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  • Enhanced Antitumor Activity in Combination with Checkpoint Inhibitors 5203 · 2020. 12. 11. · 5203 Abstract Introduction: B7-H3, a member of the B7-family of immunomodulatory molecules,
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©2020 MacroGenics, Inc. All rights reserved. MGC018, a Duocarmycin-based Antibody-drug Conjugate Targeting B7-H3, Exhibits Immunomodulatory Activity and Enhanced Antitumor Activity in Combination with Checkpoint Inhibitors Juniper A. Scribner, Michael Chiechi, Pam Li, Thomas Son, Jeff Hooley, Ying Li, Anushka De Costa, Peter Lung, Nicholas Yee-Toy, Francine Chen, Zhuangyu Pan, Bhaswati Barat, Christina Wolff, Valentina Ciccarone, James Tamura, Scott Koenig, Chet Bohac, Jon Wigginton, Paul A. Moore, Ezio Bonvini, Deryk Loo MacroGenics, Inc., Brisbane, CA and Rockville, MD Presented at the American Association for Cancer Research 2020 Virtual Annual Meeting II, June 22–24, 2020 5203 Abstract Introduction: B7-H3, a member of the B7-family of immunomodulatory molecules, is overexpressed in a wide range of solid tumors. B7-H3 tumor overexpression has been correlated with disease severity and poor outcome. MGC018 is a duocarmycin-based antibody-drug conjugate (ADC) targeting B7-H3. MGC018 exhibits a favorable preclinical profile, with strong reactivity toward tumor cells and tumor-associated vasculature, limited normal tissue reactivity, and potent antitumor activity toward B7-H3-expressing tumor xenografts. With the emergence of immune-checkpoint blockade as a promising treatment for cancer, interest has grown in understanding the potential of cytotoxic agents to promote immune surveillance or stimulate immune responses to dying cancer cells, leading to immunological memory. ADCs bearing tubulin and DNA modifying cytotoxic payloads have been reported to induce immunogenic cell death (ICD), mediate antitumor immunity in immunocompetent mouse models, and synergistically combine with checkpoint inhibitors to deliver enhanced antitumor responses. Based on those results, we investigated the immunomodulatory potential of MGC018 and the prospect to combine with checkpoint blockade to enhance antitumor responses. Methods: Syngeneic mouse models expressing human B7-H3 were employed to investigate the antitumor activity of MGC018 in an immune competent setting. Studies were conducted to assess the role of the immune system in the MGC018-mediated antitumor responses, whether MGC018 could impart antitumor memory responses in vivo, and the potential to enhance antitumor responses by combining MGC018 with PD-1 blockade. Results: MGC018 demonstrated specific, dose-dependent in vivo antitumor activity toward human B7-H3-bearing tumors in immunocompetent syngeneic mouse models. Depletion of CD8 + T cells led to reduced antitumor responses, indicating that CD8 + T cells contributed to MGC018-mediated antitumor activity. Antitumor activity in these models was enhanced when MGC018 was combined with anti-PD-1. Treatment with MGC018 alone, or in combination with anti-PD-1, led to complete antitumor responses, and the majority of mice rejected subsequent tumor rechallenge. Conclusion: MGC018, a clinical-stage therapeutic comprised of a humanized antibody targeting B7-H3, conjugated to a duocarmycin-based DNA alkylating payload, exhibits a favorable preclinical profile. Results from these syngeneic model studies support the hypothesis that the antitumor activity of the duocarmycin-based MGC018 ADC (1) mediates immunomodulatory activity, (2) is enhanced by combination with checkpoint blockade, and (3) induces immunological memory. Our findings support a clinical strategy that combines MGC018 with checkpoint blockade for the treatment of B7-H3- expressing solid cancers. Background MGC018 a Clinical-stage Anti-B7-H3 ADC Therapeutic B7-H3 ■ Member of the B7-family