ADC PK

Antibody-Auristatin Conjugates for Cancer: Antibody and Drug Pharmacokinetics and Disposition Stephen C. Alley, Ph.D. 2010 AAPS National Biotechnology Conference, San Francisco 2 Empowered antibodies  The antibody-drug conjugate (ADC) concept is to conscript antibody specificity for anticancer drug delivery  Goals  Increase activity by targeted delivery of drug  Decrease toxicity by reducing systemic exposure to drug  Today’s presentation: evaluate drug and mAb disposition in mouse xenograft models 3 Endosome Lysosome: drug release from ADC Drug escape to cytosol Receptor-mediated endocytosis ADC Antigen Cell membrane Cell death: disruption of tubulin network or DNA cleavage ADC technology Highly controlled conjugation process  Conjugates with an average of 4 drugs/mAb  Heavy and light chains remain tightly associated mAb mAb-Drug 1. Controlled reduction 2. Addition of maleimide drug-linker Auristatin drug Antigen binding site Interchain disulfides 5  An average of 4 vcMMAE drug-linkers conjugated per mAb  Dipeptide linker cleavable by lysosomal enzymes to release MMAE vcMMAE: protease cleavable drug linker 6 Tracking conjugated and free drug: brentuximab vedotin (SGN-35)  Brentuximab vedotin is being evaluated clinically for Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (sALCL)  Brentuximab vedotin is composed of the anti-CD30 mAb cAC10 and the drug linker, vcMMAE, conjugated via reduced mAb interchain disulfides 7 MMAE accumulation 0 10 20 30 40 50 60 70 80 0 200 400 600 800 1000 1200 Time (h) In tr ac el lu la r [1 4 C ]-M M AE C on ce nt ra tio n (n M )  Brentuximab vedotin demonstrated antigen-dependent cytotoxicity  MMAE released and accumulated in only CD30 positive cells cytotoxicity 0.001 0.01 0.1 1 10 100 0 25 50 75 100 CD30 positive cells CD30 negative cells Concentration (nM conjugated MMAE) % o f U nt re at ed Brentuximab vedotin active in vitro Okeley et al, Clin Cancer Res, 16, 888, 2010 8 Brentuximab vedotin active in vivo  Brentuximab vedotin or MMAE dosed to CD30 positive L540cy HL tumor xenografts in mice  Antigen dependent activity  Brentuximab vedotin more active than MMAE (MMAE dose 25-fold higher) 0 20 40 60 0 200 400 600 800 1000 Untreated B-vedotin 1 mg/kg MMAE 0.5 mg/kg IgG-vcMMAE 1 mg/kg Days post tumor implant Tu m or v ol um e (m m 3 ) dosing 9 Anti-drug immunohistochemistry detected drug in L540cy HL Tumor Xenograft  Anti-MMAE mAb used to visualize drug in tumor for both brentuximab vedotin and MMAE treated mice  Weaker signal in MMAE-treated mice suggests that ADC is detected better than free drug  This technique does not provide quantitation of drug amounts Rx: 5 mg/kg b-vedotin Rx: 0.5 mg/kg MMAE untreated Detection: anti-MMAE (brown) 10 Equal moles of injected MMAE yielded substantially different uptake and kinetics  Mass spectrometry detection of serum and tumor MMAE in L540cy HL mouse xenograft  MMAE rapidly cleared from serum, while brentuximab vedotin clears slower  Peak tumor MMAE concentrations later, higher for brentuximab vedotin than MMAE injection 0 2 4 6 8 10 0.01 0.1 1 10 100 1000 tumor serum (released) serum (conjugated) Time (d) M M AE (n M ) 0 2 4 6 8 10 0.01 0.1 1 10 100 1000 Time (d) M M AE (n M ) Rx: 2 mg/kg b-vedotinRx: 0.04 mg/kg MMAE 11 Results from drug localization studies  Brentuximab vedotin demonstrated antigen- specific cytotoxicity  MMAE delivery to tumor xenografts can be visualized by IHC and quantitated by mass spectrometry  Brentuximab vedotin delivers more MMAE to