferenceSan Francisco, CAMay 19, 2010Paul PolakisGenentech, Inc.
How do we discover, define and validate MAb targets?•Abundance•Tumor specificityTaqman•Immunization•Screen AbProteinScreenReviewCloningReviewMAbs In Vivo Toxicity•Tumor specificity•Prevalence•Tissue specificity•Abundance•Transmembrane•Plasma membraneIn Situ•Cell type•Prevalence•Tumor specificityCell Line•Screen Ab•Internalization•Localization•In Vivo testing•Counter-screen• Immunization•Epitope•Affinity•Activity•FACS•IHC•Naked•Toxinconjugated•Clone Cynogene•Clone Ab•Chimeraproduction•conjugation
Target Screening- Large scale mRNA-based search <strong>for</strong> putative TM-containing proteins.Signal intensity (Ave. Diff. x 10 -3 )0 5 10 15 20 0 1 2 3 4 5adiposeadrenaladiposebreastTACSTD1/EpCAMCRC g1cervixCNScolorectalendometriumesophagushead&neckheartkidneyliverlungmuscleneurocrineovarypancreasprostateskinSm. intestinestomachtestisthyroidurinarywbc
Naked antibodies did not workNaked <strong>Antibody</strong>Armed <strong>Antibody</strong>
Naked antibodies are likely to be ineffective againsttargets selected simply on the basis of their expression<strong>Antibody</strong> activatesor inhibits signalingNaked antibodies<strong>Antibody</strong> recruitsimmune cellsArmed antibodies<strong>Antibody</strong> specifiesdelivery of drugNKdrugInhibitionorDeathapoptosisdamagedruguptake
Bumps in the road from target identification to in vivo efficacyCopy #?Mean Tumor Volume1000800600400200vehiclecontrol ADC (6mg/kg)Targeted ADC (3mg/kg)prostate00 10 20 30Day40 50 60Single IVValidated mRNA profileEfficacious ADC
How many xenograft models should we use?More models are betterLuCAP77LuCAP35vLuCAP70LnCAPLuCAP96Patient CPatient BPatient APatient DPatient E
Expression of TENB2/TMEFF1 in prostate cancerPr1N-GlyGAGN-GlySP FS I FS II EGF TM1 40 69 135 162 227 261 301 319 342 374TenB2
RelativeTenB2 expression in human prostate explant cancer modelsIHCLuCaP35vLuCaP70 LuCaP96.1 LuCaP7789Zr-ImmunoPET89Zr-TenB2PET Day 4LuCaP 35V 70 96.1 77PET uptake (%ID/g) 4 6 7 31FACS + ++ ++ +++11
Levels of TenB2 mRNA in LuCAP77 > 20/72 Human prostate cancers70000Human prostate cancers60000tenB2 mRNAProstate <strong>Cancer</strong>50000400003000020000100000Adenocarcinoma, Gleason < 7Adenocarcinoma, Gleason < 7Adenocarcinoma, Gleason < 7Adenocarcinoma, Gleason < 7Adenocarcinoma, Gleason < 7Adenocarcinoma, Gleason < 7Adenocarcinoma, Gleason < 7Adenocarcinoma, Gleason < 7Adenocarcinoma, Gleason < 7LuCaP77Adenocarcinoma, Gleason < 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason = 7Adenocarcinoma, Gleason > 7Adenocarcinoma, Gleason > 7Adenocarcinoma, Gleason > 7Other malignant tumor (rhabdomyosarcoma)Metastatic to elsewhereLuCAP77LuCap 77naExpression ranking77 > 96.1 > 70 > 35v12
Antibodies armed with auristatinsANTIBODYDRUGMaleimidocaproylvalinecitrullinePABA MMAEMAbSONOONHOHNproteolysisONHOONOHNO“Ab-VC-MMAE”NO CH 3ONH ONOC H 3 O HON HN H 2* * **Seattle Genetics***Denotes possible sites of drug conjugation* *
