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

Abstract 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).

Abstract Background: A disintegrin and metalloprotease (ADAM) 9 is a member of the ADAM family of transmembrane proteins. ADAM9 overexpression correlates with tumor progression, metastasis and poor prognosis in multiple cancers. IMGC936, an ADAM9-targeting antibody drug conjugate (ADC), is comprised of a high-affinity humanized monoclonal antibody site-specifically coupled to DM21 at a drug-antibody ratio of 2.0. DM21 is a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker. IMGC936 is in a phase 1 dose escalation study evaluating safety and pharmacokinetics in cancer patients. The goals of this study were: 1) to explore ADAM9 expression in various solid tumors, and 2) to evaluate the activity of IMGC936 in clinically relevant patient-derived xenograft (PDX) models with ADAM9 expression similar to that observed in human tumors. Methods: ADAM9 expression was evaluated on primary patient and PDX formalin-fixed, paraffin-embedded samples with the anti-ADAM9 antibody D64B5 (Cell Signaling Technology). Expression of ADAM9 was quantitated by H-score to capture the frequency and intensity of staining. Mice bearing PDX tumors were dosed once at 8.6 mg/kg of IMGC936, which delivered 100 µg/kg of DM payload. Anti-tumor activity was defined by NCI standards: median tumor volume of treated mice over control mice (%) > 42% (inactive), ≤ 42% (active), and <10% (highly active). Results and Conclusions: ADAM9 was highly expressed in multiple tumor types. A majority of the tumor samples had medium to high levels of ADAM9 with 62% of non-small cell lung carcinoma (NSCLC), 65% of triple negative breast cancer (TNBC), 73% of gastric cancer, and 85% of pancreatic cancer samples having H- scores of 101 to 300. The remaining tumor samples had lower levels of ADAM9 expression (H-score 1 to 100) with only 1.2% of NSCLC samples being ADAM9-negative. Activity of IMGC936 was analyzed in PDX models derived from NSCLC, TNBC, pancreatic and gastric cancers. The range of ADAM9 expression was 27 to 226 by H-score, with 80% of the samples having H-scores above 101. A single dose of IMGC936 at 8.6 mg/kg was well tolerated. Across the tumor types tested, IMGC936 was active or highly active in 24 out of 35 models (69%) with complete regressions in 6 models (4 NSCLC, 2 TNBC). The 24 IMGC936 sensitive models had H-scores between 65 and 224. The 11 non-sensitive models had H-scores between 27 and 226. The data suggest that factors independent of ADAM9 expression contribute to model sensitivity toward IMGC936 and warrant further biomarker exploration. These studies demonstrate that ADAM9 is highly expressed in a large number of solid tumor indications and show that IMGC936 has activity against multiple solid tumor types with a wide range of clinically relevant ADAM9 levels. These data support the clinical evaluation of IMGC936 (NCT04622774). Citation Format: Olga Ab, Juniper A. Scribner, Kerstin Sinkevicius, Deryk Loo, Stuart W. Hicks, Krystal Watkins, Francine Z. Chen, Christopher Espelin, Marian Themeles, Ying Li, Chet Bohac, Patrick Zweidler McKay, Paul A. Moore, Callum M. Sloss, Ezio Bonvini, Eric H. Westin. IMGC936, an investigational ADAM9-targeting antibody drug conjugate, is active against patient-derived ADAM9-expressing xenograft models [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 1841.

