Preclinical Development of a Duocarmycin-based Antibody ......Preclinical Development of a...
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©2017 MacroGenics, Inc. All rights reserved. Preclinical Development of a Duocarmycin-based Antibody-Drug Conjugate Targeting B7-H3 for Solid Cancer Thomas Son, Juniper A. Scribner, Jeff Hooley, Michael Chiechi, Pam Li, Tim E. Hotaling, Anushka De Costa, Yan Chen, Francine Chen, Bhaswati Barat, Ling Huang, Valentina Ciccarone, Timur Gaynutdinov, James Tamura, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini, Deryk Loo MacroGenics, Inc., Rockville, MD and South San Francisco, CA Presented at the American Association for Cancer Research Annual Meeting 2017, April 1–5, 2017, Washington, DC http://ir.macrogenics.com/events.cfm 42 Abstract Introduction: B7-H3, a member of the B7 family of immunomodulatory molecules, is overexpressed in a wide range of solid cancers. B7-H3 overexpression has been correlated with disease severity and poor outcome in several cancer types. Proof-of-concept studies targeting B7-H3 demonstrated that auristatin-based B7-H3 ADCs exhibited potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines. Based on these preliminary results, we undertook preclinical development of a B7-H3 ADC comprised of a humanized B7-H3 mAb conjugated to a potent DNA alkylating payload. Methods: Chimeric B7-H3 mAbs were conjugated to vc-seco-DUocarmycin-hydroxyBenzamide Azaindole (DUBA) (ADC conjugated and provided by Synthon Biopharmaceuticals B.V.). In vitro and in vivo activity studies were conducted with tumor cell lines that overexpress B7-H3. Based on the potency analysis, together with the biophysical properties and immunohistochemistry (IHC) profiles of the candidates, a lead mAb was selected for preclinical development. The mAb was humanized via CDR grafting and conjugated to DUBA to yield the development candidate MGC018. In vitro and in vivo studies were then conducted with MGC018 to confirm and extend the results with the chimeric ADCs. Results: Confirming our previous data and consistent with a growing body of literature, B7-H3 mAbs exhibited strong reactivity toward carcinoma cells and the vasculature of solid cancers. Chimeric B7-H3-DUBA ADCs demonstrated specific, dose-dependent cytotoxicity toward B7-H3-positive tumor cell lines in vitro and potent antitumor activity in vivo. The humanized ADC development candidate, MGC018, retained the favorable biophysical properties and the normal tissue-versus-tumor IHC profile of the parental mAb. MGC018 displayed cytotoxicity toward B7-H3-positive tumor cell lines in vitro, with IC 50 values in the sub-nM range, and potent antitumor activity in vivo, resulting in tumor stasis and tumor regression in mice bearing B7-H3-positive human tumor xenografts, representing breast, lung and ovarian cancers. Conclusion: MGC018, a preclinical candidate comprised of a humanized mAb targeting B7-H3 conjugated to the potent DNA alkylating payload DUBA via a cleavable peptide linker, exhibited a favorable preclinical profile, with strong reactivity toward tumor cells and tumor-associated vasculature, limited normal tissue reactivity, potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing several cancer types. Our findings support further preclinical development of MGC018 to evaluate its potential as an ADC therapeutic for B7-H3-expressing solid cancers. Background B7-H3: An Attractive Cell-surface Molecule for Targeted Therapy ■ B7-H3, a member of the B7 family of immune regulators, is overexpressed on many solid cancers and displays high tumor-versus-normal tissue binding differential ■ B7-H3 