Search

ADAM metallopeptidase domain 9 (ADAM9) is a member of the ADAM family of multifunctional, multidomain type 1 transmembrane proteins. ADAM9 is overexpressed in many cancers, including non–small cell lung, pancreatic, gastric, breast, ovarian, and colorectal cancer, but exhibits limited expression in normal tissues. A target-unbiased discovery platform based on intact tumor and progenitor cell immunizations, followed by an IHC screen, led to the identification of anti-ADAM9 antibodies with selective tumor-versus-normal tissue binding. Subsequent analysis revealed anti-ADAM9 antibodies were efficiently internalized and processed by tumor cells making ADAM9 an attractive target for antibody–drug conjugate (ADC) development. Here, we describe the preclinical evaluation of IMGC936, a novel ADC targeted against ADAM9. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21-C, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker, at a drug antibody ratio of approximately 2.0. In addition, the YTE mutation (M252Y/S254T/T256E) was introduced into the CH2 domain of the antibody Fc to maximize in vivo plasma half-life and exposure. IMGC936 exhibited cytotoxicity toward ADAM9-positive human tumor cell lines, as well as bystander killing, potent antitumor activity in human cell line-derived xenograft and patient-derived xenograft tumor models, and an acceptable safety profile in cynomolgus monkeys with favorable pharmacokinetic properties. Our preclinical data provide a strong scientific rationale for the further development of IMGC936 as a therapeutic candidate for the treatment of ADAM9-positive cancers. A first-in-human study of IMGC936 in patients with advanced solid tumors has been initiated (NCT04622774).

5000 Hs700T parental cells only in the presence of 6.7 nM MGC018

5000 Hs700T B7-H3 KO/RFP cells alone in the presence of 6.7 nM MGC018

Supplementary Data

Co-culture of 5000 Hs700T parental cells and 5000 Hs700T B7-H3 KO/RFP cells in the presence of 6.7 nM MGC018

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non–small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody–drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3–positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3–positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers.

Supplementary Data from Preclinical Evaluation of IMGC936, a Next-Generation Maytansinoid-based Antibody–drug Conjugate Targeting ADAM9-expressing Tumors

Supplementary Materials and Methods

Introduction: Prostate cancer is the second most common cancer among men worldwide. In 2021, it is estimated that 248,530 men in the United States will be diagnosed with prostate cancer, and 34,130 will die from the disease. Although current treatments have success initially, development of resistance commonly leads to recurrence of an incurable castrate-resistant form of the disease. Thus, significant need for novel therapies to improve the outcome of castrate-resistant prostate cancer remains. B7-H3 (CD276), a member of the B7 family of immunomodulatory molecules, is overexpressed in primary and metastatic prostate cancer, and correlates with disease severity and poor clinical outcome. MGC018, a duocarmycin-based B7-H3 antibody-drug conjugate, is currently being evaluated in clinical studies. Here, MGC018 was explored preclinically to assess the potential for targeting B7-H3 in prostate cancer. Methods: Immunohistochemistry studies were performed to define the expression of B7-H3 in prostate cancer tissue microarrays (TMA). In vivo efficacy studies were conducted with human prostate cancer cell line-derived xenograft (CDX) models to explore the antitumor activity of MGC018 as a single agent and in combination with Poly (ADP-ribose) polymerase (PARP) and androgen receptor (AR) inhibitors. Based on the results in the CDX studies, in vivo efficacy studies were extended to a panel of metastatic prostate cancer patient-derived xenograft (PDX) models, which exhibit heterogenous expression of B7-H3 and more closely mimic the biological characteristics of patient tumors. Results: Staining of prostate tumor TMAs revealed high expression of B7-H3 in primary and metastatic prostate cancer. Of the prostate samples evaluated, 95% (38/40) of the tumor samples were positive for B7-H3 (H-score ≥ 20): 65% had H-scores greater than 200, while 20% and 10% had H-scores between 101-200 and 1-100, respectively. MGC018 demonstrated in vitro cytotoxicity toward B7-H3-positive human prostate cancer cell lines. The in vitro cytotoxicity translated to potent antitumor activity in vivo toward prostate cancer CDX models, and the antitumor activity of MGC018 was enhanced when combined with inhibitors of PARP or AR. In PDX models of metastatic prostate cancer, MGC018 was active as a single agent toward heterogeneous B7-H3-expressing tumors, and combining MGC018 with inhibitors of PARP or AR led to a greater response in some models. Conclusion: B7-H3 is frequently overexpressed in prostate cancer. MGC018 demonstrated potent antitumor activity in vivo toward CDX and PDX models of prostate cancer, and enhanced antitumor activity when combined with inhibitors of PARP or AR. These results support prostate cancer as an indication that may be responsive to ADC-based treatments directed toward B7-H3. MGC018 is being investigated in metastatic prostate cancer in a Phase 1/2 clinical study. Citation Format: Juniper A. Scribner, Francine Z. Chen, Anushka De Costa, Ying Li, Michael Chiechi, Thomas Son, Jeff Hooley, Jonathan Li, Scott Koenig, Chet Bohac, Ezio Bonvini, Paul A. Moore, Deryk Loo. Targeting B7-H3 in prostate cancer: Preclinical proof of concept with MGC018, an investigational anti-B7-H3 antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 330.