With cancer among the leading causes of death worldwide, the search for better, personalized treatments is imperative. Novel techniques such as next generation sequencing have identified many assayable genetic biomarkers associated with cancer in patient samples. The tyrosine kinase receptor cMet is one such biomarker that is upregulated in various solid tumors and associated with poor prognosis, disease progression and metastasis. While most patients with elevated cMet show increased levels through protein upregulation, a small population harbors gene amplification. These patients face worse outcomes which could be improved with therapies specifically targeting MET-amplification. Antibody-drug conjugates (ADCs) are a modality designed to selectively deliver highly potent cytotoxic agents to tumors. cMet is an attractive target for ADCs which may address the unmet treatment need for patients with tumors harboring MET amplification. Since dimerization of cMet receptors by ligand HGF leads to agonistic proliferative events, a carefully selected antibody should be chosen to avoid triggering activation. As previously described, we identified and humanized an antibody with minimal agonism. Introducing an additional disulfide in the hinge region while maintaining the IgG1 isotype further reduced agonism as measured in vitro in both cell proliferation and phosphorylation signaling assays, while retaining high affinity to human and cynomolgus cMet, and acceptable expression and biophysical properties. To assess potential toxicity due to normal tissue expression, we measured binding of our antibody to normal hepatocytes from humans and cynos. Here we found very low expression and binding versus tumor cell lines. Next, we demonstrated that the cytotoxic activity of disulfide-cleavable maytansinoid ADCs prepared from the hinge-variant cMet antibody were equivalent to the parental form in in vivo models. In a MET-amplified xenograft model of gastric cancer, Hs746T, both parental and modified hucMet-sSPDB-DM4 ADCs demonstrated tumor eradication and comparable plasma clearance at 5 mg/kg. Similar results were found in the MET-amplified NSCLC model EBC-1 at 2.5 mg/kg. Conjugation to the newly-described dipeptide-linked maytansinoid DM21 further improved anti-tumor activity in both Hs746T and EBC-1 models, with a 2-fold decrease in minimally-efficacious dose. The activity of hucMet27Gv1.3Hinge-L-DM21 was durable, with a single dose yielding full regressions and tumor-free survivors in both models (EBC-1, 1.25 mg/kg dose, 6/6 TFS d49; Hs746T, 2.5 mg/kg dose, 8/8 TFS d55). Taken together, these data demonstrate compelling cMet-targeted activity of hucMet27Gv1.3Hinge-L-DM21 in MET-amplified models of NSCLC and gastric cancer with a wide margin of safety. These data merit further exploration of this ADC as a novel treatment option for patients with MET-amplified tumors. Citation Format: Katharine C. Lai, Min Li, Kathryn Selvitelli, Surina Sikka, Steven Boulé, L Cristina Gavrilescu, Stuart W. Hicks, Kerry Donahue. Preclinical evaluation of a new, non-agonist ADC targeting MET-amplified tumors with a peptide-linked maytansinoid [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 4817.

The majority of antibody-drug conjugates (ADCs) with maytansinoid payloads in development are linked via lysine residues and target an average drug-antibody ratio (DAR) of 3.5. These ADCs demonstrate substantial preclinical potency while maintaining good biophysical and pharmacological properties. However, some targets are expressed in normal tissues at levels that result in target-mediated drug disposition (TMDD), which can impact systemic and tumor ADC exposure and tumor penetration. For targets with TMDD, lowering the DAR of the ADCs may be advantageous. Since ADC tolerability is generally determined by the payload dose, lower DAR ADCs can be dosed at a higher antibody concentration, resulting in an increased conjugate exposure which may improve efficacy by saturating TMDD and/or increasing tumor delivery and penetration of the conjugate. In preclinical cell line xenograft studies, lysine-conjugated 2.0 and 3.5 DAR ADCs had comparable efficacy when dosed by payload concentration. However, the effect of TMDD cannot be captured with these models because the antibody in the ADCs does not cross-react with murine target antigen expressed on normal tissues. To supplement our understanding of the factors that influence ADC activity, we generated a cross-reactive model system that utilizes a chimeric anti-murine folate receptor α (FRα) antibody that binds with similar affinity to mouse and human FRα. Using this cross-reactive system, where the target is also expressed in normal tissues, 2.0 DAR conjugates were more efficacious than 3.5 DAR conjugates when dosed at matched payload concentrations in multiple xenograft models, suggesting that lower DAR can be an effective strategy to compensate for TMDD. Additional studies using xenograft models with varying antigen expression levels are ongoing, and studies designed to assess different conjugation methods of generating lower DAR ADCs are planned. This work highlights some of the many factors that impact ADC activity and demonstrates that the DAR of an ADC should be optimized for each target. Citation Format: Kerstin W. Sinkevicius, Leanne Lanieri, Jenny Lee, Steven Boule, Nicholas C. Yoder, Stuart W. Hicks, Jan Pinkas, Jose F. Ponte, Richard J. Gregory. The potential benefit of lower drug-antibody ratio (DAR) on antibody-maytansinoid conjugate in vivo efficacy [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 219.