of immune regulators ■ Overexpressed on solid cancers, with high tumor-versus-normal tissue binding differential ■ Overexpression correlated with disease severity and poor outcome in multiple cancers MGC018 ■ Comprised of a humanized antibody targeting B7-H3 (MGA017) ■ Conjugated to a duocarmycin-based DNA alkylating payload via native cysteines ■ Potent antitumor activity in mouse models toward B7-H3-expressing human tumor xenografts at clinically relevant dose levels, despite model limitations – Rapid clearance (half-life of ADC ~40 hours) due to degradation by rodent-specific carboxylesterase CES1c — not present in primates or humans ■ Favorable safety and toxicokinetics in a repeat-dose GLP toxicology study in cynomolgus monkeys, a validated toxicology species for MGC018 ■ Phase I/II clinical study in advanced solid cancers in progress (NCT03729596) Immunomodulatory Activity of ADC’s ■ Have been reported to induce immunogenic cell death (Gerber et al., Biochem Pharm 2016) ■ Synergistically combine with checkpoint inhibitors to deliver enhanced antitumor responses ■ Mediate antitumor immunity in immunocompetent mouse models Objectives ■ Evaluate MGC018 to mediate immunomodulatory activity ■ Determine whether treatment with MGC018 is enhanced by combination with checkpoint blockade ■ Assess whether MGC018 induces immunological memory Introduction Targeting B7-H3 with MGC018: Potential for Immunomodulation Tumor Cells MGC018 CD4 T Cells CD8 T Cells Tumor Vasculature Immature DC Direct Killing of Tumor Cells Lymph Node CD8 T Cells Mature DC Granzyme B and Perforin Tumor Antigens DAMPs: CRT, HMGB1, ATP TCR MHCII TCR MHCI DC Adapted from Gerber et al., Biochem Pharm 2016 Tumor Antigens DAMPs: Damage-associated molecular patterns. Duocarmycin-based Linker Payload 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) vc-seco-DUocarmycin-hydroxyBenzamide Azaindole (DUBA) Structure MGA017 MGC018 Duocarmycin Payload huIgG1 (wild type Fc) Anti-B7-H3 Drug-antibody ratio 2.68 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 ■ Trastuzumab-vc-seco-DUBA (SYD985) in Phase 3 clinical trial in locally advanced or metastatic breast cancer (Byondis, B.V.) DUBA Linker Payload provided and conjugated by Byondis, B.V. B7-H3 is Highly Expressed on a Range of Solid Cancers SCCHN Pancreatic Ca Bladder Ca Ovarian Ca Anal Ca Mesothelioma Kidney Ca Melanoma Prostate Ca Colorectal Ca Sarcoma Lung Ca Breast Ca Gastric Ca Glioblastoma 0 10 20 30 40 50 60 70 80 90 100 Percent of Tumors with Indicated H-Score 1-100 101-200 201-300 H-Score 16% 14% 84% 22% 32% 38% 53% 16% 42% 15% 34% 15% 26% 6% 26% 39% 30% 43% 14% 48% 28% 24% 40% 24% 66% 28% 58% 16% 18% 50% 22% 18% 37% 46% 51% 38% 25% 44% 15% 33% 53% 84% 16% ■ B7-H3 is expressed on tumor epithelium and tumor-associated vasculature FFPE staining with goat polyclonal antibody (R&D Systems). Independent specimens ranged from 19-189 for each indication. MGC018 Antitumor Activity Toward Patient-derived Xenograft (PDX) Models H-score = 180 H-score = 130 H-score = 180 H-score = 240 0 500 1000 1500 2000 2500 Vehicle Study Day Mean Tumor Volume (mm 3 ) MGC018 3 mg/kg x 2 Control ADC 3 mg/kg x 2 0 10 20 30 0 500 1000 1500 2000 2500 Vehicle Study Day Mean Tumor Volume (mm 3 ) MGC018 3 mg/kg x 2 Control ADC 3 mg/kg x 2 0 500 1000 1500 Vehicle Study Day Tumor Volume (mm 3 ) Mean Tumor Volume (mm 3 ) MGC018 3 mg/kg x 3 Control ADC 3 mg/kg x 3 Vehicle MGC018 3 mg/kg x 2 Control ADC 3 mg/kg x 2 Breast Cancer PDX CTG-0869 Head and Neck Cancer PDX CTG-0790 Pancreatic Cancer PDX CTG-0305 Prostate Cancer PDX CTG-2441 Dosing Dosing Dosing Dosing 0 500 1000 1500 2000 Study Day 0 10 20 30 0 10 20 30 -10 0 10 20 30 ■ MGC018 displays antitumor activity toward heterogeneous B7-H3 expressing PDX models representing a range of solid cancers Results Syngeneic Mouse Model System Stably transfected human B7-H3 into mouse