the tumor than MMAE dosing  Next: evaluation of drug and mAb 12 Tracking mAb and drug: dual radiolabeled SGN-75  SGN-75, being evaluated clinically in NHL and RCC, is composed of the anti-CD70 mAb h1F6 and the drug linker mcMMAF  Two radiolabels used to track mAb (3H) and drug (14C) biodistribution simultaneously 14C auristatin drug 3H propionate 13 mcMMAF: drug linker with no built-in cleavage site  Complete mAb degradation by lysosomal proteases yields cys-mcMMAF as the released drug 14  Tumor uptake kinetics are different for mAb and released drug  Shift in time for peak released drug concentration likely due to time required for internalization and lysosomal degradation 0 2 4 6 8 10 0 100 200 300 0 50 100 mAb released drug Time (d) R el ea se d dr ug (n M ) Antibody (nM ) Alley et al, J Pharmacol Exp Ther, 330, 932, 2009 mAb and drug accumulated in 786-O RCC mouse xenograft 15 Released drug concentrations higher in tumor than normal tissues  Released drug in 786-O tumor peaks at 2 d, while mouse normal tissues fall from start  Tumor released drug concentrations often more than 100 fold higher than normal tissues 0 2 4 6 8 10 0.1 1 10 100 1000 tumor serum liver lung kidney intestine spleen muscle Time (d) R el ea se d dr ug (n M ) Alley et al, J Pharmacol Exp Ther, 330, 932, 2009 16 Tumor exposure to released drug higher than normal tissues  Exposure was calculated from 0 to 10 days for 786-O tumor and mouse tissues  Tumor to normal ratios were often greater than 100:1 kid ne y liv er sp lee n he art mu sc le br ain bo ne m arr ow lun g pa nc rea s bla dd er ov ary sto ma ch int es tin e c on ten ts int es tin e 0 200 400 600 800 1000 2500 Tu m or to n or m al e xp os ur e Alley et al, J Pharmacol Exp Ther, 330, 932, 2009 17 Results from drug and mAb localization  Dual radiolabeled SGN-75 allowed simultaneous tracking of drug and mAb  Peak tumor concentrations for mAb and drug were at 1-2 d post dose, while normal tissues peaked soon after the dose  Absolute tumor concentrations of released drug were higher than normal tissues, yielding tumor to normal exposure ratios often in excess of 100:1 18 Novel delivery formats  While mAbs have been shown to be effective drug delivery formats, are there better formats?  Impact of delivery vehicle size  mAbs are ~150 kDa and may have trouble penetrating tumors  Alternative scaffolds exists from 10-50 kDa: better tumor drug delivery?  Next: diabody-drug conjugates vs ADCs  PK  Tumor drug delivery  Normal tissue delivery 19 Diabodies: ~50 kDa scFv dimers  Engineered cysteines allow conjugation S84C S14C VH VL CDRs 20  Dipeptide linker cleavable by lysosomal enzymes to release MMAF  IgG and diabody conjugates made with 4 drugs per backbone  IgG conjugated via reduced interchain disulfides (average of 4 drugs)  Diabody conjugated via reduced engineered cysteines (homogeneous 4 drugs) vcMMAF: protease cleavable drug linker 21 Diabody conjugate achieved activity in vivo at higher dose than ADC  cAC10 (anti-CD30) diabody and mAb conjugates evaluated in Karpas 299 (ALCL) mouse xenografts 0 20 40 60 0 200 400 600 800 Single dose 0/9 CRs 7/9 CRs 6/9 CRs Days post tumor implant Tu m or v ol um e (m m 3 ) 0 20 40 60 0 200 400 600 800 Single dose 1/8 CRs 8/8 CRs 8/8 CRs Days post tumor implant Tu m or v ol um e (m m 3 ) Untreated cAC10 diabody-vcF4, 0.72 mg/kg (1x) cAC10 diabody-vcF4, 2.16 mg/kg (3x) cAC10 diabody-vcF4, 7.2 mg/kg (10x) cAC10-vcF4, 0.66 mg/kg (0.33x) cAC10-vcF4, 2.0 mg/kg (1x) cAC10-vcF4, 6.0 mg/kg (3x) Untreated Kim et al, Mol Cancer Ther, 7, 2486, 2008 22 Diabody cleared from serum faster than mAb  Lack of Fc region causes rapid diabody clearance  Surprisingly, diabody drug conjugate cleared slower than unconjugated diabody 0 10 20 30 40 0.01 0.1 1 10 100 1000 cAC10-vcMMAF cAC10 diabody cAC10 diabody-vcMMAF Time (d) Se ru m c on ce nt ra tio n ( µ g/ m L) Kim et al, Mol Cancer Ther, 7, 2486, 2008 23  Released drug concentrations evaluated with 3H- labeled drug  Released drug concentration in the tumor peaked earlier for diabody, consistent with more rapid clearance  Smaller diabody size may allow better normal tissue penetration Tumor 0 10 20 30 40 50 0 100 200 300 Time (h) R el ea se d dr ug (n M ) Liver 0 10 20 30 40 50 0 50 100 150 Time (h) R el ea se d dr ug (n M ) Kidney 0 10 20 30 40 50 0 25 50 75 Time (h) R el ea se d dr ug (n M ) cAC10-vcF 2.0 mg/kg cAC10 diabody-vcF 0.72 mg/kg Kim et al, Mol Cancer Ther, 7, 2486, 2008 Diabody yielded less released drug in tumor, more in normal tissues 24 Released drug tumor to normal exposure was higher for ADC  0 to 2 day exposure to released drug was higher for IgG for all tissues Se ru m Liv er Int es tin e Ki dn ey Sp lee n St om ac h Bl ad de r 0 20 40 60 80 100 0.72 mg/kg cAC10 diabody-vcF4 2.0 mg/kg cAC10-vcF4 tu m or to n or m al e xp os ur e Kim et al, Mol Cancer Ther, 7, 2486, 2008 25  Maximum tumor uptake achieved by molecules close to size of IgGs: PK compensates for poorer tumor penetration  Minimum tumor uptake achieved by molecules of ~25 kDa: better tumor penetration does not compensate for poor PK Schmidt and Wittrup, Mol Cancer Ther, 8, 2861, 2009 Modeling of tumor uptake versus size 26 Conclusions  Auristatin ADCs improve anti-tumor activity and tumor localization of drug compared to dosing with free drug  By monitoring both mAb and drug, auristatin ADCs achieve higher tumor localization versus normal tissues, with tumor to normal tissue exposure ratios often >100:1  Delivery vehicle size plays a role in drug delivery, with IgG format yielding higher tumor over normal drug delivery versus diabody format  PK and tumor penetration are both important 27 Acknowledgements ADCs in the Lab: Nathan Ihle Svetlana Doronina Brian Toki Damon Meyer Robert Lyon Michael Sun Jocelyn Aker Dennis Benjamin Nicole Okeley Amy Zhang Mechthild Jonas Django Sussman Che-Leung Law Peter Senter ADCs in the Clinic: Eric Sievers Dana Kennedy Carmel Lynch Jonathan Drachman Nancy Whiting Antibody-Auristatin Conjugates for Cancer: Antibody and Drug Pharmacokinetics and Disposition Empowered antibodies ADC technology Highly controlled conjugation process Slide Number 5 Slide Number 6 Brentuximab vedotin active in vitro Brentuximab vedotin active in vivo Anti-drug immunohistochemistry detected drug in L540cy HL Tumor Xenograft Equal moles of injected MMAE yielded substantially different uptake and kinetics Results from drug localization studies Tracking mAb and drug: dual radiolabeled SGN-75 mcMMAF: drug linker with no built-in cleavage site Slide Number 14 Released drug concentrations higher in tumor than normal tissues Tumor exposure to released drug higher than normal tissues Results from drug and mAb localization Novel delivery formats Diabodies: ~50 kDa scFv dimers Slide Number 20 Diabody conjugate achieved activity in vivo at higher dose than ADC Diabody cleared from serum faster than mAb Slide Number 23 Released drug tumor to normal exposure was higher for ADC Slide Number 25 Conclusions Acknowledgements