Epithelial Cell Adhesion Molecule (EpCAM) is a glycosylated, 40-kDa type I transmembrane protein that plays a role in cell adhesion and cell signaling. EpCAM is an attractive target for antibody drug conjugate (ADC) development due to its overexpression on a variety of tumors of epithelial origin, including lung, colon, breast, ovarian, prostate and pancreatic cancers. In addition, EpCAM is enriched on tumor-initiating cells (TICs), which are often resistant to conventional cancer therapies. As such, EpCAM-targeted therapies may lead to more durable responses. However, EpCAM is also expressed on a variety of normal epithelia, thus limiting its utility as an ADC target due to potential toxicity. We aim to overcome this limitation by developing an EpCAM-targeting Probody™ drug conjugate (PDC). A Probody therapeutic is an antibody engineered with a mask that blocks the antigen binding site. Probody therapeutics can be selectively activated by tumor associated proteases releasing an active antibody with restored antigen binding activity. Therefore, an EpCAM-targeting PDC could have the anti-tumor potency of an ADC, while limiting binding to healthy tissues and minimizing toxicities. Here, we describe the development of EpCAM-targeted PDCs, based on a novel human/cynomolgus cross-reactive anti-EpCAM antibody. Probody molecules were successfully conjugated to either the maytansine-derived microtubule disruptor, DM4, linked via a hindered disulfide hydrophilic linker (sulfo-SPDB) or the ultra-potent DNA alkylating payload, DGN549. The antigen binding and in vitro cytotoxicity of intact PDCs were dramatically reduced compared to the corresponding ADC, but could be restored following in vitro proteolytic activation. Furthermore, EpCAM-targeting PDCs displayed compelling and specific anti-tumor activity in xenograft mouse models. In addition, the tolerability and pharmacokinetics (PK) of a selection of the EpCAM PDCs were compared to the EpCAM-targeting ADC in cynomolgus monkeys. EpCAM-targeting PDCs were better tolerated than the corresponding EpCAM-targeting ADC even at higher dose levels and displayed longer half-lives and greater exposure. Therefore, EpCAM PDCs showed greatly improved tolerability and PK profiles compared to the EpCAM ADC. The studies presented herein support an anti-EpCAM PDC as a promising novel therapeutic to target a wide range of EpCAM-expressing cancers with the potential to overcome the associated on-target toxicities. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Yimao Liu, Rui Wu, Cristina Gavrilescu, Jason Sagert, Kimberly Tipton, Shouchun Liu, Chanty Chan, Steven Boulé, Alan Wilhelm, Jacquelynn Lucas, Bahar Matin, Jean-Michel Lecerf, Marian Themeles, Ashley Morneault, Tara Drake, Sadiqa Yancey, Neeraj Kohli, Christopher Espelin, John Follit, Kerry A. Donahue, Tom Chittenden, Cynthia Guidi, Stuart W. Hicks. Development of a probody-drug conjugate targeting EpCAM for the treatment of solid tumors [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 213.