colorectal cancer cell lines (MC38 and CT26) Tumor cell lines grown in vitro Human B7-H3 expressing tumor cell lines grown in vitro Tumor originating in an inbred strain Image adapted from Taconic Tumor cells implanted in mice of same inbred strain ■ Tumor cells are originally derived from the same genetic background as the implanted mouse ■ Mice have an intact and fully functioning immune system ■ Syngeneic models provide an effective approach for studying how cancer therapies perform in the presence of a functional immune system ■ While transduced human B7-H3 may be immunogenic in the mouse, the model provides proof-of-concept for the ADC’s ability to enhance adaptive responses Murine Colorectal Cancer Cell Lines Engineered to Overexpress Human B7-H3 Relative Expression of Human B7-H3 on the Cell Surface % of Max 100 80 60 40 20 0 10 0 10 1 10 2 10 3 10 4 PE Anti-B7-H3 mAb Negative control Unstained control % of Max 100 80 60 40 20 0 10 0 10 1 10 2 10 3 10 4 PE CT26/huB7-H3 MC38/huB7-H3 QFACS MC38/huB7-H3 Murine Colorectal Cancer CT26/huB7-H3 Murine Colorectal Cancer Antibody Binding Capacity 4.08E+04 5.37E+04 Syngeneic Mouse Model C57BL/6 BALB/c Antibody Binding Capacity determined by Bangs QFACS Kit. MGC018 Mediates Apoptosis of Murine Tumor Cells Caspase 3/7 Activation of MC38/huB7-H3 Tumor Cells 2 12 24 36 48 60 0 20000 40000 60000 Time (hours) Apoptotic Cell Count/Well MGC018 (3.3 nM) MGA017 (3.3 nM) Untreated 2 hour 48 hour 60 hour Untreated MGA017 (3.3 nM) MGC018 (3.3 nM) MGC018 Modulates Damage-associated Molecular Patterns Calreticulin Expression on the Surface of MC38/huB7-H3 Tumor Cells Isotype control Calreticulin-AF647 10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4 10 1 10 2 10 3 10 4 Untreated SYD978 Calreticulin (+) 10 0 10 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 MGC018 Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 10 4 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 10 4 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 24 Hour 72 Hour 48 Hour Counts Counts Counts Counts Counts Counts Counts Counts Counts ■ MGC018 and SYD978 (seco-DUBA Payload) induced calreticulin translocation to the surface of MC38/huB7-H3 murine tumor cells ■ Analysis of other DAMPs are currently underway Antitumor Activity of MGC018 is Dependent on Human B7-H3 Expression MC38/huB7-H3 MC38 Parental CT26/huB7-H3 CT26 Parental 0 10 20 30 40 50 Vehicle MGC018 10 mg/kg Vehicle MGC018 10 mg/kg Vehicle MGC018 10 mg/kg Vehicle MGC018 10 mg/kg Day (post inoculation) 0 10 20 30 40 50 Day (post inoculation) Day (post inoculation) Mean Tumor Volume (mm 3 ) Mean Tumor Volume (mm 3 ) 0 10 20 30 40 50 Day (post inoculation) 0 10 20 30 40 50 0 500 1000 1500 2000 2500 3000 Mean Tumor Volume (mm 3 ) 0 500 1000 1500 2000 2500 3000 Mean Tumor Volume (mm 3 ) Dosing Dosing Dosing Dosing 0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000 ■ Parental xenografts lacking human B7-H3 expression are insensitive to MGC018 Antitumor Activity is Dependent on Targeted Delivery of Payload by MGC018 MC38/huB7-H3 CT26/huB7-H3 0 10 20 30 40 0 500 1000 1500 2000 2500 3000 3500 4000 Day (post inoculation) Mean Tumor Volume (mm 3 ) 0 10 20 30 40 50 60 0 500 1000 1500 2000 2500 3000 3500 Vehicle MGA017 10 mg/kg + SYD978 0.0948 mg/kg Day (post inoculation) Mean Tumor Volume (mm 3 ) MGA017 10 mg/kg MGC018 10 mg/kg SYD978 0.0948 mg/kg Dosing Dosing ■ No antitumor activity with MGA017, SYD978 (seco-DUBA payload) or combination of MGA017 + SYD978 ■ Antitumor activity of SYD978 requires conjugation to B7-H3-targeting mAb MGC018 Increases Infiltration of Lymphocytes into MC38/huB7-H3 Tumor Xenografts Untreated MGC018 10 mg/kg Untreated MGC018 10 mg/kg 0 10 20 30 40 Mean Positive Expression (%) Treatment Mean Positive Cell Count/ Total Viable Area (mm 2 ) CD4 CD8 Granzyme B H&E Stain B7-H3 Expression CD4 DAPI CD8 DAPI Granzyme