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Drug conjugation to carriers George Badescu Next-Generation Drug Conjugates: The Holistic View of Developing Superior Drug Conjugates Cambridge, 24 February 2016 Enabling better biopharmaceuticals • • • Immunogenicity assessment Deimmunised proteins & humanised mAbs Manufacturing cell line development Antibody creation to GMP manufacture • • • Manufacturing process development GMP antibody & protein manufacture Fill/finish into vials for clinical studies • • • Proprietary site-specific linker technology Half-life extension & ADCs development Novel toxin payload development ADC components Antibody Linker Cytotoxic payload • • • ADC creation to GMP manufacture Toxin payloads & linker-payloads for ADCs Reagent manufacturing & conjugation Custom chemistry An integrated solution for creation, selection and manufacture of next generation ADCs & other biopharmaceuticals 2 Enabling better biopharmaceuticals Proprietary technologies to produce product candidates with improved properties Design and create ABZENA inside • • • Composite Human Antibodies™ Composite Proteins™ ThioBridge™ ADCs Test and select Develop and manufacture Services for selection of product candidates with greater probability of successful development & commercialisation Expertise & capabilities to develop efficient and flexible manufacturing processes and produce products for clinical development ADC - antibody drug conjugate 5 3 ThioBridge™ Disulfide bridging conjugation 4 ThioBridge™ reagents attach drugs at accessible disulfides mAb Fab 5 ThioBridge™ conjugation has been applied to a wide range of types of disulfide-containing proteins Category Protein Activity Enzymes Asparaginase Amylase Cytokines Interferon α-2a and α-2b Interferon β Hormones Leptin Erythropoietin Peptides Octreotide Tumour imaging ligand Blood proteins Coagulation factors (VIIa, VIII & IX) Antibodies mAb Fab Novel formats Scaffolds Homogeneous ADC created from an anti-HER2 antibody fragment (Fab) using ThioBridge™-MMAE ThioBridge™ Fab drug conjugate SDS-PAGE analysis of reaction mixture ThioBridge Drug conjugate Site-specific conjugation Conversion nearly 100% Low reagent consumption Homogeneous product (DAR 1) Simple purification and characterisation ThioBridge™ anti-HER2 Fab-MMAE conjugate is bridged at the disulfide 100% 100% 100% 100% Complete re-bridging of the reduced disulfide was observed for this conjugation reaction so structural integrity of the Fab was maintained Using ThioBridge™ can improve homogeneity 100 Crude reaction mixture Av DAR=3.95 Clinical grade product Av DAR=3.8 • Competitive inhibitor 80 % 60 40 • Lower tolerability 20 • Accelerated clearance • High instability 0 • High hydrophobicity 0% 012345678 012345678 ThioBridge™ Brentuximab-vc-PAB-MMAE Maleimide conjugation to reduced disulfide bonds ADCETRIS® 9 DAR 4 Conjugation optimisation using Design of Experiments (DoE) • Screening for factors influencing yield and determine factor interactions • Successfully achieved conversion to greater than 90% DAR 4 with simple optimisations in reagent concentrations and pH • A powerful approach for optimisation of conjugation > 90% conversion to DAR 4 HIC-UV of unpurified reaction: mAU WVL:280 nm intensity 94 % DAR 4 Extrapolated range Experimental range Example of a surface model: response surface plot (2 factors) for % DAR 4 achieved 5 3 4.0 5.0 6.0 7.0 8.0 9.0 10.0 minutes 11.0 12.0 13.0 mi 14.0 n 15.0 ThioBridge™ conjugates were stable in different sera Method: Alexa Fluor 488 conjugated to trastuzumab using ThioBridge™ or maleimide chemistry with average of 2 disulfides reduced. Conjugates incubated in sera from