overexpression has been correlated with disease severity and poor outcome in many cancer types ■ MacroGenics is targeting B7-H3 by two modalities: – Enoblituzumab (MGA271) 1 : a humanized Fc-enhanced mAb with enhanced ADCC – B7-H3 x CD3 DART (MGD009): a humanized Fc-bearing bispecific DART molecule for redirected T-cell killing ■ A B7-H3 ADC may provide a complementary mechanism of action: – Proof-of-concept studies demonstrated that auristatin-based B7-H3 ADCs exhibited potent cytotoxicity in vitro and antitumor activity in vivo (AACR 2015: Abstract# 1201 2 ) ■ Objective: Develop a B7-H3 ADC clinical candidate – Identify a lead humanized anti-B7-H3 mAb – Conduct preclinical development studies with B7-H3 ADC based on the DUBA 3 DNA alkylating payload Lead Candidate Selection and Humanization ■ MG.Ab.02 selected as lead candidate based on: – Tumor v. Normal tissue reactivity – Reduced liver reactivity – In vitro potency – In vivo anti-tumor activity – Favorable CMC properties ■ The murine mAb was humanized by replacing mouse variable region heavy chain (VH) and mouse variable region light chain (VL) framework sequences with human germline framework sequences hMG.Ab.02-DUBA = MGC018 SPR Analysis Antibody Human B7-H3 Cyno B7-H3 K D On Rate Off Rate K D On Rate Off Rate MG.Ab.02 18.2 nM 5.5E+05 1.0E-02 11.8 nM 5.7E+05 6.7E-03 hMG.Ab.02 20.3 nM 6.9E+05 1.4E-02 17.0 nM 5.9E+05 1.0E-02 Binding of human and B7-H3 V1-C1(6.25-100 nM) to mAbs captured on F(ab)2 GAH Fc surface (normalized; 1:1 binding fit). In Vitro Cytotoxicity Calu-6 Lung Cancer 0.01 1 100 10000 0 2000 4000 6000 8000 10000 12000 mAb [pM] RFU (Alamar Blue) 0.0001 0.01 1 100 10000 0 2000 4000 6000 8000 10000 MDA-MB-468 Breast Cancer mAb [pM] RFU (Alamar Blue) MG.Ab.02-DUBA hMG.Ab.02-DUBA (MGC018) Anti-Tumor Activity Calu-6 Lung Cancer 0 20 40 60 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 Study Day 0 20 40 60 Study Day 3 mg/kg 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 10 mg/kg Tumor Volume (mm 3 ) Tumor Volume (mm 3 ) Vehicle (PBS) MG.Ab.02-DUBA Control mAb-DUBA hMG.Ab.02-DUBA (MGC018) Anti-B7-H3 2+ Treatment Dose (mg/kg) T/C (%) MG.Ab.02-DUBA 3 37 MG.Ab.02-DUBA 10 10 hMG.Ab.02-DUBA (MGC018) 3 27 hMG.Ab.02-DUBA (MGC018) 10 7 Control mAb-DUBA 3 67 Control mAb-DUBA 10 67 ■ Humanized MG.Ab.02-DUBA (MGC018): – Retains binding affinity for human and cyno B7-H3 – Retains potency toward B7-H3-positive tumor cells in vitro and in vivo MGC018 Exhibits Potent Anti-Tumor Activity MDA-MB-468 Breast Cancer 0 20 40 60 80 100 120 0 200 400 0 20 40 60 80 100 120 0 200 400 0 20 40 60 80 100 120 0 200 400 3 mg/kg 6 mg/kg 3 mg/kg x 3 Vehicle (PBS) Control mAb-DUBA MGC018 Study Day Study Day Study Day Tumor Volume (mm 3 ) Tumor Volume (mm 3 ) Tumor Volume (mm 3 ) Anti-B7-H3 2+ Treatment Dose (mg/kg) T/C (%) CR MGC018 3 51 1/5 MGC018 6 1 4/5 MGC018 3 x 3 2 3/5 Control mAb-DUBA 3 43 0/5 Control mAb-DUBA 6 41 0/5 Control mAb-DUBA 3 x 3 53 0/5 A375.S2 Melanoma 0 20 40 60 0 200 400 600 800 1000 1200 0 20 40 60 0 200 400 600 800 1000 1200 Tumor Volume (mm 3 ) Tumor Volume (mm 3 ) 1 mg/kg 3 mg/kg Study Day Study Day Vehicle (PBS) Control mAb-DUBA MGC018 Anti-B7-H3 3+ Treatment Dose (mg/kg) T/C (%) CR MGC018 1 16 1/7 MGC018 3 1 5/7 Control mAb-DUBA 1 70 1/7 Control mAb-DUBA 3 33 0/7 MGC018 Exhibits Potent Anti-Tumor Activity PA-1 Ovarian Cancer 0 20 40 60 0 500 1000 1500 2000 0 20 40 60 0 500 1000 1500 2000 0 20 40 60 0 500 1000 1500 2000 3 mg/kg 6 mg/kg 10 mg Vehicle (PBS) Control mAb-DUBA MGC018 Study Day Study Day Study Day Tumor Volume (mm 3 ) Tumor Volume (mm 3 ) Tumor Volume (mm 3 ) Anti-B7-H3 2+ Treatment Dose (mg/kg) T/C (%) CR