Antibody-drug conjugates (ADC) are a novel way to deliver potent cytotoxic compounds to cells expressing a specific antigen. Four ADC targeting CD19, including SAR3419 (coltuximab ravtansine), have entered clinical development. Here, we present huB4-DGN462, a novel ADC based on the SAR3419 anti-CD19 antibody linked via sulfo-SPDB to the potent DNA-alkylating agent DGN462. huB4-DGN462 had improved in vitro anti-proliferative and cytotoxic activity compared to SAR3419 across multiple B-cell lymphoma and human acute lymphoblastic leukemia cell lines. In vivo experiments using lymphoma xenografts models confirmed the in vitro data. The response of B-cell lymphoma lines to huB4-DGN462 was not correlated with CD19 expression, the presence of BCL2 or MYC translocations, TP53 inactivation or lymphoma histology. In conclusion, huB4-DGN462 is an attractive candidate for clinical investigation in patients with B-cell malignancies.

Abstract Purpose: c-Met dysregulation and/or overexpression are associated with tumor progression, metastasis and poor prognosis in numerous cancers. Despite strong pre-clinical evidence that blocking c-Met activity inhibits tumor cell growth and metastasis, targeted therapies have thus far failed to deliver an effective treatment option to the majority of patients. To address patients with both c-Met over-expressing and MET amplified tumors, we designed an antibody-drug conjugate (ADC) comprised of a humanized anti-c-Met monoclonal antibody linked to a highly potent indolinobenzodiazepine DNA-alkylating payload (DGN549) to enable activity against not only MET amplified but also c-Met over-expressing tumors. Experimental Design: Panels of monoclonal antibodies (Abs) against c-Met were generated and screened for antagonistic and agonistic activity in the presence or absence of the c-Met ligand, HGF. Lead Abs were humanized and conjugated to DGN549 either through lysine (Drug-to-Ab ratio (DAR) = 2.5) or engineered cysteine residues (DAR 2.0). Abs were also conjugated via lysine residues to the potent anti-microtubule maytansine derivative, DM4, using a sulfo-SPDB linker (DAR 3.5). Binding and cytotoxicity of ADCs were tested in vitro on normal and cancer cell lines with varying c-Met levels. Expression of c-Met was evaluated in patient tumors and xenografts along with normal human tissues using the CONFIRM immunohistochemistry assay. In vivo efficacy of anti-c-Met-DGN549 and anti-c-Met-DM4 ADCs was tested in both MET amplified and c-Met over-expressed (but non-amplified) xenograft tumor models. Results: A humanized anti-c-Met antibody, hucMet27, was identified which exhibits low c-Met agonist activity. Conjugates of hucMet27 were prepared with two different payloads, DGN549 and DM4, and in vitro and in vivo activity were determined. Both DGN549 and DM4 conjugates of hucMet27 bound with similar sub-nanomolar affinity to c-Met-expressing cells. hucMet27-DGN549 conjugates exhibited potent cytotoxicity against a large panel of c-Met expressing cell lines. By contrast, the potency of the hucMet27-DM4 conjugate was restricted mainly to cell lines harboring MET amplification, despite all cell lines demonstrating sensitivity to the unconjugated payload. When tested in mice bearing human xenograft tumors, both hucMet27-DGN549 and hucMet-DM4 conjugates were highly active in a MET amplified model, whereas hucMet27-DGN549 was more potent in inducing regressions in a model with c-Met over-expression without MET amplification. Conclusion: hucMet27-DGN549 exhibits compelling c-Met targeted anti-cancer activity in vitro and in vivo, and represents a promising therapeutic strategy to deliver a potent cytotoxic agent to tumor cells bearing a wide range of c-Met expression. Citation Format: Katharine C. Lai, Asli Muvaffak, Min Li, Marian Themeles, Surina Sikka, Kerry Donahue, Stuart W. Hicks, Angela Romanelli, Thomas Chittenden. In vitro and in vivo activity of a novel c-Met-targeting antibody-drug conjugate using a DNA-alkylating, indolinobenzodiazepine payload [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 45. doi:10.1158/1538-7445.AM2017-45