B DAPI ■ MGC018 treatment increases CD4 and CD8 infiltration and Granzyme B expression CD8 + T Cells Contribute to MGC018-mediated Antitumor Response MGC018 dosing Anti-CD8 dosing Vehicle MGC018 10 mg/kg + Anti-CD8 8 mg/kg MGC018 10 mg/kg Anti-CD8 8 mg/kg 0 20 40 60 10 30 50 0 500 1000 1500 2000 2500 3000 3500 MGC018 dosing Anti-CD8 dosing Day (post inoculation) Mean Tumor Volume (mm 3 ) MC38/huB7-H3 0 10 20 30 40 50 0 500 1000 1500 2000 2500 3000 CT26/huB7-H3 Mean Tumor Volume (mm 3 ) Day (post inoculation) ■ Anti-CD8 depletes CD8 + T cells and attenuates MGC018 antitumor activity Anti-CD8 antibody from BioXCell: Clone 53-6.7. Antitumor Activity of MGC018 in Combination with Anti-PD-1 in CT26 Model: Induction of Immunological Memory 0 10 20 30 40 50 60 0 500 1000 1500 2000 2500 MGC018 dosing Rechallenge Anti-PD-1 dosing Vehicle MGC018 10 mg/kg + Anti-PD-1 20 mg/kg Day (post inoculation) Mean Tumor Volume (mm 3 ) MGC018 10 mg/kg Anti-PD-1 20 mg/kg CT26/huB7-H3 CR animals selected for tumor rechallenge Survival of Animals Challenged with CT26 Parent Days (post rechallenge) Percent Survival 0 20 40 60 80 100 120 0 25 50 75 100 Naive Animals MGC018 + Anti-PD-1 CR Animals MGC018 CR Animals ■ Antitumor activity of MGC018 is enhanced by anti-PD-1 ■ MGC018 and MGC018 + anti-PD-1 induces immunological memory CR defined as tumors < 24 mm 3 . Anti-PD-1 antibody from BioXCell: Clone RMP1-14. Antitumor Activity of MGC018 in Combination with Anti-PD-1 in MC38 Model: Induction of Immunological Memory MGC018 dosing Rechallenge Anti-PD-1 dosing 0 10 20 30 40 50 0 500 1000 1500 2000 2500 3000 3500 Vehicle MGC018 10 mg/kg + Anti-PD-1 20 mg/kg Day (post inoculation) Mean Tumor Volume (mm 3 ) MGC018 10 mg/kg Anti-PD-1 20 mg/kg Survival of Animals Challenged with MC38/huB7-H3 Days (post rechallenge) Percent Survival MC38/huB7-H3 A. 0 25 50 75 100 MGC018 + Anti-PD-1 CR Animals MGC018 CR Animals Naive Animals CR animals selected for tumor rechallenge 0 10 20 30 40 50 60 Rechallenge Dosing MC38/huB7-H3 Survival of Animals Challenged with MC38/huB7-H3 Survival of Animals Challenged with MC38 Parent CR animals selected for tumor rechallenge 0 10 20 30 40 50 60 70 80 90 0 500 1000 1500 2000 2500 Day (post inoculation) Mean Tumor Volume (mm 3 ) 0 10 20 30 40 50 0 25 50 75 100 Days (post rechallenge) Percent Survival Naive animals MGC018 CR animals 0 10 20 30 40 50 0 25 50 75 100 Days (post rechallenge) Percent Survival Naive animals MGC018 CR animals B. N=15 N=15 MGC018 10 mg/kg ■ Antitumor activity of MGC018 is enhanced by anti-PD-1 ■ MGC018 and MGC018 + anti-PD-1 induces immunological memory CR defined as tumors < 16 mm 3 . Anti-PD-1 antibody from BioXCell: RMP1-14. Conclusions ■ MGC018 demonstrated in vivo antitumor activity toward heterogeneous B7-H3- expressing PDX models – TNBC, Pancreatic, Prostate, Head and Neck Squamous Cell Carcinoma ■ MGC018 demonstrated specific, dose-dependent in vivo antitumor activity toward human B7-H3-expressing tumors in immunocompetent syngeneic mouse models – MGC018 induced translocation of calreticulin, a component of damage-associated molecular patterns (DAMPs) – MGC018 treatment increased infiltration of CD4 + and CD8 + lymphocytes into the tumor ■ Depletion of CD8 + T cells led to reduced antitumor responses, indicating that CD8 + T cells contributed to MGC018-mediated antitumor activity ■ Antitumor activity was enhanced when MGC018 was combined with anti-PD-1 leading to more complete antitumor responses ■ MGC018 alone or in combination with anti-PD-1 led to complete responses and subsequent tumor rejection, indicative of immunological memory Our findings support a clinical strategy that combines MGC018 with checkpoint blockade for the treatment of B7-H3-expressing solid cancers Acknowledgements DUBA linker payload provided and conjugated by Byondis, B.V. Presenter Contact Information: [email protected]