MGC018 3 57 1/6 MGC018 6 9 2/6 MGC018 10 11 3/6 Control mAb-DUBA 3 111 0/6 Control mAb-DUBA 6 89 0/6 Control mAb-DUBA 10 84 0/6 MGC018 Exhibits Favorable PK Profile Single-Dose Cynomologous Monkey PK Study 0 200 400 600 0.01 0.1 1 10 100 1000 Time (hours) 0 200 400 600 Time (hours) 0 200 400 600 Time (hours) Concentration (μg/mL) Total IgG Intact ADC 0.01 0.1 1 10 100 1000 Concentration (μg/mL) Total IgG Intact ADC 0.01 0.1 1 10 100 1000 Concentration (μg/mL) Total IgG Intact ADC 10 mg/kg 3 mg/kg 1 mg/kg 0 200 400 600 0.01 0.1 1 10 100 1000 Time (hours) Concentration (μg/mL) 10 mg/kg Male 10 mg/kg Female 3 mg/kg Male 3 mg/kg Female 1 mg/kg Male 1 mg/kg Female Intact MGC018 PK Parameters for Intact MGC018 Species Dose (mg/kg) Route T ½ (hr) C max (µg/mL) AUC Last (hr*µg/mL) Cyno 1 IV 46.5 20.5 561 Cyno 3 IV 62.7 113.5 3798 Cyno 10 IV 57.3 331.0 17978 MGC018: Observations in Cynomolgus Monkeys A Single Intravenous Infusion of 1, 3 & 10 mg/kg of MGC018 was Well Tolerated in Male Cynomolgus Monkeys ■ Body weight: no change ■ Red blood cell parameters & platelets: no change ■ White blood cell populations – Transient mild-to-moderate increases in neutrophil counts in individual animals at 10 mg/kg ■ Coagulation – Mild to moderate increases in fibrinogen concentration at 10 mg/kg ■ Liver – Mild, transient increases in ALT/AST at doses of ≥3 mg/kg in ~ half of animals – No correlative histopathological findings in liver Conclusions ■ MGC018 (hMG.Ab.02–DUBA ADC): – Favorable tumor-versus-normal tissue IHC profile – Potent in vitro cytotoxicity and in vivo antitumor activity toward B7-H3-positive tumor cell lines representing a range of solid cancers – Cross-reactive with cynomolgus monkey B7-H3 with equivalent K D values – Favorable pharmacokinetic properties in cynomolgus monkeys – Well tolerated in cynomolgus monkeys at a single dose administration up to 10 mg/kg The preclinical profile supports continued development of MGC018 as a therapeutic ADC for the treatment of B7-H3-positive cancers References 1. Loo D, Alderson R, Chen F et al., Clin Cancer Res 18(14) 2012. 2. Loo D, Scribner J, Son T et al., AACR Annual Meeting Abstract(1201) 2015. 3. Dokter W, Ubink R et al., Mol Cancer Ther 13(11) 2014 Acknowledgment DUBA ADCs are conjugated and provided by Synthon Biopharmaceuticals B.V. Introduction Targeting B7-H3 via Multiple Mechanisms of Action S–S Redirected T-cell Killing Tumor Cells B7-H3 ADC NK Cells T Cells Tumor Vasculature Macrophages MGD009 Enoblituzumab Direct Killing of Tumor Cells ADCC and Presentation of Tumor Antigens via Fc-mediated Interactions Duocarmycin-based Linker Payload Structure Drug-antibody ratio ~2.70 O O O s N O O N H N H O O N H O O N I N O O NH O H 2 N N CL O N N HN O OH O OH Cleavable Peptide Self-elimination Module Proteolytic Cleavage and Release of Toxin O N O N O N HN OH Active Toxin (DUBA) Duocarmycins ■ DNA alkylating agents – cell cycle independent ■ Fully synthetic – picomolar activity in vitro ■ Retain potency in multi-drug resistant lines ■ Cleavable peptide linker – facilitates bystander effect ■ Anti-HER2-DUBA (SYD985) in Phase 1 clinical study (Synthon Biopharmaceuticals) DUBA Linker Payload provided and conjugated by Synthon Biopharmaceuticals, B.V. B7-H3 is Highly Expressed on a Range of Solid Cancers Breast Cancer Prostate Cancer Melanoma Kidney Cancer Glioblastoma Colon Cancer Lung Cancer FFPE tumor specimens stained with Goat pAb Ovarian Cancer 19/19 100% 19/19 100% Kidney Cancer Head and Neck 77/78 99% 75/78 96% Glioblastoma 65/66 98% 63/66 95% Thyroid Cancer 34/35 97% 33/35 94% Mesothelioma 41/44 93% 39/44 89% Melanoma 132/146 90% 94/146 