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

Background. CD19 is a cell surface membrane protein expressed in most mature and immature B cell neoplasms, making it a promising target for antibody-drug conjugate (ADC) therapy for B cell malignancies. Here we describe the preclinical activity of a novel CD19-targeting ADC, based on the potent indolinobenzodiazepine DNA-alkylating payload DGN462. Methods. The humanized anti-CD19 antibody, huB4, was conjugated to DGN462 via a cleavable disulfide linker, sulfo-SPDB. In vitro activity of the huB4-DGN462 ADC or the unconjugated DGN462 toxin was evaluated in 54 lymphoma cell lines [27 diffuse large B cell lymphomas (DLBCL); 10 mantle cell lymphomas; 6 marginal zone lymphomas; 5 anaplastic large T-cell lymphomas; 6 others]. Cell proliferation/viability after 72h of exposure was measured using a MTT assay. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation in respect to controls using the Promega ApoTox-Glo Triplex Assay. Gene expression profiling (GEP) was performed with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES u003e |2|, P |1.2|; P Results. huB4-DGN462 was cytotoxic against a broad panel of 48 B cell lymphoma cell lines (median IC50 100 pM; 95%CI, 38-214). The cytotoxic activity was not limited by P53, BCL2, MYC or CDKN2A status, or associated with DLBCL cell of origin. Consistent with overall lower CD19 expression, huB4-DGN462 was significantly (p-value=0.007) less active in eight T cell-derived lymphomas (median IC50 of 1.75 nM (95%CI, 0.5-5.75)) than in B cell lymphomas. huB4-DGN462 induced caspase 3/7 activation in 48/54 cell lines (89%) consistent with an apoptotic mechanism of action. huB4-DGN462 demonstrated compelling anti-tumor activity after a single intravenous dose in two diffuse large B-cell lymphoma cell line xenograft models: DoHH2 (a subcutaneous model) and Farage (a disseminated model). In the DoHH2 model, huB4-DGN462 resulted in a significant, dose-dependent tumor growth delay and survival benefit at 1.7 mg Ab/kg compared to a non-targeted control DGN462 ADC. In the disseminated Farage model, a significant dose-dependent increase in survival was observed in mice treated with as low as 0.17 mg Ab/kg of huB4-DGN462. At 1.7 mg Ab/kg, the life span was increased u003e400% compared to untreated mice. Conclusions. The novel ADC huB4-DGN462 presented strong preclinical anti-lymphoma activity, which provides evidence for further study. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Alberto J. Arribas, Roberta Pittau Bordone, Andrea Rinaldi, Georg Stussi, Emanuele Zucca, Davide Rossi, Anastasios Stathis, Min Li, Alan Wilhem, Kate Lai, Qifeng Qiu, Stuart Hicks, Callum Sloss, Francesco Bertoni. A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models [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 2651. doi:10.1158/1538-7445.AM2017-2651

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 IC 50 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