Comparison of anti-tenB2vc-MMAE in human prostate cancer explant modelsAll single IV dose of 5mg/kgTumor volume (mm 3 )1000800600400200900700500300100LuCAP 35v00 10 20 30 40 50DaySingle IV0 10 20 30 40 50Single IVLuCap96.1DayTenB2vcMMAETenB2vcMMAEExpression ranking8007006005004003002001001000800600400200TenB2: 77 > 96.1 > 70 > 35v00 10 20 30 40 50 60Single IV00 10 20 30 40 50 60Single IVLuCap77LuCap70DayDayTenB2vcMMAETenB2vcMMAE
MRP4 (multi drug resistance protein 4)Affymetrix probes covering all known drug resistance efflux transportersProstate cancerProstate normalMRP4MRP4MRP4Prostate <strong>Cancer</strong>LuCAP xenografts96.177 35v 70MRP4IHC w/ Anti-MRP4immunoblot w/ Anti-MRP4
Mucin 16 (CA0772) ovarian cancer targetOvCAR-3 (cell line)OVCAR-3Xenograft<strong>Cancer</strong>IHC+Serous24/25Endometrioid16/20Clear cell 5/10Mucinous 0/8IHC Serous3+ 62+ 91+ 90+ 1TumorStroma• Strong membranousstaining in serous OvCa
Structure of the MUC16 antigen and MAb binding sites.3A5 reacts with the mucin repeats that dominate the MUC16 sequence; 11D10recognizes a unique epitope in the extracellular domain.3A511D10TMH 2 N17 copies of mucin repeatCOOHFACS: OvCAR-3 Scatchard: OvCAR-3 Cell killing: OvCAR-311D103A5MAb(HuFcchimera)Affinity(pM)Sites percell11D10 52 31,7133A5 (#1) 433 243,4323A5 (#2) 288 107,760Cell viabilityArmed3A5Armed11D10<strong>Antibody</strong> amount
OvCAR3-luciferase intraperitoneal ovarian tumor modelDAY0 14 28 DAY 0 14 281.E+101.E+10Anti-Ragweedvc-MMAE11D10-vc-MMAE1.E+103A-5vc-MMAELuminescence1.E+081.E+061.E+041.E+081.E+061.E+041.E+081.E+061.E+041.E+020 7 14 21 28 35 42 49 56 63Daydosing at 2.5 mg/kg 1/wk x 4wk at d 0,7,14 and 211.E+021.E+020 7 14 21 28 35 42 49 56 63 0 7 14 21 28 35 42 49 56 63DayDay0/10 5/10
Endothelin B receptor: A target <strong>for</strong> melanomamelanoma
Targeting broadly expressed antigens with armed antibodies can be dangerousExpression of CD44 mRNA in human tissuesnormal skin“At the highest dose levelachieved in this study (140mg/m2), one patient developedtoxic epidermal necrolysis aftertwo infusions and died. Massiveapoptosis of skin keratinocyteshad occurred”
Melanocytes and EDNBR staining in normal skinα-MART-1melanomaAnti-EDNBR IHCα-IgG nonimmuneαEDNBR
Lots of modelsEndogenous EDNBR expression on melanoma lines526mel 537mel 888mel 928mel 1300melA375A2058COLO829G361Hs-294-TMalme3mSK23lowSKmel5 Skmel5-PRC SKmel28 WM-266-4medUACC257hiUACC257-NCI60α-mouse 2 0 Alexa 4882µg/ml EDNBR Mab 5E9.7.1
Increased cell killing in vitro with increased EtBR copy numberCopies/cell:1,582 9,410 33,939A2058(low)WM-266-4(med)UACC257(high)180000015000001200000900000600000300000A2058 WM-266-4 UACC257PBScontrol-vc-MMAE5E9-vc-MMAE00.001 0.01 0.1 1 10Ab concentration (ug/ml)100000090000080000070000060000050000040000030000020000010000000.001 0.01 0.1 1 10Ab concentration (ug/ml)PBScontrol-vc-MMAE5E9-vc-MMAE120000011000001000000900000800000700000600000500000400000300000200000100000PBScontrol-vc-MMAE5E9-vc-MMAE00.001 0.01 0.1 1 10Ab concentration (ug/ml)24
