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Antibody Drug Conjugates Dr Philip W. Howard Head of Targeted Therapeutics Spirogen Therapeutic Antibodies • Many tumours express antigens that can be targeted by antibodies • These antibodies offer the prospect of selectively targeting tumour cells Key Characteristics of an ADC Linker Stable in in vivo circulation Cleaved inside target cell Stable upon storage Antibody Drug High Tumour Specificity High Potency High Avidity Linkable High Antigen Expression Water Soluble Antigen and Antibody Requirements for ADC • Antigen – tumour specific or overexpressed on tumour cells vs. normal tissue – antigen should be highly expressed on surface of tumour cells • Antibody – affinity of the antibody for the target antigen should not be compromised by forming an ADC – The antigen-ADC complex must be internalized Targeting Members of the Her Receptor Family Figure 15.38b The Biology of Cancer (© Garland Science 2007) Myeloid and haematopoietic Targets A + - *: #6@'J @'5/ From Teicher (2009) Curr Can Drug Targ 9:982 •At least 16 current targets on non-solid cancer cells •Epitopes are important •Not all internalise at the same rate IL2Ra Epitopes are important Not all internalize at the same rate Fc!R1 NCAM From Teicher (2009) Curr Can Drug Targ 9:982 A + - *: #6@'J @'5/ Targets – solid tumours From Teicher (2009) Curr Can Drug Targ 9:982 LeY MUC1 CA125 HER2 NCAM Bi-specific targeting vehicles may reduce toxicity in normal •At least 19expressing current only targets tissue one on of the solid tumours plus some targets two surface antigens not found directly on tumours Syndecan Endoglin From Teicher (2009) Curr Can Drug Targ 9:982 Stromal Target Stromal Target Some Approved Therapeutic Antibodies For Oncology Antibody Brand Name Approv Date Target Mechanism Indication Market ($B) Bevacizumab Avastin 2004 VEGF Ligand depletion Denosumab Prolia; Xgeva 2010 Rank ligand Ligand depletion Colorectal Ca Bone Mets (SRE) Ipilumumab Yervoy 2011 CTLA-4 T-Cell upregulation Melanoma Alemtuzumab Campath 2001 CD52 ADCC ± CMC Cetuximab Erbitux 2004 EGFr ↓EGFr signaling CLL Colorectal; H & Neck Ca 3.2 (2010) Panitumumab 2006 EGFr ↓EGFr signaling Colorectal Ca 0.23 (2009) Rituximab Vectibix Rituxin; MabThera 1997 CD20 ADCC ± CMC NHL; RA 6.7 (2010) Ofatumumab Arzerra 2009 CD20 ADCC ± CMC CLL Ibritumomab Zevalin 2002 CD20 90Y-RIT NHL Tositumomab Bexxar 2003 CD20 131I-RIT NHL HER2 ↓HER2 signaling; ADCC Breast Ca Trastuzumab Herceptin 1998 6.8 (2010) 0.1 (2008) 5.5 (2010) Do we Need ADCs? • Unfortunately, many therapeutic antibodies lack in vivo efficacy even when combined with established chemotherapeutic agents. • Even in the case of successful agents such as Herceptin, patients relapse and the tumours become resistant. • Fortunately, the resistance mechanism does not affect surface expression of Her2 Antibody Modification to Enable Conjugation • Reduce native disulphide bonds and conjugate to maleimide linker • Functionalize lysines with thiol containing chains and conjugate to maleimide linker • Conjugate to lysines with a succinimide linker • Engineer new thiols at specific positions and conjugate to maleimide linkers. Conjugation to Thiols ADC-drugs linked to reduced inter-chain cysteines Non homogenous drug load (DAR = 0-8) Drug linked to single engineered cysteine Homogenous drug load (DAR=2) • Using engineered thiols reduces Drug Antibody Ratio • Not a problem with potent warheads • Too many warheads can cause aggregation and loss of specificity Conjugation Chemistries Maleimide Approach Iodoacetamide Approach Characterization DAR Determination Hydrophobic Interaction Chromatography (HIC) Aditya Wakankar et al, mAbs, 2011; 3;2: 164-175 Does not work for all Spirogen ADCs Characterization DAR Determination LC/ESI-TOF MS Alexandru C. Lazar et al, Rapid Commun. Mass Spectrom. 