Abstract Naratuximab emtansine (IMGN529) is an investigational CD37-targeting antibody-drug conjugate (ADC) that has shown both preclinical and clinical activity in DLBCL . We have shown that rituximab, an anti-CD20 monoclonal antibody, enhances the preclinical activity of IMGN529. The combination of IMGN529 and rituximab is more active than either agent alone, and this benefit is associated with an increase in apoptosis and cell death, resulting in enhanced potency in DLBCL models. Efficacy of ADCs, such as IMGN529, relies on the internalization, intracellular trafficking and degradation of the ADC leading to the release of the cytotoxic catabolite. To explore the mechanism underlying the enhanced activity of the combination, we investigated the effect of rituximab treatment on the binding, internalization and processing of IMGN529 in vitro. To evaluate the effect on the internalization of IMGN529, DLBCL cell lines were incubated with fluorescently-labeled K7153A antibody (the CD37 targeting moiety of IMGN529) alone or in combination with rituximab or a non-targeting control antibody. K7153A internalization was similar whether incubated alone or with the non-targeting control. However, when cells were co-incubated with K7153A and rituximab, the internalization of K7153A significantly increased. This increase was also observed with other anti-CD20 antibodies suggesting an anti-CD20 class effect. Radiolabeled processing studies were performed to determine if the observed increase in internalization led to a corresponding increase in antibody degradation, which is required for the release of the cytotoxic agent in IMGN529. By trace-labeling the K7153A antibody with tritiated propionate (3H-K7153A), we were able to follow binding, uptake, and degradation of the antibody component of IMGN529 in DLBCL cell lines. Following pulse exposure, the amount of 3H-K7153A degraded after 24 hours remained the same whether treated alone or in combination with other B-cell targeting antibodies. However, when combined with rituximab the percentage of degraded 3H-K7153A increased as much as 3-fold and a similar increase of 3H catabolite was generated. When compared, the combination of K7153A and rituximab produced considerably more catabolite (~6-fold, p-value <0.0001) than the combination of rituximab and a CD19-targeting antibody. These findings suggest a novel mechanism where the increased potency of IMGN529 and rituximab can be explained by CD20 binding resulting in an increase in internalization and degradation of IMGN529, leading to generation of greater amounts of cytotoxic agent. Overall, these data provide a biological rationale for the enhanced activity of the rituximab plus IMGN529 combination, further supporting the clinical development strategy of this combination in DLBCL. Citation Format: Stuart W. Hicks, Katharine C. Lai, Yong Yi, Prerak Shah, Cristina L. Gavrilescu, Joe Ponte, Callum M. Sloss, Angela Romanelli. Increased internalization and processing of the CD37-targeting antibody-drug conjugate, naratuximab emtansine (IMGN529), in the presence of rituximab leads to enhanced potency in diffuse large B-cell lymphoma models [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 1073. doi:10.1158/1538-7445.AM2017-1073

Naratuximab emtansine (IMGN529) is an investigational antibody-drug conjugate consisting of a CD37-targeting antibody conjugated to the maytansine-derived microtuble disruptor, DM1. IMGN529 has shown promising preclinical and clinical activity in non-Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL). Due to the aggressive nature of the disease, DLBCL is often treated with combination therapies to maximize clinical outcomes; therefore, we investigated the potential of combining IMGN529 with both standard-of-care and emerging therapies against multiple oncology-relevant targets and pathways. The strongest enhancement in potency was seen with anti-CD20 antibodies, including rituximab. The combination of IMGN529 and rituximab was more potent than either agent alone, and this combinatorial benefit was associated with increased apoptotic induction and cell death. Additional studies revealed that rituximab treatment increased the internalization and degradation of the CD37-targeting antibody moiety of IMGN529. The combination of IMGN529 and rituximab was highly efficacious in multiple xenograft models, with superior antitumor efficacy seen compared to either agent alone or treatment with R-CHOP therapy. These findings suggest a novel mechanism whereby the potency of IMGN529 can be enhanced by CD20 binding, which results in the increased internalization and degradation of IMGN529 leading to the generation of greater amounts of cytotoxic catabolite. Overall, these data provide a biological rationale for the enhanced activity of IMGN529 in combination with rituximab and support the ongoing clinical evaluation of IMGN529 in combination with rituximab in patients with relapsed and/or refractory DLBCL.

Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼ 100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.