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non–small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody–drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3–positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3–positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers.

Introduction: Squamous cell carcinoma of the head and neck (SCCHN) is the seventh most common cancer worldwide, accounting for ~ 4% of all cancers in the United States. Although PD-1-directed therapy has efficacy in SCCHN, 85-95% of patients progress following initial response. B7-H3 is a member of the B7 family of immunomodulatory molecules, is overexpressed in SCCHN, and correlates with disease severity and poor clinical outcome. Furthermore, consistent with its putative coinhibitory function, B7-H3 expression in SCCHN is inversely correlated with the number of tumor infiltrating CD8+ T-cells. We are developing therapeutics targeting B7-H3, including enoblituzumab, an Fc-engineered anti-B7-H3 monoclonal antibody, and MGC018, a duocarmycin-based B7-H3 ADC, both of which are currently being evaluated in clinical studies. Here the potential of MGC018 was explored in preclinical models as a proof of concept for targeting B7-H3 in SCCHN. Methods: Immunohistochemistry studies were performed to define the expression of B7-H3 in SCCHN tissue microarrays (TMA). Single and repeat-dose in vivo efficacy studies were conducted in CD-1 nude mice with cell line-derived SCCHN human tumor xenografts to explore the relationship between Cmax, exposure and antitumor activity, and to define the minimal efficacious dose in these models. Based on results in these cell-derived xenograft (CDX) studies, in vivo efficacy studies were extended to a panel of SCCHN patient-derived xenograft (PDX) models, which more closely mimic the biological characteristics of the patient tumor and exhibit heterogenous expression of B7-H3. Results: Analysis of B7-H3 expression on a SCCHN TMA confirmed and extended previously reported expression of B7-H3 in SCCHN. Of the SCCHN samples evaluated, 90% (36/40) of the tumor samples were positive for B7-H3: 35% (14/40) had H-scores greater than 200, with the remaining 22 samples equally distributed between the H-score range of 101-200 and 1-100 (~ 28% each). MGC018 demonstrated specific, dose-dependent in vitro cytotoxicity toward SCCHN human tumor cell lines. The in vitro cytotoxicity translated to potent antitumor activity in vivo against SCCHN CDX models, with a single administration of 3 mg/kg resulting in complete responses in 7/7 mice in the FaDu model. In the PDX setting (H-scores 120-283), repeat dose administration every week or two weeks with MGC018 at 3 mg/kg/dose, led to regressions and/or stable disease in 10/18 models, and a delay in tumor growth in 5 additional models. Conclusion: B7-H3 is frequently overexpressed in SCCHN. At clinically relevant dose levels, MGC018 demonstrated potent antitumor activity in vivo toward SCCHN CDX mouse models and the majority of SCCHN PDX mouse models examined. These results support SCCHN as a potential indication that may be responsive to ADC-based treatments directed toward B7-H3. Citation Format: Juniper A. Scribner, Francine Z. Chen, Ying Li, Michael Chiechi, Thomas Son, Jeff Hooley, Scott Koenig, Paul A. Moore, Ezio Bonvini, Chet Bohac, Deryk Loo. Targeting B7-H3 in squamous cell carcinoma of the head and neck: Preclinical proof-of-concept with the investigational anti-B7-H3 antibody-drug conjugate, MGC018 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 950.