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  • ©2020 MacroGenics, Inc. All rights reserved.

    MGC018, a Duocarmycin-based Antibody-drug Conjugate Targeting B7-H3, Exhibits Immunomodulatory Activity and Enhanced Antitumor Activity in Combination with Checkpoint Inhibitors

    Juniper A. Scribner, Michael Chiechi, Pam Li, Thomas Son, Jeff Hooley, Ying Li, Anushka De Costa, Peter Lung, Nicholas Yee-Toy, Francine Chen, Zhuangyu Pan, Bhaswati Barat, Christina Wolff, Valentina Ciccarone, James Tamura, Scott Koenig, Chet Bohac, Jon Wigginton, Paul A. Moore, Ezio Bonvini, Deryk Loo

    MacroGenics, Inc., Brisbane, CA and Rockville, MD

    Presented at the American Association for Cancer Research 2020 Virtual Annual Meeting II, June 22–24, 2020

    5203

    AbstractIntroduction: B7-H3, a member of the B7-family of immunomodulatory molecules, is overexpressed in a wide range of solid tumors. B7-H3 tumor overexpression has been correlated with disease severity and poor outcome. MGC018 is a duocarmycin-based antibody-drug conjugate (ADC) targeting B7-H3. MGC018 exhibits a favorable preclinical profile, with strong reactivity toward tumor cells and tumor-associated vasculature, limited normal tissue reactivity, and potent antitumor activity toward B7-H3-expressing tumor xenografts. With the emergence of immune-checkpoint blockade as a promising treatment for cancer, interest has grown in understanding the potential of cytotoxic agents to promote immune surveillance or stimulate immune responses to dying cancer cells, leading to immunological memory. ADCs bearing tubulin and DNA modifying cytotoxic payloads have been reported to induce immunogenic cell death (ICD), mediate antitumor immunity in immunocompetent mouse models, and synergistically combine with checkpoint inhibitors to deliver enhanced antitumor responses. Based on those results, we investigated the immunomodulatory potential of MGC018 and the prospect to combine with checkpoint blockade to enhance antitumor responses.

    Methods: Syngeneic mouse models expressing human B7-H3 were employed to investigate the antitumor activity of MGC018 in an immune competent setting. Studies were conducted to assess the role of the immune system in the MGC018-mediated antitumor responses, whether MGC018 could impart antitumor memory responses in vivo, and the potential to enhance antitumor responses by combining MGC018 with PD-1 blockade.

    Results: MGC018 demonstrated specific, dose-dependent in vivo antitumor activity toward human B7-H3-bearing tumors in immunocompetent syngeneic mouse models. Depletion of CD8+ T cells led to reduced antitumor responses, indicating that CD8+ T cells contributed to MGC018-mediated antitumor activity. Antitumor activity in these models was enhanced when MGC018 was combined with anti-PD-1. Treatment with MGC018 alone, or in combination with anti-PD-1, led to complete antitumor responses, and the majority of mice rejected subsequent tumor rechallenge.

    Conclusion: MGC018, a clinical-stage therapeutic comprised of a humanized antibody targeting B7-H3, conjugated to a duocarmycin-based DNA alkylating payload, exhibits a favorable preclinical profile. Results from these syngeneic model studies support the hypothesis that the antitumor activity of the duocarmycin-based MGC018 ADC (1) mediates immunomodulatory activity, (2) is enhanced by combination with checkpoint blockade, and (3) induces immunological memory. Our findings support a clinical strategy that combines MGC018 with checkpoint blockade for the treatment of B7-H3-expressing solid cancers.

    BackgroundMGC018 a Clinical-stage Anti-B7-H3 ADC TherapeuticB7-H3■■Member of the B7-family of immune regulators■■Overexpressed on solid cancers, with high tumor-versus-normal tissue binding differential■■Overexpression correlated with disease severity and poor outcome in multiple cancers

    MGC018■■Comprised of a humanized antibody targeting B7-H3 (MGA017)■■Conjugated to a duocarmycin-based DNA alkylating payload via native cysteines■■Potent antitumor activity in mouse models toward B7-H3-expressing human tumor xenografts at clinically relevant dose levels, despite model limitations

    – Rapid clearance (half-life of ADC ~40 hours) due to degradation by rodent-specific carboxylesterase CES1c — not present in primates or humans

    ■■Favorable safety and toxicokinetics in a repeat-dose GLP toxicology study in cynomolgus monkeys, a validated toxicology species for MGC018■■Phase I/II clinical study in advanced solid cancers in progress (NCT03729596)

    Immunomodulatory Activity of ADC’s■■Have been reported to induce immunogenic cell death (Gerber et al., Biochem Pharm 2016)■■Synergistically combine with checkpoint inhibitors to deliver enhanced antitumor responses■■Mediate antitumor immunity in immunocompetent mouse models

    Objectives■■Evaluate MGC018 to mediate immunomodulatory activity ■■Determine whether treatment with MGC018 is enhanced by combination with checkpoint blockade■■Assess whether MGC018 induces immunological memory

    Introduction

    Targeting B7-H3 with MGC018: Potential for Immunomodulation

    Tumor Cells

    MGC018

    CD4T Cells

    CD8T Cells

    TumorVasculature

    Immature DC

    Direct Killing of Tumor Cells

    Lymph Node

    CD8 T Cells

    Mature DC

    Granzyme B and Perforin

    Tumor AntigensDAMPs: CRT, HMGB1, ATP

    TCR MHCII

    TCR MHCI

    DCAdapted from Gerber et al., Biochem Pharm 2016

    Tumor Antigens

    DAMPs: Damage-associated molecular patterns.