Abstract Antibody-drug conjugates (ADCs) are hybrid biotherapeutics that combine the targeting specificity of monoclonal antibodies with chemically conjugated, highly potent small molecule chemotherapeutics. Using established research scale approaches, the amount of antibody material needed to prepare candidate ADCs far exceeds the quantities required for initial in vitro screening. The need to scale up production across many antibodies slows down early lead selection efforts and wastes material. We have therefore developed methods for conjugating multiple antibodies with ADC payloads in parallel at the 50-150 μg scale in 96-well plates. Pilot reactions show that antibodies can be titrated to different final drug:antibody ratios (DARs) with different payloads, and that differences in pH can alter the reaction kinetics with useful effects. We show that 96-well centrifugal ultrafiltration enables highly parallel ADC purification while maintaining the rigorous removal of residual cytotoxic impurities observed with more established methods such as gel filtration chromatography. In addition, we describe characterization of microscale ADCs using a single chromatographic assay requiring ∼5 μg of material. The resulting platform reduces the required input quantity of antibody required for in vitro ADC screening by at least 5-10 fold. It also enables much higher conjugation throughput with concomitant decrease in time needed to generate and characterize ADCs. To assess the utility of the platform for ranking candidate antibodies, we compare in vitro cytotoxicity results for a panel of ADCs produced by both microscale and research scale methods. We also present three example screens in which antibody libraries of 10-85 members against different targets were conjugated using microscale methods and the resulting ADCs ranked by in vitro potency. For each antibody library, the screen size, conjugation conditions, and target DAR range were adjusted to suit the target biology, antibody type,and payload class. For example, we present evidence suggesting that, for targets in which functional antibody activity is not observed, normalization of DAR to the 2-6 range is adequate for screening. Across the screens, the success rates for producing ADC in quantity and quality suitable for screening were in the 75-90% range, using 200-600 μg of input antibody. Cytotoxic potencies ranging over 2-3 orders of magnitude were observed in the resulting ADC libraries, suggesting that microscale conjugation can rapidly focus ADC discoverycampaigns on high potency molecules. For early stage antibody and ADC screening efforts, we find that microscale conjugation methods yield ADCs that can substitute for traditionally prepared conjugates. We expect these methods will be applicable across many different ADC targets and payloads, and possibly applicable more generally to conjugated macromolecule therapeutic or diagnostic reagents. Citation Format: Nicholas C. Yoder, Kalli C. Catcott, Molly A. McShea, Carl Uli Bialucha, Parmita Saxena, Chen Bai, Kathy L. Miller, Thomas G. Gesner, Mikias Woldegiorgis, Stuart W. Hicks, Megan E. Lewis, Michael S. Fleming, Hans K. Erickson, Seth E. Ettenberg, Thomas A. Keating. Microscale methods for preparation and screening of antibody-drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5442. doi:10.1158/1538-7445.AM2015-5442

Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.

Several bacterial species have evolved specialized secretion systems to deliver bacterial effector proteins into eukaryotic cells. These effectors have the capacity to modulate host cell pathways in order to promote bacterial survival and replication. The spatial and temporal context in which the effectors exert their biochemical activities is crucial for their function. To fully understand effector function in the context of infection, we need to understand the mechanisms that lead to the precise subcellular localization of effectors following their delivery into host cells. Recent studies have shown that bacterial effectors exploit host cell machinery to accurately target their biochemical activities within the host cell.

S-Palmitoylation of G protein-coupled receptors (GPCRs) is a prevalent modification, contributing to the regulation of receptor function. Despite its importance, the palmitoylation status of the β(1)-adrenergic receptor, a GPCR critical for heart function, has never been determined. We report here that the β(1)-adrenergic receptor is palmitoylated on three cysteine residues at two sites in the C-terminal tail. One site (proximal) is adjacent to the seventh transmembrane domain and is a consensus site for GPCRs, and the other (distal) is downstream. These sites are modified in different cellular compartments, and the distal palmitoylation site contributes to efficient internalization of the receptor following agonist stimulation. Using a bioorthogonal palmitate reporter to quantify palmitoylation accurately, we found that the rates of palmitate turnover at each site are dramatically different. Although palmitoylation at the proximal site is remarkably stable, palmitoylation at the distal site is rapidly turned over. This is the first report documenting differential dynamics of palmitoylation sites in a GPCR. Our results have important implications for function and regulation of the clinically important β(1)-adrenergic receptor.