64% Prostate Cancer 88/99 89% 51/99 52% Pancreas Cancer 69/78 88% 45/78 58% Bladder 134/156 86% 123/156 79% Lung Cancer 324/379 85% 300/379 79% Breast Cancer 189/249 76% 156/249 63% Ovarian Cancer 59/79 75% 36/79 46% Fixed Tumor MicroArray IHC Summary of Samples Screened B7-H3 Positive 2+ or Above Potential Indications: ■ Membrane expression on tumor epithelium and tumor-associated vasculature Results B7-H3-DUBA ADCs Exhibit Potent In Vitro Cytotoxicity 1 100 10000 0 2000 4000 6000 8000 Calu-6 NSCLC mAb [pM] RFU (Alamar Blue) Cold mAb Competition Confirms ADC Specificity MG.Ab.02-DUBA MG.Ab.02-DUBA + 6.7 nM MG.Ab.02 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 0 2000 4000 6000 8000 10000 NCI-H1703 NSCLC mAb [pM] RFU (Alamar Blue) 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 1 10 100 1000 10000 100000 0 2000 4000 6000 8000 10000 Calu-6 NSCLC mAb [pM] RFU (Alamar Blue) 0 1000 2000 3000 4000 5000 MDA-MB-468 Breast Cancer mAb [pM] RFU (Alamar Blue) 0 2000 4000 6000 8000 PA-1 Ovarian Cancer mAb [pM] RFU (Alamar Blue) 0 2000 4000 6000 8000 10000 LN-229 Glioblastoma mAb [pM] RFU (Alamar Blue) 0 2000 4000 6000 8000 A375.S2 Melanoma mAb [pM] RFU (Alamar Blue) 0 2000 4000 6000 8000 10000 Hs700T Pancreatic Cancer mAb [pM] RFU (Alamar Blue) 0 2000 4000 6000 8000 Raji (Negative B7-H3 Expression) B Cell Lymphoma mAb [pM] RFU (Alamar Blue) MG.Ab.01-DUBA MG.Ab.02-DUBA MG.Ab.03-DUBA Relative Potency Across Multiple Tumor Types IC 50 (pM) * Breast Cancer Melanoma Ovarian Cancer Non-Small Cell Lung Cancer Pancreatic Cancer Glioblastoma MDA-MB-468 A375.S2 PA-1 Calu-6 NCI-H1703 Hs700T LN-229 Antibody Binding Sites ** 4.20E+05 7.50E+05 6.10E+05 8.50E+05 8.10E+05 2.10E+05 8.12E+05 MG.Ab.01-DUBA 87 86 161 ND ND 1187 ND MG.Ab.02-DUBA 140 27 160 116 1523 373 920 MG.Ab.03-DUBA 647 130 471 132 1763 661 1412 *Alamar Blue cytotoxicity assay; **Antibody Binding Sites determined by Bangs QFACS Kit ■ Activity observed against a range of B7-H3-positive tumor lines DUBA ADCs Exhibit Fast PK in Rodents Exposure to Intact ADC in Mice Limited by Rodent-specific Carboxyesterase CES1c Total mAb and Intact ADC 0 100 200 300 0 10 20 30 Time (hours) Concentration (μg/mL) Total MG.Ab.02 Intact MG.Ab.02-DUBA mAb Dose (mg/kg) Route T ½ (hr) C max (µg/mL) AUC Last (hr*µg/mL) Total IgG 5 IP 78.0 21.2 2635 Intact ADC 5 IP ND 5.9 45 Total IgG determined with anti-human IgG capture/detection ELISA. Intact ADC determined with anti-hapten capture/anti-human IgG detection ELISA. ■ DUBA ADC activity in rodents may under-predict activity in humans chB7-H3-DUBA ADCs Exhibit Anti-Tumor Activity Calu-6 Lung Cancer 0 10 20 30 40 50 0 500 1000 1500 2000 Vehicle (PBS) MG.Ab.02-DUBA MG.Ab.03-DUBA Study Day Tumor Volume (mm 3 ) 0 10 20 30 40 50 0 500 1000 1500 2000 Study Day Tumor Volume (mm 3 ) Control mAb-DUBA MG.Ab.01-DUBA 3 mg/kg 10 mg/kg Anti-B7-H3 2+ Treatment Dose – QW (mg/kg) T/C (%) MG.Ab.01-DUBA 3 40 MG.Ab.01-DUBA 10 12 MG.Ab.02-DUBA 3 22 MG.Ab.02-DUBA 10 4 MG.Ab.03-DUBA 3 44 MG.Ab.03-DUBA 10 8 Control mAb-DUBA 3 66 Control mAb-DUBA 10 37 S375.S2 Melanoma 0 20 40 60 80 100 0 20 40 60 80 100 0 500 1000 1500 2000 0 500 1000 1500 2000 Vehicle (PBS) MG.Ab.02-DUBA MG.Ab.03-DUBA Study Day Tumor Volume (mm 3 ) Study Day Tumor Volume (mm 3 ) Control mAb-DUBA MG.Ab.01-DUBA 3 mg/kg 10 mg/kg Anti-B7-H3 3+ Treatment Dose – QW (mg/kg) T/C (%) CR MG.Ab.01-DUBA 3 17 4/7 MG.Ab.01-DUBA 10 0 7/7 MG.Ab.02-DUBA 3 0 7/7 MG.Ab.02-DUBA 10 6 6/7 MG.Ab.03-DUBA 3 3 5/7 MG.Ab.03-DUBA 10 0 7/7 Control mAb-DUBA 3 28 1/7 Control mAb-DUBA 10 21 2/7
Transcript of Preclinical Development of a Duocarmycin-based Antibody ......Preclinical Development of a...