    Duocarmycin-based Linker Payload

    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)

    vc-seco-DUocarmycin-hydroxyBenzamide Azaindole (DUBA) Structure

    MGA017

    MGC018

    DuocarmycinPayload

    huIgG1(wild type Fc)

    Anti-B7-H3

    Drug-antibody ratio 2.68

    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■■Trastuzumab-vc-seco-DUBA (SYD985) in Phase 3 clinical trial in locally advanced or metastatic breast cancer (Byondis, B.V.)

    DUBA Linker Payload provided and conjugated by Byondis, B.V.

    B7-H3 is Highly Expressed on a Range of Solid Cancers

    SCCH

    N

    Panc

    reat

    ic Ca

    Bladd

    er Ca

    Ovar

    ian Ca

    Anal

    Ca

    Meso

    theli

    oma

    Kidne

    y Ca

    Melan

    oma

    Pros

    tate

    Ca

    Color

    ecta

    l Ca

    Sarco

    ma

    Lung

    Ca

    Brea

    st Ca

    Gastr

    ic Ca

    Gliob

    lasto

    ma

    0

    10

    20

    30

    40

    50

    60

    70

    80

    90

    100

    Perc

    ent

    of T

    umor

    s w

    ith

    Indi

    cate

    d H

    -Sco

    re 1-100101-200

    201-300

    H-Score

    16% 14%

    84%

    22%

    32% 38%

    53%

    16%

    42%

    15%

    34%

    15%

    26%

    6%

    26%

    39%

    30%43%

    14%

    48%

    28%

    24%

    40%

    24%

    66%

    28%

    58% 16%18%

    50%

    22%

    18%

    37%

    46%51%

    38%

    25%

    44%

    15%

    33%

    53%

    84%

    16%

    ■■B7-H3 is expressed on tumor epithelium and tumor-associated vasculatureFFPE staining with goat polyclonal antibody (R&D Systems). Independent specimens ranged from 19-189 for each indication.

    MGC018 Antitumor Activity Toward Patient-derived Xenograft (PDX) Models

    H-score = 180

    H-score = 130

    H-score = 180

    H-score = 240

    0

    500

    1000

    1500

    2000

    2500 Vehicle

    Study Day

    Mea

    n Tu

    mor

    Vol

    ume

    (mm

    3 )

    MGC018 3 mg/kg x 2

    Control ADC 3 mg/kg x 2

    0 10 20 300

    500

    1000

    1500

    2000

    2500 Vehicle

    Study Day

    Mea

    n Tu

    mor

    Vol

    ume

    (mm

    3 )

    MGC018 3 mg/kg x 2

    Control ADC 3 mg/kg x 2

    0

    500

    1000

    1500 Vehicle

    Study Day

    Tum

    or V

    olum

    e (m

    m3 )

    Mea

    n Tu

    mor

    Vol

    ume

    (mm

    3 )

    MGC018 3 mg/kg x 3

    Control ADC 3 mg/kg x 3

    Vehicle

    MGC018 3 mg/kg x 2

    Control ADC 3 mg/kg x 2

    Breast Cancer PDXCTG-0869

    Head and Neck Cancer PDXCTG-0790

    Pancreatic Cancer PDXCTG-0305

    Prostate Cancer PDXCTG-2441

    Dosing

    Dosing

    Dosing

    Dosing

    0

    500

    1000

    1500

    2000

    Study Day

    0 10 20 30

    0 10 20 30 -10 0 10 20 30

    ■■MGC018 displays antitumor activity toward heterogeneous B7-H3 expressing PDX models representing a range of solid cancers

    Results

    Syngeneic Mouse Model SystemStably transfected human

    B7-H3 into mouse colorectalcancer cell lines(MC38 and CT26)

    Tumor cell linesgrown in vitro

    Human B7-H3expressing tumor cell

    lines grown in vitro

    Tumor originatingin an inbred strain

    Image adapted from TaconicTumor cells implanted in

    mice of same inbred strain

    ■■Tumor cells are originally derived from the same genetic background as the implanted mouse■■Mice have an intact and fully functioning immune system■■Syngeneic models provide an effective approach for studying how cancer therapies perform in the presence of a functional immune system■■While transduced human B7-H3 may be immunogenic in the mouse, the model provides proof-of-concept for the ADC’s ability to enhance adaptive responses