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 immunological 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. Citation Format: Juniper A. Scribner, Michael Chiechi, Pam Li, Thomas Son, Jeff Hooley, Ying Li, Anushka De Costa, Peter Lung, Nicholas Yee-Toy, Francine Chen, Bhaswati Barat, Christina Wolff, Valentina Ciccarone, James Tamura, Scott Koenig, Chet Bohac, Jon Wigginton, Paul A. Moore, Ezio Bonvini, Deryk Loo. MGC018, a duocarmycin-based antibody-drug conjugate targeting B7-H3, exhibits immunomodulatory activity and enhanced antitumor activity in combination with checkpoint inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5203.

Dysregulation of ADAM9, a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression correlates with poor prognosis in multiple cancers. We have shown that ADAM9 is overexpressed in multiple solid tumor indications and that anti-ADAM9 antibodies are efficiently internalized and degraded by tumor cell lines making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe IMGC936, the first ADAM9-targeting ADC to enter preclinical development. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable peptide linker at a drug-antibody ratio of two. To maximize the potential for IMGC936 activity, the M252Y/S254T/T256E (YTE) mutation was introduced into the CH2 domain of the antibody to increase in vivo plasma half-life and exposure. In vitro studies demonstrated targeted cytotoxicity of IMGC936 across a panel of ADAM9-positve tumor cell lines with activity at least 2 logs greater than a non-targeting conjugate. Consistent with the in vitroactivity, an anti-ADAM9-DM21 conjugate displayed compelling anti-tumor activity in multiple xenograft models representing non-small cell lung, gastric and colorectal cancers. For example, in the EBC-1 non-small cell lung cancer subcutaneous xenograft model with only moderate ADAM9 expression (H-score of 130), anti-ADAM9-DM21 not only induced tumor growth delay but produced complete and durable remissions in 6/6 mice following a single intravenous dose of 8.6 mg Ab/kg (100 ug DM21/kg). IMGC936 demonstrated a favorable pharmacokinetic profile with good conjugate stability in non-human primates. Importantly, IMGC936 was well-tolerated following repeat dosing in cynomolgus monkeys with no ADAM9 target-related toxicities identified at doses exceeding the levels required for anti-tumor activity in murine xenograft models. Based on the totality of the preclinical data, IMGC936 represents a promising therapeutic candidate to target a wide range of ADAM9-expressing tumors. Citation Format: Stuart Hicks, Deryk Loo, Kerstin Sinkevicius, Juniper Scribner, Bhaswati Barat, Nicholas Yoder, Christopher Espelin, Marian Themeles, Francine Chen, Jacquelynn Lucas, Jennifer Brown, Bahar Matin, Megan Fuller, Jenny Lee, Paulin Salomon, Juliet Costoplus, Sadiqa Yancey, Gundo Diedrich, Sergey Gorlatov, Thomas Son, Michael Chiechi, Pam Li, Michael Spliedt, Valentina Ciccarone, Jeff Hooley, Nadia Gantt, James Tamura, Kerry Donahue, Paul Moore, Syd Johnson, Thomas Chittenden, Richard Gregory, Ezio Bonvini. IMGC936, a first-in-class ADAM9-targeting antibody-drug conjugate, demonstrates promising anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1533.