©2017 MacroGenics, Inc. All rights reserved.
Preclinical Development of a Duocarmycin-based Antibody-Drug Conjugate Targeting B7-H3 for Solid CancerThomas Son, Juniper A. Scribner, Jeff Hooley, Michael Chiechi, Pam Li, Tim E. Hotaling, Anushka De Costa, Yan Chen, Francine Chen, Bhaswati Barat,
Ling Huang, Valentina Ciccarone, Timur Gaynutdinov, James Tamura, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini, Deryk Loo MacroGenics, Inc., Rockville, MD and South San Francisco, CA
Presented at the American Association for Cancer Research Annual Meeting 2017, April 1–5, 2017, Washington, DC
http://ir.macrogenics.com/events.cfm
42
AbstractIntroduction: B7-H3, a member of the B7 family of immunomodulatory molecules, is overexpressed in a wide range of solid cancers. B7-H3 overexpression has been correlated with disease severity and poor outcome in several cancer types. Proof-of-concept studies targeting B7-H3 demonstrated that auristatin-based B7-H3 ADCs exhibited potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines. Based on these preliminary results, we undertook preclinical development of a B7-H3 ADC comprised of a humanized B7-H3 mAb conjugated to a potent DNA alkylating payload.
Methods: Chimeric B7-H3 mAbs were conjugated to vc-seco-DUocarmycin-hydroxyBenzamide Azaindole (DUBA) (ADC conjugated and provided by Synthon Biopharmaceuticals B.V.). In vitro and in vivo activity studies were conducted with tumor cell lines that overexpress B7-H3. Based on the potency analysis, together with the biophysical properties and immunohistochemistry (IHC) profiles of the candidates, a lead mAb was selected for preclinical development. The mAb was humanized via CDR grafting and conjugated to DUBA to yield the development candidate MGC018. In vitro and in vivo studies were then conducted with MGC018 to confirm and extend the results with the chimeric ADCs.
Results: Confirming our previous data and consistent with a growing body of literature, B7-H3 mAbs exhibited strong reactivity toward carcinoma cells and the vasculature of solid cancers. Chimeric B7-H3-DUBA ADCs demonstrated specific, dose-dependent cytotoxicity toward B7-H3-positive tumor cell lines in vitro and potent antitumor activity in vivo. The humanized ADC development candidate, MGC018, retained the favorable biophysical properties and the normal tissue-versus-tumor IHC profile of the parental mAb. MGC018 displayed cytotoxicity toward B7-H3-positive tumor cell lines in vitro, with IC50 values in the sub-nM range, and potent antitumor activity in vivo, resulting in tumor stasis and tumor regression in mice bearing B7-H3-positive human tumor xenografts, representing breast, lung and ovarian cancers.
Conclusion: MGC018, a preclinical candidate comprised of a humanized mAb targeting B7-H3 conjugated to the potent DNA alkylating payload DUBA via a cleavable peptide linker, exhibited a favorable preclinical profile, with strong reactivity toward tumor cells and tumor-associated vasculature, limited normal tissue reactivity, potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing several cancer types. Our findings support further preclinical development of MGC018 to evaluate its potential as an ADC therapeutic for B7-H3-expressing solid cancers.
BackgroundB7-H3: An Attractive Cell-surface Molecule for Targeted Therapy■■ B7-H3, a member of the B7 family of immune regulators, is overexpressed on many solid cancers and displays high tumor-versus-normal tissue binding differential■■ B7-H3 overexpression has been correlated with disease severity and poor outcome in many cancer types■■ MacroGenics is targeting B7-H3 by two modalities:
– Enoblituzumab (MGA271)1: a humanized Fc-enhanced mAb with enhanced ADCC – B7-H3 x CD3 DART (MGD009): a humanized Fc-bearing bispecific DART molecule for redirected T-cell killing
■■ A B7-H3 ADC may provide a complementary mechanism of action: – Proof-of-concept studies demonstrated that auristatin-based B7-H3 ADCs exhibited potent cytotoxicity in vitro and antitumor activity in vivo (AACR 2015: Abstract# 12012)
■■ Objective: Develop a B7-H3 ADC clinical candidate – Identify a lead humanized anti-B7-H3 mAb – Conduct preclinical development studies with B7-H3 ADC based on the DUBA3 DNA alkylating payload
Lead Candidate Selection and Humanization ■■ MG.Ab.02 selected as lead candidate based on:
– Tumor v. Normal tissue reactivity – Reduced liver reactivity
– In vitro potency – In vivo anti-tumor activity – Favorable CMC properties
■■ The murine mAb was humanized by replacing mouse variable region heavy chain (VH) and mouse variable region light chain (VL) framework sequences with human germline framework sequences
hMG.Ab.02-DUBA = MGC018SPR Analysis
AntibodyHuman B7-H3 Cyno B7-H3
KD On Rate Off Rate KD On Rate Off RateMG.Ab.02 18.2 nM 5.5E+05 1.0E-02 11.8 nM 5.7E+05 6.7E-03
hMG.Ab.02 20.3 nM 6.9E+05 1.4E-02 17.0 nM 5.9E+05 1.0E-02
Binding of human and B7-H3 V1-C1(6.25-100 nM) to mAbs captured on F(ab)2 GAH Fc surface (normalized; 1:1 binding fit).