2005; 19: 1806-1814 Linkers: Stability and Cleavage • Linkers must be stable in circulation • But readily cleaved inside the target cell LINKER RELEASE MECHANISM HALF LIFE (MOUSE SERUM) HYDRAZONE Designed for degradation in acidic compartments within the cell 2 days PEPTIDE Val-Cit Designed for serum stability and to be enzymatically hydrolysed ((esp. by lysosomal proteases such as cathepsin B) 30 days PEPTIDE (Phe Lys) Designed for serum stability and to be enzymatically hydrolysed ((esp. by lysosomal proteases such as cathepsin B) 12days DISULPHIDE (Non hindered) Designed to be cleaved through dislulfide exchange with an intracellular thiol, such as glutathione 2 days DISULPHIDE (Hindered) Designed to be cleaved through disulphide exchange with an intracellular thiol, such as glutathione 5 days THIOETHER Non-reducible and stable to proteolytic degradation 7 days Mylotarg Acute Myeloid Leukaemia • Voluntarily withdrawn after follow-up trial: • No survival benefit in controlled study • Increased mortality and toxicity (myelosuppression) • Premature release, hydrazone linker? Trastuzumab DM1 Genentech/Immunogen Cleavable Disulphide Linker • Endocytic cleavage of disulphide very inefficient • Disulphide designed to cleave under reducing conditions • Endocytic pathway now thought to be oxidizing • Disulphide linker unstable in circulation • Only 11% of disulphide remaining after 7 days Trastuzumab MCC-DM1 • MCC linker was found to be significantly more robust than the disulphide linker – Negligible loss of MCC-DM1 in serum after 7 days • The MCC linker is an example of a non-cleavable linker – The antibody is degraded by proteases leaving the drug-linker intact • But Maytansinoid-linker molecule still active and potent Trastuzumab MCC-DM1 Her2 Positive Breast Cancer • Phase I – 24 patients (median 4 prior chemotherapeutic agents) – 73% clinical benefit rate (OR + Stable disease at 6 months) • Phase II (Burris et al.) – 108 patients; Her2 +ve metastatic breast cancer all received trastuzumab – 26% overall response rate (after 12 months) – 4.6 months overall median progression free survival • Phase II (TDM4450g) – Previously untreated patients with Her2 +ve metastatic or advanced breast cancer – Treated patients benefited from an extra 5 months of life compared to those treated with Herceptin + chemotherapy Adcetris (Brentuximab) Seattle Genetics • Robust protease labile linker with self-immolative spacer Adcetris (Brentuximab) • Adcetris brentuximab velodotin CD30 Hodgkin lymphoma data (102 patients) • 32% CR (duration of response 20.5 months) – CR= Disappearance of all evidence of disease • 40% PR (duration of response 3.5 months) – PR = Regression of measurable disease and no new sites • 73% ORR (duration of response 6.7 months) – ORR of 30% considered a meaningful response (FDA Seattle genetics discussions) Approved by FDA The ADC Field is Dependent on a Narrow Range of Warheads • Calicheamycin – DNA cutting agent – Narrow therapeutic window (1-0.1 nM) • Maytansinoids – Potent (picomolar) – Tubulin binder – PGP substrate • Auristatin – Potent (nanomolar – 10 pM) – Tubulin binder – PGP substrate • Doxorubicin – Insufficient potency ( M) Name Mylotarg (Gemtuzumab ozogamicin) Ab Target Drug Linker Indication Current Phase MA 2000 Withdrawn 2010 CD33 Calicheamicin Hydrazone Acute Myeloid Leukaemia CD30 Auristatin E Valine-citruline Relapsed/refractory HL ALCL Approved Post-transplant HL August 2011 Her2 Maytansinoid DM1 Thioether Metastatic breast CD22 Calicheamicin Hydrazone CD56 Maytansinoid DM1 