    Murine Colorectal Cancer Cell Lines Engineered to Overexpress Human B7-H3

    Relative Expression of Human B7-H3 on the Cell Surface

    % o

    f Max

    100

    80

    60

    40

    20

    0100 101 102 103 104

    PEAnti-B7-H3 mAb Negative control Unstained control

    % o

    f Max

    100

    80

    60

    40

    20

    0100 101 102 103 104

    PE

    CT26/huB7-H3MC38/huB7-H3

    QFACSMC38/huB7-H3

    Murine Colorectal CancerCT26/huB7-H3

    Murine Colorectal CancerAntibody Binding Capacity 4.08E+04 5.37E+04

    Syngeneic Mouse Model C57BL/6 BALB/c

    Antibody Binding Capacity determined by Bangs QFACS Kit.

    MGC018 Mediates Apoptosis of Murine Tumor CellsCaspase 3/7 Activation of MC38/huB7-H3 Tumor Cells

    2 12 24 36 48 600

    20000

    40000

    60000

    Time (hours)

    Apo

    ptot

    ic C

    ell C

    ount

    /Wel

    l

    MGC018 (3.3 nM)MGA017 (3.3 nM)Untreated

    2 hour 48 hour 60 hour

    Untreated

    MGA017(3.3 nM)

    MGC018 (3.3 nM)

    MGC018 Modulates Damage-associated Molecular PatternsCalreticulin Expression on the Surface of MC38/huB7-H3 Tumor Cells

    Isotype control Calreticulin-AF647

    101 102 103 104

    101 102 103 104

    101 102 103 104

    101 102 103 104

    101 102 103 104

    101 102 103 104

    Untreated

    SYD978

    Calreticulin (+)

    100

    100

    100

    101 102 103 104 100 101 102 103 104 100 101 102 103 104

    MGC018

    Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647

    Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647

    Calreticulin-AF647 Calreticulin-AF647 Calreticulin-AF647

    100

    80

    60

    40

    20

    0

    100

    80

    60

    40

    20

    0

    100

    80

    60

    40

    20

    0

    104

    100

    80

    60

    40

    20

    0

    100

    80

    60

    40

    20

    0

    100

    80

    60

    40

    20

    0

    104

    100

    80

    60

    40

    20

    0

    100

    80

    60

    40

    20

    0

    100

    80

    60

    40

    20

    0

    24 Hour 72 Hour48 Hour

    Coun

    ts

    Coun

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    ■■MGC018 and SYD978 (seco-DUBA Payload) induced calreticulin translocation to the surface of MC38/huB7-H3 murine tumor cells■■Analysis of other DAMPs are currently underway

    Antitumor Activity of MGC018 is Dependent on Human B7-H3 Expression

    MC38/huB7-H3

    MC38Parental

    CT26/huB7-H3

    CT26Parental

    0 10 20 30 40 50

    Vehicle

    MGC018 10 mg/kg

    Vehicle

    MGC018 10 mg/kg

    Vehicle

    MGC018 10 mg/kg

    Vehicle

    MGC018 10 mg/kg

    Day (post inoculation)

    0 10 20 30 40 50Day (post inoculation)

    Day (post inoculation)

    Mea

    n Tu

    mor

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    ume

    (mm

    3 )M

    ean

    Tum

    or V

    olum

    e (m

    m3 )

    0 10 20 30 40 50

    Day (post inoculation)0 10 20 30 40 50

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    0

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    ■■Parental xenografts lacking human B7-H3 expression are insensitive to MGC018

    Antitumor Activity is Dependent on Targeted Delivery of Payload by MGC018

    MC38/huB7-H3 CT26/huB7-H3

    0 10 20 30 400

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    0 10 20 30 40 50 600

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    MGA017 10 mg/kg + SYD978 0.0948 mg/kg

    Day (post inoculation)

    Mea

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    (mm

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    MGA017 10 mg/kg

    MGC018 10 mg/kg

    SYD978 0.0948 mg/kg

    Dosing Dosing

    ■■No antitumor activity with MGA017, SYD978 (seco-DUBA payload) or combination of MGA017 + SYD978■■Antitumor activity of SYD978 requires conjugation to B7-H3-targeting mAb

    MGC018 Increases Infiltration of Lymphocytes into MC38/huB7-H3 Tumor Xenografts

    Untreated MGC018 10 mg/kg

    Untreated MGC018 10 mg/kg0

    10

    20

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    Mean Positive Expression (%)

    Treatment

    Mea

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    siti

    ve C

    ell C

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    tal V

    iabl

    e A

    rea

    (mm

    2 ) CD4

    CD8

    Granzyme B

    H&E Stain B7-H3 Expression

    CD4 DAPI

    CD8 DAPI

    Granzyme B DAPI

    ■■MGC018 treatment increases CD4 and CD8 infiltration and Granzyme B expression

    CD8+ T Cells Contribute to MGC018-mediated Antitumor Response

    MGC018 dosing

    Anti-CD8 dosing

    Vehicle

    MGC018 10 mg/kg +Anti-CD8 8 mg/kg

    MGC018 10 mg/kg

    Anti-CD8 8 mg/kg

    0 20 40 6010 30 500

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    MGC018 dosing

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    (mm

    3 )

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    0 10 20 30 40 500

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    Day (post inoculation)

    ■■Anti-CD8 depletes CD8+ T cells and attenuates MGC018 antitumor activityAnti-CD8 antibody from BioXCell: Clone 53-6.7.