In Vitro CytotoxicityCalu-6
Lung Cancer
0.01 1 100 100000
2000
4000
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10000
12000
mAb [pM]
RFU
(Ala
mar
Blu
e)
0.0001 0.01 1 100 100000
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10000
MDA-MB-468Breast Cancer
mAb [pM]
RFU
(Ala
mar
Blu
e)
MG.Ab.02-DUBAhMG.Ab.02-DUBA (MGC018)
Anti-Tumor ActivityCalu-6 Lung Cancer
0 20 40 600
200400600800
10001200140016001800200022002400
Study Day0 20 40 60
Study Day
3 mg/kg
0200400600800
10001200140016001800200022002400
10 mg/kg
Tum
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olum
e (m
m3 )
Tum
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e (m
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Vehicle (PBS)
MG.Ab.02-DUBAControl mAb-DUBA
hMG.Ab.02-DUBA (MGC018)
Anti-B7-H3 2+
Treatment Dose (mg/kg) T/C (%)
MG.Ab.02-DUBA 3 37MG.Ab.02-DUBA 10 10hMG.Ab.02-DUBA (MGC018) 3 27hMG.Ab.02-DUBA (MGC018) 10 7Control mAb-DUBA 3 67Control mAb-DUBA 10 67
■■ Humanized MG.Ab.02-DUBA (MGC018): – Retains binding affinity for human and cyno B7-H3 – Retains potency toward B7-H3-positive tumor cells in vitro and in vivo
MGC018 Exhibits Potent Anti-Tumor ActivityMDA-MB-468 Breast Cancer
0 20 40 60 80 100 1200
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4003 mg/kg 6 mg/kg 3 mg/kg x 3
Vehicle (PBS)Control mAb-DUBAMGC018
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Treatment Dose (mg/kg) T/C (%) CR
MGC018 3 51 1/5MGC018 6 1 4/5MGC018 3 x 3 2 3/5Control mAb-DUBA 3 43 0/5Control mAb-DUBA 6 41 0/5Control mAb-DUBA 3 x 3 53 0/5
A375.S2 Melanoma
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m3 )
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Vehicle (PBS)Control mAb-DUBAMGC018
Anti-B7-H3 3+
Treatment Dose (mg/kg) T/C (%) CR
MGC018 1 16 1/7MGC018 3 1 5/7Control mAb-DUBA 1 70 1/7Control mAb-DUBA 3 33 0/7
MGC018 Exhibits Potent Anti-Tumor ActivityPA-1 Ovarian Cancer
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m3 )
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Treatment Dose (mg/kg) T/C (%) CR
MGC018 3 57 1/6
MGC018 6 9 2/6
MGC018 10 11 3/6
Control mAb-DUBA 3 111 0/6
Control mAb-DUBA 6 89 0/6
Control mAb-DUBA 10 84 0/6
MGC018 Exhibits Favorable PK Profile Single-Dose Cynomologous Monkey PK Study
0 200 400 6000.01
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Time (hours)
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Conc
entr
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n (µ
g/m
L) Total IgGIntact ADC
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entr
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Conc
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atio
n (µ
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L)
10 mg/kg Male10 mg/kg Female3 mg/kg Male3 mg/kg Female1 mg/kg Male1 mg/kg Female
Intact MGC018
PK Parameters for Intact MGC018Species Dose
(mg/kg) Route T½ (hr)
Cmax (µg/mL)
AUC Last (hr*µg/mL)
Cyno 1 IV 46.5 20.5 561
Cyno 3 IV 62.7 113.5 3798
Cyno 10 IV 57.3 331.0 17978
MGC018: Observations in Cynomolgus MonkeysA Single Intravenous Infusion of 1, 3 & 10 mg/kg of MGC018 was Well Tolerated in Male Cynomolgus Monkeys■■ Body weight: no change■■ Red blood cell parameters & platelets: no change■■ White blood cell populations