ADAM9, also known as MDC9 or meltrin-γ, is a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, which have been implicated in cytokine and growth factor shedding, and cell migration. Dysregulation of ADAM9 has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression has been shown to correlate with poor prognosis in prostate, renal, and pancreatic cancers. Using an immunization approach in which antibodies were raised to fetal progenitor and stem-like cancer cell lines followed by screening on tumor and normal tissues, we identified ADAM9 as a promising cell surface tumor target. FFPE-IHC expression analysis revealed that ADAM9 is overexpressed in multiple solid tumor indications relative to corresponding normal tissues. The overexpression of ADAM9 in tumors coupled with its restricted expression in normal tissues make ADAM9 an attractive target for antibody-drug conjugate (ADC) therapy. Here, we describe two ADCs both of which are based on a high affinity anti-ADAM9 antibody to selectively target ADAM9-expressing tumors. The first ADC utilizes the maytansine-derived microtubule disruptor, DM4, linked via a hindered disulfide hydrophilic linker (sulfo-SPDB). The second ADC exploits an ultra-potent DNA alkylating payload, DGN549, which is conjugated to two engineered cysteines via a peptide linker. Both conjugates bound with similar subnanomolar affinity to ADAM9-expressing cells. In vitro cytotoxicity studies showed that anti-ADAM9 ADCs can kill a broad panel of ADAM9-positve tumor cell lines, including lung, pancreatic, renal, prostate, and colon tumor cell lines. In particular, the anti-ADAM9-DGN549 conjugate was extremely potent with IC50 values ranging from 0.1 to 65 pM and was at least 2 logs more active than a non-targeting conjugate. Surprisingly, efficient in vitro cytotoxicity was observed at ADAM9 expression levels as low as a few thousand cell surface receptors per cell. Consistent with their in vitro activity, both anti-ADAM9 ADCs displayed compelling anti-tumor activity in xenograft models. In a CaLu3 non-small cell lung cancer xenograft model, anti-ADAM9-DM4 induced tumor growth delay at a single 1.25 mg Ab/kg dose. In the same model, a single intravenous dose of 0.25 mg Ab/kg of the anti-ADAM9-DGN549 produced durable complete remissions in 8/8 mice. A non-targeting DGN549 ADC was inactive even when dosed at 10 times that of the anti-ADAM9 ADC, demonstrating that targeted delivery of DGN549 through ADAM9 binding is required for activity. These data demonstrate that anti-ADAM9 ADCs exhibit antitumor activity against a broad panel of ADAM9-positive malignancies and cause durable remissions in preclinical models at doses expected to be clinically achievable. Anti-ADAM9 ADCs represent a promising therapeutic strategy to target a wide range of ADAM9-expressing tumors. Citation Format: Stuart W. Hicks, Nicholas C. Yoder, Deryk Loo, Asli Muvaffak, Yinghui Zhou, Megan E. Fuller, Molly A. McShea, Marian Themeles, Katherine H. Mucciarone, Juniper A. Scribner, Bhaswati Barat, Thomas Sun, James Tamura, Francine Z. Chen, Kerry A. Donahue, Tom Chittenden. Novel antibody-drug conjugates targeting ADAM9-expressing solid tumors demonstrate potent preclinical activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 37. doi:10.1158/1538-7445.AM2017-37

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 antibody-drug conjugates (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 Biopharmecuticals 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. Citation Format: Thomas Son, Juniper A. Scribner, Jeff Hooley, Michael Chiechi, Pam Li, Timothy E. Hotaling, Anushka De Costa, Yan Chen, Francine Chen, Bhaswati Barat, Valentina Ciccarone, Timur Gaynutdinov, James Tamura, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini, Deryk Loo. Preclinical development of a duocarmycin-based antibody-drug conjugate targeting B7-H3 for solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 42. doi:10.1158/1538-7445.AM2017-42

Introduction: A target-unbiased approach based on intact cell immunizations with fetal progenitor cells and cancer stem cells, followed by an immunohistochemistry (IHC) screen for cancer-specific candidates, led to the identification of anti-ADAM9 (a disintegrin and metalloproteinase) mAbs with highly differential tumor-versus-normal tissue binding. ADAM9 is a cell surface protein over-expressed in multiple tumors, with a possible role in promotion and progression of cancer through multiple mechanisms, including modulation of adhesion and migration as well as processing of tumorigenic and pro-angiogenic factors. In this preclinical study, we performed target/mAb validation and evaluated the therapeutic potential of anti-ADAM9 antibody-drug conjugates (ADCs) toward ADAM9-expressing solid cancers. Methods: IHC was performed with anti-ADAM9 mAbs to confirm and extend available data of human normal and tumor tissue expression. Epitope mapping studies were conducted to define epitope-specificity. mAbs were also screened to identify those that efficiently internalized into tumor cells. In vitro cellular processing studies were performed to further evaluate the mAbs as ADC candidates. Selected mAbs were converted to ADCs via chemical conjugation to potent anti-microtubule (DM4) or DNA alkylating (DGN549) agents; in vitro cytotoxicity studies were conducted with tumor cell lines representing human cancer types that overexpress ADAM9. A lead mAb was then selected for humanization and affinity maturation to yield a development candidate. Results: Anti-ADAM9 mAbs exhibited strong reactivity toward the tumor epithelium of solid cancers, including pancreatic, kidney, prostate, bladder, breast, colon, lung, and ovarian cancer, but limited reactivity toward normal tissues. Anti-ADAM9 mAbs were efficiently internalized and processed by tumor cell lines, including lines with only modest ADAM9 expression. Anti-ADAM9 ADCs exhibited specific, dose-dependent cytotoxicity toward ADAM9-positive cancer cell lines in vitro, with IC50 values in the sub-nanomolar range. Humanization and affinity maturation of the lead mAb yielded a development candidate that retains potent antitumor activity toward ADAM9-positive tumor cell lines and equivalent, high affinity binding to both human and cynomolgus monkey ADAM9. Conclusion: ADAM9 is a cell surface antigen that is over-expressed on a wide range of solid cancers. Anti-ADAM9 mAbs that were strongly reactive with representative tumors exhibited high affinity for the antigen and were efficiently internalized and processed by ADAM9-bearing tumor cells. Anti-ADAM9 ADCs demonstrated dose-dependent cytotoxicity in vitro toward a panel of ADAM9-positive tumor cell lines. Our findings demonstrate that an ADC targeting ADAM9 may serve as a potential therapeutic for ADAM9-expressing solid tumors. Citation Format: Juniper A. Scribner, Bhaswati Barat, Stuart W. Hicks, Nicholas C. Yoder, Thomas Son, Lusiana Widjaja, Gundo Diedrich, Sergey Gorlatov, Jeff Hooley, Ann Easton, Peter Lung, Anushka De Costa, Francine Chen, Michael Chiechi, Pam Li, Monica Licea, Timothy E. Hotaling, Michael Spliedt, Valentina Ciccarone, Nadia Gantt, James Tamura, Megan E. Fuller, Molly McShea, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini, Deryk Loo. Target validation, antibody discovery and preclinical data supporting ADAM9 as an antibody-drug conjugate therapeutic target for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 38. doi:10.1158/1538-7445.AM2017-38