different species at 37 ⁰C for 96 h; analysed using SEC 50 LU ThioBridge™ conjugate: 0 h 30 10 0 PBS 5.0 7.0 9.0 11.0 13.0 100 LU 15.0 17.0 19.0 min ThioBridge™ conjugate: 96 h 80 50 LU Maleimide conjugate: 0 h 30 10 0 PBS 5.0 7.0 9.0 11.0 13.0 100 LU 15.0 17.0 19.0 min Maleimide conjugate: 96 h 80 60 human 60 human 40 mouse 40 mouse 20 rat 20 rat monkey 0 5.0 7.0 9.0 11.0 13.0 15.0 17.0 19.0 min monkey 0 5.0 7.0 9.0 11.0 13.0 15.0 17.0 19.0 min ThioBridge™ conjugates were more stable than maleimide conjugates in human and animal sera 11 ThioBridge™ conjugates were more stable in vivo Distribution of Alexa fluor 488 ADC species in blood pool 48 h after i.v. administration to SCID mice based on analysis of serum by SEC with fluorescence detection 100 Peak area (%) 80 ThioBridge TM Maleimide 60 40 20 0 Intact ADC Cross-conjugated & dissociated species Aggregates 12 Different ThioBridge™ brentuximab ADCs had similar in vitro potency in Karpas 299 cells ThioBridge™ brentuximab ADCs (DAR 4) were prepared with four different reagents with the same linker-payload. MMAE Adcetris ThioBridge™ ADC 1 ThioBridge™ ADC 2 ThioBridge™ ADC 3 ThioBridge™ ADC 4 Cell Viability [%] 100 50 0 10 0 10 1 10 2 10 3 10 4 Conc (pM) The four different ThioBridge™ brentuximab ADCs with MMAE and a cleavable linker had similar potencies in vitro in Karpas 299 cells We thank Dr Karpas of Cambridge University for supply of the Karpas 299 cell line ThioBridge™ brentuximab ADCs with different linker structures have different efficacy profiles in vivo ThioBridge™ ThioBridge™ ThioBridge™ ThioBridge™ ADC 1 ADC 2 ADC 3 ADC 4 Adcetris™ • Karpas-299 xenograft model at 0.5 and 1 mg/kg (single dose, i.v. administration) • The flexibility of the ThioBridge™ technology allows quick screening of reagent configurations and selection of optimal ADC ThioBridge™ brentuximab ADCs with different linker structures have different efficacy profiles in vivo ThioBridge™ ThioBridge™ ADC 1ADC 1 ThioBridge ADC 2ADC 2 ThioBridge TM 3 Tumour volume (mm ) 3 Tumour volume (mm ) 2000 1000 0 2000 1000 10 20 30 40 50 20 30 40 3 1000 40 Days post treatment Adcetris Control ADC Vehicle 50 • Two ThioBridge™ brentuximab ADCs (DAR 4) with structurally different linkers were tested in Karpas-299 xenograft model at 0.4 mg/kg (Q4Dx4, i.v. administration) • Different ThioBridge™ reagents generated ADCs with different in vivo efficacy profiles 2000 1000 0 0 30 20 50 Vehicle 0.4 mg/kg, Q4Dx4 Dosing 3000 Tumour volume (mm ) 3 10 Adcetris Adcetris™ 2000 20 ThioBridge ADC 1 Days post treatment Vehicle 0.4 mg/kg, Q4Dx4 Dosing 10 40 Days 0 Control ADC 0 60 0 10 20 30 40 50 60 70 80 Days post treatment 3000 ThioBridge ADC 2 80 0 0 0 Tumour volume (mm ) 100 Vehicle 0.4 mg/kg, Q4Dx4 Dosing 3000 Percent survival Vehicle 0.4 mg/kg, Q4Dx4 Dosing 3000 Kaplan-Meier Plot TM 0 10 20 30 40 Days post treatment 50 In Vivo Imaging of ThioBridge™ anti-PSMA Minibody Variants Maleimide control ThioBridge™ PEG 6u ThioBridge™ PEG 36u • Excellent radiolabeling efficiency and immunoreactivity for all three conjugates ImaginAb Inc: Jean Gudas, VP of R&D Michael Torgov, Ph.D Tove Olafsen, Ph.D 16 ThioBridge™ conjugates of an antibody fragment with desferrioxamine and 89Zr – images at 24 h Maleimide control ThioBridge™ PEG 6u ThioBridge™ PEG 36u 24 h Nude mice • Maleimide conjugation of desferrioxamine on cysteine residues destabilized the protein and resulted in rapid clearance through the kidneys • Disulfide bridging through ThioBridge™ stabilized the antibody fragment and reduced kidney clearance ImaginAb Inc: Jean Gudas, VP of R&D Michael Torgov, Ph.D Tove Olafsen, Ph.D 17 CyPEG™ Stable conjugation of thiols 18 CyPEG™ Cysteine