– Transient mild-to-moderate increases in neutrophil counts in individual animals at 10 mg/kg
■■ Coagulation – Mild to moderate increases in fibrinogen concentration at 10 mg/kg
■■ Liver – Mild, transient increases in ALT/AST at doses of ≥3 mg/kg in ~ half of animals – No correlative histopathological findings in liver
Conclusions■■ MGC018 (hMG.Ab.02–DUBA ADC):
– Favorable tumor-versus-normal tissue IHC profile – Potent in vitro cytotoxicity and in vivo antitumor activity toward B7-H3-positive tumor cell lines representing a range of solid cancers – Cross-reactive with cynomolgus monkey B7-H3 with equivalent KD values – Favorable pharmacokinetic properties in cynomolgus monkeys – Well tolerated in cynomolgus monkeys at a single dose administration up to 10 mg/kg
The preclinical profile supports continued development of MGC018 as a therapeutic ADC for the treatment of B7-H3-positive cancers
References1. Loo D, Alderson R, Chen F et al., Clin Cancer Res 18(14) 2012. 2. Loo D, Scribner J, Son T et al., AACR Annual Meeting Abstract(1201) 2015. 3. Dokter W, Ubink R et al., Mol Cancer Ther 13(11) 2014
AcknowledgmentDUBA ADCs are conjugated and provided by Synthon Biopharmaceuticals B.V.
Introduction
Targeting B7-H3 via Multiple Mechanisms of Action
S–S
Redirected T-cell Killing
Tumor Cells
B7-H3 ADC
NK Cells
T Cells
Tumor Vasculature
Macrophages
MGD009
Enoblituzumab
Direct Killing of Tumor Cells
ADCC and Presentation ofTumor Antigens via
Fc-mediated Interactions
Duocarmycin-based Linker PayloadStructure
Drug-antibody ratio ~2.70
OO
OsN O
ONH
NH
O
ONH
OO
NI
NOO
NHOH2N
N
CL
ON
N
HNO
OH
O OHCleavable Peptide
Self-elimination Module
Proteolytic Cleavageand Release of Toxin
O
NO
NO
N
HN
OH
Active Toxin (DUBA)
Duocarmycins■■ DNA alkylating agents – cell cycle independent■■ Fully synthetic – picomolar activity in vitro■■ Retain potency in multi-drug resistant lines■■ Cleavable peptide linker – facilitates bystander effect■■ Anti-HER2-DUBA (SYD985) in Phase 1 clinical study (Synthon Biopharmaceuticals)
DUBA Linker Payload provided and conjugated by Synthon Biopharmaceuticals, B.V.
B7-H3 is Highly Expressed on a Range of Solid CancersBreast Cancer
Prostate Cancer
Melanoma
Kidney Cancer Glioblastoma Colon Cancer
Lung Cancer
FFPE tumor specimens stained with Goat pAb
Ovarian Cancer
19/19 100% 19/19 100%
Kidney Cancer
Head and Neck
77/78 99% 75/78 96%
Glioblastoma 65/66 98% 63/66 95%
Thyroid Cancer 34/35 97% 33/35 94%
Mesothelioma 41/44 93% 39/44 89%
Melanoma 132/146 90% 94/146 64%
Prostate Cancer 88/99 89% 51/99 52%
Pancreas Cancer 69/78 88% 45/78 58%
Bladder 134/156 86% 123/156 79%
Lung Cancer 324/379 85% 300/379 79%
Breast Cancer 189/249 76% 156/249 63%
Ovarian Cancer 59/79 75% 36/79 46%
Fixed Tumor MicroArray
IHC Summary of Samples Screened
B7-H3 Positive 2+ or AbovePotential Indications:
■■ Membrane expression on tumor epithelium and tumor-associated vasculature
Results
B7-H3-DUBA ADCs Exhibit Potent In Vitro Cytotoxicity
1 100 100000
2000
4000
6000
8000