Disulphide CD74 Doxorubicin Thioether GPNMB Auristatin E Valine-citruline SAR-3419 CD19 Maytansinoid DM4 SGN-75 CD70 Auristatin F Hindered disulphide Maleimidocaproyl Breast melanoma Relapsed/refractory NHL Relapsed/refractory NHL, Renal carcinoma IMGN-388 αv-integrin Thioether Solid tumours I MDX-1203 CD70 Valine-citruline NHL, renal I MN-IC CA9 Solid tumours I BIIB-015 Cripto Hindered disulphide Breast I BT-062 CD138 Hindered disulphide Multiple myeloma I BAY-79-4620 MN carbonic anhydride IX Auristatin E Valine-citruline Solid tumours I AGS-16M8F AGS-16 Auristatin F Maleimidocaproyl Renal I SGN-35 Brentuximab vedotin Trastuzumab-DM1 Trastuzumab-emtansine CMC-544 Inotuzumab ozogamicin IMGN-901 Lorvotuzumab mertansine Milatuzumabdoxorubicin CDX-011 Glembatumumab veditin Maytansinoid DM4 Duocarmycin MED 2460 Auristatin Maytansinoid DM4 Maytansinoid DM4 Relapsed/refractory NHL Multiple myeloma Solid tumours: SCLC, Merkel cell, ovarian Multiple myeloma III III II II II II I Pyrrolobenzodiazepines (PBDs) 9 8 10 11 11a N H A B 7 1 N C 6 O 3 2 Naturally Occurring PBDs OH Me H OMe H N OMe H N N N NH2 O N N H O Tomaymycin Prothracarcin OH N Me H N MeO O DC-81 H N O HO O Porothramycin N MeO NMe2 O O Anthramycin HO OMe H H N O Me Me MeHN O O HO OH H N Sibiromycin OH Mode of Action PBDs form a covalent, aminal linkage with the N2 of guanine in the minor groove of DNA O N HN H N .. 2 2 O N N DN A N R' A H N R' N C R" N O N DN A H B O H N H N 11 N HN R" PBD Dimers (In Vitro Activity) Molecule (Control) L428 786-O HEL HL-60 MCF-7 SG2057 0.001 0.001 0.000017 0.000035 0.021 SG2202 <0.0001 <0.0001 <0.0001 <0.0001 0.000016 MMAE 0.288 1.397 1.268 0.162 0.700 In Vivo Dimer Activity SG2000 15 Solvent Control Cisplatin 4mg/kg (i.v.; days 0,4,8) 10 SJG136 0.2mg/kg (i.v.; days 0,4,8) 5 Mean Relative Tumour Volume CH1:CisR 0 SG2000 20 Solvent Control Cisplatin 4mg/kg (i.v.; days 0,4,8) SJG136 0.2mg/kg (i.v.; days 0,4,8) 10 0 0 10 20 30 40 50 0 10 Day 20 30 40 50 Day SG2285, LOXIMVI 3 mg/kg SG2057, 0.075 mg/Kg 200 Mean RTV Mean Relative Tumour Volume CH1 Solvent SG 2285 150 100 50 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Time (days) DNA Cross-Link – Comparison to Other Agents Cisplatin SJG-136 Yondelis SG2000 Clinical Trial Status • Phase I Trials Complete • SG2000 is in Phase II clinical trials at four centres in the U.S.A. for the treatment of cisplatin refractory ovarian cancer. • A Phase II leukaemia trial is in preparation in the UK for Q1 2012 New PBD ADC Warheads Compound IC50 (pM) X IC50 (pM) Y SG-3202 4.1 3.3 SG-3192 6.4 11.4 SG-3198 9.3 7.3 SG-2963 16.1 18.5 SG-2219 10.1 27.3 SG-2962 16.4 18.5 Linking to Antibodies Maleimide Succinimide etc. For Thiols, Amines and Phenols Self-immolative Moiety. PAB, cinnamyl or cyclizing Enzyme labile Trigger. Emphasis on avoiding lipophilic or reactive groups PBD-ADC Xenograft Standard ADC 10 mg/Kg PBD-ADC 1mg/Kg Key Advantages of PBDs as ADC Warheads • • • • Potency in picomolar range Alternative Mode of Action Non-Distortive of DNA Proven as mono-therapies in oncology – Demonstrable Therapeutic index • • • • Robust and scalable synthesis Modulation of solubility Compatible with multiple linker system chemistries Broad intellectual property protection Confidential Summary • Antigen – Tumour specific and highly expressed • Antibody – Humanized – Must be internalized – Engineered thiols groups • Linker – Must be robust (i.e. Val-Cit or MCC or one of your own devising!) • Warhead – As potent as possible • Conjugation – Reproducible DAR (2-4) • Characterisation – Access to ESI-TOF or HIC instrumentation Acknowledgements • • • • • Prof. John Hartley Dr Mahendra Deonarian Dr John Adair Dr Christina von Bulow Marina Folarin