Expression levels in tumor models are within the range of human melanomaA2058UACC257melanoma
ETBR Expression in Cell Lines versus Tumor SamplesUACC-257A2058ETBR Staining Intensity0 1+ 2+ 3+A2058UACC-257
Some targets remain effective at very low copy numberA2058Copies/cell=1,582A2058UACC257Copies/cell=UACC25733,939In vivo efficacy with α-EtBr-vc-MMAEMean Tumor Volume (mm 3 )1800140010006002000A2058vehicleEtBr-vcE(1mg/kg)Control-vcE(6mg/kg)EtBr-vcE(3mg/kg)EtBr-vcE(6mg/kg)0 4 8 12 16 20 24 28 32 36 40 44 48DayMean Tumor Volume (mm 3 )UACC25712001000Control-vcE(4mg/kg)Vehicle800600400200EtBr-vcE(1mg/kg)0EtBr-vcE(4mg/kg)0 20IV treatments40 60 80 100Day
Efficacy/SafetyFour ways <strong>for</strong> armed antibodies to kill cellsdrug1. 2.drugNon specific uptakeof intact ADC/TDCdamagedruguptakedamagedruguptakeCells with target3. 4.damagedrugdrugLiver, lung, kidney, etcdamageUptake of releasedfree drugdrugdrugBone marrow cells
Stability impacts SafetyTwo types of armed antibody instabilityPK Her-MC-vc-PAB-MMAE of antibody (8 D/Ab), and Her-MC-vc-PAB-MMAE antibody-drug (5 D/Ab) in Rats conjugate2 mg/kg100Conc. µg/ml101Herceptin-vcMMAE(5:1 drug:MAb ratio)Total MAb0.1Herceptin-vcMMAE(8:1 drug:MAb ratio)0.010 7 14 21 28daysTime (days)1. The drug is released from intact antibodyMAb-drug conjugate2. The drug is released upon catabolism of the antibody
<strong>Antibody</strong> doses are normalized to drug equivalenceTumor volume
Strategies to Control Stoichiometry of Cysteine-Directed<strong>Antibody</strong> <strong>Drug</strong> Conjugation* * ** ** ** *Conventional ADCTHioMAb drug conjugate (TDC)Thiol reactive drugsare conjugated toreduced disulfidesEngineeredCysteine Thiol(A118C)2 420681<strong>Drug</strong>s / antibody<strong>Drug</strong>s / antibody31
In Vivo Efficacy is Retained With TDC FormatOVCAR-3 xenograft tumors in mammary fat padADC (1.5 mg/kg = 71 µg/m 2 )Tumor volume (mm 3 )vehicleTDC (1.5 mg/kg = 35 µg/m 2 )3 mg/kg (69 or 141 µg/m 2 )6 mg/kg (139 or 283 µg/m 2 )Days after single IV dose32
TDC is Better Tolerated than ADC by RatsDay 5 PMN(1000/µL)Weight change (g)TDC 36 mg/kgDay 5 AST(U/L)ADC 16 mg/kgmg/kg:V16 25 36 68 100ADCTDC
Cumulative toxicity from long-lived intact ADC/TDCAcute toxicity <strong>for</strong>m unstable ADC/TDCDLTADCTDC03 6 9 12 15 18 21mg/kg <strong>Antibody</strong>-MMAE
In vivo efficacy of an antibody armed with a novel DNA Damaging Agent1500tumor volume (mm 3 )1000500vehiclecontrolsAnti-cd22-mk-DDA(1MPK)Anti-cd22-mk-DDA(5MPK)00 5 10 15 20 25 30Single IVDay
• GNE ADC Team– Susan Spencer– Kelly Flagella– Jeffrey Gorrell– Bonnee Rubinfeld– Jyoti Asundi– Kedan Lin• ThioMab / TDC Team– Jagath Junutula– Sunil Bhakta– Helga Raab– Richard VandlenAcknowledgments‣Our colleagues at Seattle Genetics and Nerviano• <strong>Antibody</strong> Technology– Mark Dennis– Yvonne Chen– Bob Kelley• In Vivo safety/efficacy– Suzanna Clark– Shang-Fan Yu– Sarajane Ross– Douglas Leipold– Will Leach– Jeffery Lau– Nic Van Bruggen36
Change language