Calu-6NSCLC
mAb [pM]
RFU
(Ala
mar
Blu
e)
Cold mAb CompetitionConfirms ADC Specificity
MG.Ab.02-DUBAMG.Ab.02-DUBA + 6.7 nM MG.Ab.02
1 10 100 1000 10000 100000
1 10 100 1000 10000 100000 1 10 100 1000 10000 100000
1 10 100 1000 10000 100000 1 10 100 1000 10000 1000000
2000
4000
6000
8000
10000NCI-H1703
NSCLC
mAb [pM]
RFU
(Ala
mar
Blu
e)
1 10 100 1000 10000 100000
1 10 100 1000 10000 100000
1 10 100 1000 10000 1000000
2000
4000
6000
8000
10000Calu-6NSCLC
mAb [pM]
RFU
(Ala
mar
Blu
e)
0
1000
2000
3000
4000
5000MDA-MB-468Breast Cancer
mAb [pM]
RFU
(Ala
mar
Blu
e)
0
2000
4000
6000
8000PA-1
Ovarian Cancer
mAb [pM]
RFU
(Ala
mar
Blu
e)
0
2000
4000
6000
8000
10000LN-229
Glioblastoma
mAb [pM]
RFU
(Ala
mar
Blu
e)
0
2000
4000
6000
8000A375.S2
Melanoma
mAb [pM]
RFU
(Ala
mar
Blu
e)
0
2000
4000
6000
8000
10000Hs700T
Pancreatic Cancer
mAb [pM]
RFU
(Ala
mar
Blu
e)
0
2000
4000
6000
8000
Raji (Negative B7-H3 Expression)B Cell Lymphoma
mAb [pM]
RFU
(Ala
mar
Blu
e)
MG.Ab.01-DUBA MG.Ab.02-DUBA MG.Ab.03-DUBA
Relative Potency Across Multiple Tumor Types
IC50 (pM) *Breast Cancer Melanoma Ovarian
CancerNon-Small Cell
Lung CancerPancreatic
Cancer Glioblastoma
MDA-MB-468 A375.S2 PA-1 Calu-6 NCI-H1703 Hs700T LN-229Antibody Binding Sites ** 4.20E+05 7.50E+05 6.10E+05 8.50E+05 8.10E+05 2.10E+05 8.12E+05
MG.Ab.01-DUBA 87 86 161 ND ND 1187 ND
MG.Ab.02-DUBA 140 27 160 116 1523 373 920
MG.Ab.03-DUBA 647 130 471 132 1763 661 1412
*Alamar Blue cytotoxicity assay; **Antibody Binding Sites determined by Bangs QFACS Kit
■■ Activity observed against a range of B7-H3-positive tumor lines
DUBA ADCs Exhibit Fast PK in RodentsExposure to Intact ADC in Mice Limited by Rodent-specific Carboxyesterase CES1c
Total mAb and Intact ADC
0 100 200 3000
10
20
30
Time (hours)
Conc
entr
atio
n (µ
g/m
L)
Total MG.Ab.02Intact MG.Ab.02-DUBA mAb Dose
(mg/kg) Route T½ (hr)
Cmax (µg/mL)
AUC Last (hr*µg/mL)
Total IgG 5 IP 78.0 21.2 2635
Intact ADC 5 IP ND 5.9 45
Total IgG determined with anti-human IgG capture/detection ELISA. Intact ADC determined with anti-hapten capture/anti-human IgG detection ELISA.
■■ DUBA ADC activity in rodents may under-predict activity in humans
chB7-H3-DUBA ADCs Exhibit Anti-Tumor ActivityCalu-6 Lung Cancer
0 10 20 30 40 500
500
1000
1500
2000
Vehicle (PBS)
MG.Ab.02-DUBAMG.Ab.03-DUBA
Study Day
Tum
or V
olum
e (m
m3 )
0 10 20 30 40 500
500
1000
1500
2000
Study Day
Tum
or V
olum
e (m
m3 )
Control mAb-DUBAMG.Ab.01-DUBA
3 mg/kg 10 mg/kg
Anti-B7-H3 2+
Treatment Dose – QW (mg/kg) T/C (%)
MG.Ab.01-DUBA 3 40MG.Ab.01-DUBA 10 12MG.Ab.02-DUBA 3 22MG.Ab.02-DUBA 10 4MG.Ab.03-DUBA 3 44MG.Ab.03-DUBA 10 8Control mAb-DUBA 3 66Control mAb-DUBA 10 37
S375.S2 Melanoma
0 20 40 60 80 100 0 20 40 60 80 1000
500
1000
1500
2000
0
500
1000
1500
2000
Vehicle (PBS)
MG.Ab.02-DUBAMG.Ab.03-DUBA
Study Day
Tum
or V
olum
e (m
m3 )
Study Day
Tum
or V
olum
e (m
m3 )
Control mAb-DUBAMG.Ab.01-DUBA
3 mg/kg 10 mg/kg
Anti-B7-H3 3+
Treatment Dose – QW (mg/kg) T/C (%) CR
MG.Ab.01-DUBA 3 17 4/7MG.Ab.01-DUBA 10 0 7/7MG.Ab.02-DUBA 3 0 7/7MG.Ab.02-DUBA 10 6 6/7MG.Ab.03-DUBA 3 3 5/7MG.Ab.03-DUBA 10 0 7/7Control mAb-DUBA 3 28 1/7Control mAb-DUBA 10 21 2/7