thiol conjugation and stabilisation schematic CyPEG™ Reagent = Payload/fluorescent dye or other functional molecule Reduced or cys-engineered antibody Efficient Michael addition conjugation with a mono-sulfone reagent followed by stabilisation (‘locking’) of the formed conjugate with NaBH4 CyPEG™ conjugation to an antibody mimetic thiol was efficient • CyPEG™ PEGylation of an Affibody* was compared with other thiol-reactive PEG reagents • CyPEG™ reactivity was comparable to the maleimide reagent • CyPEG™ was more reactive than vinyl sulfone, acrylate and haloacetamide reagents PEGylation with CyPEG™ was successfully achieved with a 1:1 ratio of reagent to protein * Anti-Her2 Affibody® with single free cysteine; a PEG size of 5 kDa was used for all reagents CyPEG™ Stabilisation with borohydride did not affect mAb binding affinity by ELISA • Borohydride locking step stabilises the conjugate preventing retro-Michael reactions • Mild borohydride treatment an established method for stabilising bioconjugates, e.g., PEG-GSCF (N-terminal amine conjugation with PEG-aldehyde) • Antibody shown to retain full binding efficiency post borohydride treatment 4 A630 (nm) 3 2 1 0 0.001 0.01 0.1 1 10 100 1000 10000 [Compound] (ng/mL) Native Trastuzumab, n=1 Trastuzumab + Reagent + NaBH4 Trastuzumab + NaBH4 ADC preparation example: CyPEG™ val-cit-PAB-MMAE addition to partially reduced trastuzumab - DAR profile by HIC • CyPEG™ trastuzumab ADC DAR profile under 3 h; average DAR (0-8) = 4.0 31.20 26.20 30 % Area at 280 nm 25 17.30 20 15 9.20 7.00 10 1.50 3.70 1.10 5 2.80 0.00 0 pH 7.0 Conjugation reaction pH DAR 0 DAR 1 DAR 2 DAR 3 DAR 4 DAR 5 DAR 6 DAR 7 Typical profile for mono-cysteine conjugation observed (e.g., malemide conjugation) DAR 8 DAR >8 HIC purified DAR 4 CyPEG™ADC analysed by native SEC-MS Intact mass for Trastuzumab 148250 Da Mass ion for Locked CyPEGTM ADC 158175 Da Compound = CyPEGTM val-cit-PAB-MMAE TOF MS ES+ Mass by LC/MS (Da) CyPEG™ trastuzumab ADC DAR 4 158175 Da trastuzumab 148250 Da Mass difference (ADC – ADC) Mass of conjugated reagent 9925 Da 4 x 2481 = 9924 Da Deconvoluted MS allowed for determination of the intact mass and confirmed full borohydride stabilisation CyPEG™ DAR 4 ADC Stability versus equivalent maleimide based ADC: Incubation in PBS with excess HSA, 7 d, 37 °C • ADCs purified to DAR 4 using HIC and then incubated in PBS with HSA (20 mg/mL; 50 mol equivalents to ADC) over 7 d • HIC used to determine the DAR profile after 7 d % change in DAR 4 at 7 d CyPEG™ ADC 0 Maleimide ADC 30 CyPEG™ DAR 4 ADC showed excellent stability in presence of HSA whereas the maleimide ADC degraded by 3̴ 0% to DAR < 4 variants HiPEG™ Covalent conjugation at polyhistidine motifs 25 HiPEG™: Covalent conjugation at polyhistidine motifs • • • • Utilises terminal or intra-protein polyhistidines as site for covalent conjugation Conjugation to N or C-terminal histidines reduces interference with protein binding Stoichiometrically efficient and predictable conjugation process His-tag available for purification post-conjugation ADC services & technologies provided by Abzena Abzena provides one of the broadest range of services to support the creation and development of ADCs of any company in the industry supported by a wealth of proprietary technologies to produce a better product and a strong manufacturing background Antibody Services Conjugation Payloads Manufacture Immunogenicity assessment ThioBridge™ CyPEG™ Over 20 prepared payloads 15,000 L GMP Antibody Antibody humanisation HiPEG™ Custom synthesis 100g Toxin 50g linker Manufacturing cell line development Cysteine Payload-linker reagents Lysine 50g Toxin-linker 10g ADC Others 27