Introduction: Monoclonal antibodies (mAbs) were generated via a target-unbiased approach based on intact cell immunization with cell lines, fetal progenitor cells, and cancer stem cells. An immunohistochemical screen for cancer-specific candidates identified a panel of anti-B7-H3 (CD276) mAbs with highly differential tumor-versus-normal tissue binding. B7-H3 expression was observed in tumor epithelium as well as tumor-associated vasculature and stroma. Consistent with our findings, B7-H3 has been reported to be overexpressed in a growing number of solid cancers, including breast, lung, pancreatic, prostate, kidney, and colon cancer, as well as melanoma and glioblastoma. Furthermore, overexpression of B7-H3 has been correlated with disease severity and poor outcome in a number of these cancer types. A humanized version of an anti-B7-H3 mAb engineered with an enhanced Fc domain (enoblituzumab or MGA271) and a humanized Dual-Affinity Re-Targeting (DART®) protein that recognizes both B7-H3 and CD3 and redirects T cells to kill B7-H3-expressing cells (MGD009) are being investigated in Phase 1 clinical studies. In this nonclinical study, we evaluated the therapeutic potential of anti-B7-H3 antibody-drug conjugates (ADCs) toward B7-H3-expressing solid cancers. Methods: A panel of anti-B7-H3 mAbs was screened for internalization and a subset of mAbs that were efficiently internalized by tumor cells was identified. These mAbs were converted to ADCs via chemical conjugation; in vitro and in vivo activity studies were then conducted with a range of tumor cell lines representing human cancer types that overexpress B7-H3. Results: The anti-B7-H3 ADCs exhibited specific, dose-dependent cytotoxicity toward B7-H3-positive tumor cell lines in vitro, including breast, lung, ovarian, pancreatic, and prostate cancer lines, with IC50 values generally in the sub-nM range. Cytotoxicity was not observed with cell lines lacking B7-H3 expression. The anti-B7-H3 ADCs exhibited potent antitumor activity in vivo, resulting in tumor stasis and tumor regression in mice bearing B7-H3-positive human breast, lung, and ovarian tumor xenografts. Conclusion: Anti-B7-H3 ADCs exhibited dose-dependent cytotoxicity in vitro and potent antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing cancer types that overexpress B7-H3. Our findings demonstrate that ADCs targeting B7-H3 may serve as potential therapeutics for B7-H3-expressing solid cancers. Citation Format: Deryk Loo, Juniper A. Scribner, Thomas Son, Jeff Hooley, Timothy Hotaling, Michael Chiechi, Pam Li, Anushka De Costa, Yan Chen, Ann Easton, Francine Z. Chen, Bhaswati Barat, Valentina Ciccarone, James Tamura, Mark Kubik, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini. Anti-B7-H3 antibody-drug conjugates as potential therapeutics for solid cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1201.