    Antitumor Activity of MGC018 in Combination with Anti-PD-1 in CT26 Model: Induction of Immunological Memory

    0 10 20 30 40 50 600

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    MGC018 dosing

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    Anti-PD-1 dosing

    Vehicle

    MGC018 10 mg/kg +Anti-PD-1 20 mg/kg

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    (mm

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    MGC018 10 mg/kg

    Anti-PD-1 20 mg/kg

    CT26/huB7-H3

    CR animals selected for tumor rechallenge

    Survival of AnimalsChallenged with CT26 Parent

    Days (post rechallenge)

    Perc

    ent

    Surv

    ival

    0 20 40 60 80 100 1200

    25

    50

    75

    100 Naive Animals

    MGC018 + Anti-PD-1CR Animals

    MGC018 CR Animals

    ■■Antitumor activity of MGC018 is enhanced by anti-PD-1■■MGC018 and MGC018 + anti-PD-1 induces immunological memory

    CR defined as tumors < 24 mm3. Anti-PD-1 antibody from BioXCell: Clone RMP1-14.

    Antitumor Activity of MGC018 in Combination with Anti-PD-1 in MC38 Model: Induction of Immunological Memory

    MGC018 dosing

    Rechallenge

    Anti-PD-1 dosing

    0 10 20 30 40 500

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    MGC018 10 mg/kg +Anti-PD-1 20 mg/kg

    Day (post inoculation)

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    n Tu

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    (mm

    3 )

    MGC018 10 mg/kg

    Anti-PD-1 20 mg/kg

    Survival of AnimalsChallenged with MC38/huB7-H3

    Days (post rechallenge)

    Perc

    ent

    Surv

    ival

    MC38/huB7-H3A.

    0

    25

    50

    75

    100

    MGC018 + Anti-PD-1CR Animals

    MGC018 CR Animals

    Naive Animals

    CR animals selected for tumor rechallenge

    0 10 20 30 40 50 60

    Rechallenge

    Dosing

    MC38/huB7-H3

    Survival of AnimalsChallenged with MC38/huB7-H3

    Survival of AnimalsChallenged with MC38 Parent

    CR animals selected fortumor rechallenge

    0 10 20 30 40 50 60 70 80 900

    500

    1000

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    3 )

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    Perc

    ent

    Surv

    ival

    Naive animalsMGC018 CR animals

    0 10 20 30 40 500

    25

    50

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    100

    Days (post rechallenge)

    Perc

    ent

    Surv

    ival

    Naive animalsMGC018 CR animals

    B.N=15

    N=15

    MGC018 10 mg/kg

    ■■Antitumor activity of MGC018 is enhanced by anti-PD-1■■MGC018 and MGC018 + anti-PD-1 induces immunological memory

    CR defined as tumors < 16 mm3. Anti-PD-1 antibody from BioXCell: RMP1-14.

    Conclusions■■MGC018 demonstrated in vivo antitumor activity toward heterogeneous B7-H3-expressing PDX models

    – TNBC, Pancreatic, Prostate, Head and Neck Squamous Cell Carcinoma■■MGC018 demonstrated specific, dose-dependent in vivo antitumor activity toward human B7-H3-expressing tumors in immunocompetent syngeneic mouse models

    – MGC018 induced translocation of calreticulin, a component of damage-associated molecular patterns (DAMPs) – MGC018 treatment increased infiltration of CD4+ and CD8+ lymphocytes into the tumor

    ■■Depletion of CD8+ T cells led to reduced antitumor responses, indicating that CD8+ T cells contributed to MGC018-mediated antitumor activity■■Antitumor activity was enhanced when MGC018 was combined with anti-PD-1 leading to more complete antitumor responses■■MGC018 alone or in combination with anti-PD-1 led to complete responses and subsequent tumor rejection, indicative of immunological memory

    Our findings support a clinical strategy that combines MGC018 with checkpoint blockade for the treatment of B7-H3-expressing solid cancers

    AcknowledgementsDUBA linker payload provided and conjugated by Byondis, B.V.

    Presenter Contact Information: [email protected]


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