Background The presence of regulatory T cells (Tregs) in solid tumors attenuates the ability of immune cells to mediate an effective response to cancer. Expansion and activation of intratumoral Tregs is a proposed mechanism of PD-1/PD-L1 checkpoint inhibition resistance and elimination of these cells could facilitate an immune response to tumors. Single cell transcriptomic and flow cytometric analysis of tumor-associated immune cells revealed enhanced expression of CD30 on an activated subset of Tregs, suggesting that treatment with an anti-CD30 molecule could selectively deplete this subset of intratumoral Tregs. Moreover, anti-PD1 treatment of intratumoral T cells selectively enhanced expression of CD30 on Tregs compared with either CD4+ or CD8+ T cells, suggesting compatibility with immune checkpoint inhibitor therapy. The anti-CD30 antibody-drug conjugate (ADC) brentuximab vedotin is approved for the treatment of advanced classical Hodgkin lymphoma and other CD30-expressing lymphomas. It is currently being tested in combination with pembrolizumab for the ability to deplete Tregs and increase immune checkpoint activity in anti-PD-1-refractory metastatic melanoma and NSCLC (NCT04609566). Methods Preliminary analysis of IHC and RNA-seq data from paired biopsies in responder patients suggests increased tumor-infiltrating CD8+ T cells post treatment. SGN-35T, an anti-CD30 ADC with the same antibody (cAC10) and cytotoxic payload (monomethyl auristatin E) as brentuximab vedotin, contains a novel tripeptide linker comprised of D-leucine-alanine-glutamate (DLAE) and was developed to improve the tolerability profile while leveraging the known activity of brentuximab vedotin. Results In addition to cytotoxic activity on CD30-expressing tumor cells, SGN-35T depleted CD30-expressing Tregs in vitro, while in contrast CD8+ T cells were not affected. This may be due to the variable expression of drug efflux transporters which differs across T cell subsets and activation states, with Tregs demonstrating limited efflux capacity in contrast to naive CD8+ T cells and memory CD8+ T cells. Conclusions Thus, the cytotoxic activity of SGN-35T on CD30-expressing Tregs is likely a combination of the enrichment of CD30 expression on activated Tregs coupled with reduced drug efflux capacity. Together, these data support future clinical investigation of SGN-35T in combination with anti-PD-1/PD-L1 checkpoint inhibitors in solid tumors.

Description of additional methods and procedures used in the study.

Supplementary Figure Legends and Supplementary Table. Supplementary Table. EGFRvIII receptors/cell quantification of U251vIII and D317

Predicted topology for SLC46A3 (S1); Effect of siRNA on mRNA expression of SLC46A3 and CD70 in 786-0 cells (S2); Effect of siRNA on CD70 protein expression in 786-0 cells (S3); Anti-CD70 Ab-DSG-Maytansine and anti-CD70 Ab-Cys-Maytansine are potent against CD70 expressing 786-0 Cells (S4); Lysine-MCC-DM1 concentration time course in lysosome and total cell lysate (S5); Confluency of 786-0 cells transfected with siRNA (S6).

Abstract Antibody-drug conjugates (ADCs) employing the vedotin drug linker are effective anti-cancer agents in multiple indications including Hodgkin lymphoma, cervical cancer, and bladder cancer. While vedotin ADCs have demonstrated efficacy for the treatment of a wide variety of solid and hematologic cancers, some side effects, including dose-limiting neutropenia, are commonly observed. To improve the tolerability while leveraging the known activity of brentuximab vedotin, an ADC approved for the treatment of advanced classical Hodgkin lymphoma and other CD30-expressing lymphomas, alternative ADC structural components are under exploration. A novel cleavable linker containing a tripeptide molecule comprised of D-leucine-alanine-glutamate (DLAE) was developed and used to generate SGN-35T, an anti-CD30 ADC with the same mAb backbone and cytotoxic monomethyl auristatin E (MMAE) payload as brentuximab vedotin. SGN-35T was investigated for both anti-tumor activity and effects on hematopoiesis using in vitro and in vivo assays. SGN-35T displayed comparable activity to brentuximab vedotin against three lymphoma cell lines in vitro, and complete or near-complete tumor growth inhibition in five lymphoma xenograft models including a CD30-heterogeneous model. SGN-35T led to cell cycle arrest at the G2/M phase leading to apoptosis, in concordance with the established mechanism of action of MMAE. Introduction of the tripeptide linker in SGN-35T maintained the induction of immunogenic cell death hallmarks observed with brentuximab vedotin and other vedotin-based ADCs. SGN-35T demonstrated reduced potency on hematopoiesis compared to brentuximab vedotin in a human bone marrow colony-forming unit assay. In cynomolgus monkey toxicity studies, administration of SGN-35T had a reduced impact to neutrophils counts at matched dose levels and a two-fold improvement in tolerability compared to brentuximab vedotin, with a highest non-severely toxic dose (HNSTD) of 6 mg/kg versus an HNSTD of 3 mg/kg for brentuximab vedotin when administered Q3Wx4. Together, these data demonstrate that SGN-35T exhibits anti-tumor activity in preclinical models and may reduce the severity of hematopoietic toxicities while retaining anti-tumor activity observed with brentuximab vedotin. A Phase 1, first-in-human study is planned to evaluate the safety and anti-tumor activity of SGN-35T in lymphoid malignancies. Citation Format: Kevin J Hamblett, Steven Jin, Roma Yumul, Yufei Chen, John Kwon, Jessica Simmons, Clark Henderson, Hao Sun, Jason P Schrum, Andrea R Lim, Noah A Bindman, Melissa Conerly, Astrid Clarke. SGN-35T: A novel CD30-directed antibody-drug conjugate for the treatment of lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C132.

Supplementary Figure S3. Serum Concentrations of AMG 595 and control conjugate in mice bearing U251vIII xenografts

SGN-35C is an antibody drug conjugate composed of an anti-CD30 antibody conjugated to a camptothecin-derived topoisomerase 1 (TOP1) inhibitor payload. SGN-35C was designed to leverage the antibody backbone from brentuximab vedotin (BV) and the novel mechanism of action of camptothecin-derived ADCs. Here, we evaluated the mechanism of action, cytotoxicity, and safety in in vitro and in vivo models. To characterize the mechanism of action of SGN-35C in vitro and in vivo experiments were performed in CD30-positive anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma (HL) cell lines, and versions of these cell lines that are resistant to BV. SGN-35C binding, internalization, induction of DNA damage, cell cycle analysis, and in vitro cytotoxicity were evaluated. To assess if payload released in CD30-positive cells could kill adjacent CD30-negative cells, an in vitro bystander assay of CD30-positive and CD30-negative co-cultures was developed. The in vivo antitumor activity of SGN-35C was evaluated in animals bearing subcutaneous lymphoma models. SGN-35C antitumor activity was evaluated in a xenograft model of heterogeneous CD30 expression consisting of a mixture of a BV-resistant Karpas-299 cell line mixed with parental Karpas-299 cells to assess bystander activity in vivo. The tolerability of SGN-35C was assessed in a non-human primate model. SGN-35C binding to CD30-positive cells was similar to the unconjugated anti-CD30 antibody and demonstrated internalization into CD30-expressing cells. Treatment of cells with SGN-35C for 24 hours led to an increase of the DNA damage marker phospho-γH2A.X, which induced an increase in the fraction of cells in S-phase, consistent with the mechanism of camptothecin-based TOP1 inhibitors. Cytotoxicity was observed in ALCL and HL cell lines treated with SGN-35C, including an ALCL cell line resistant to BV. In a mixture of CD30-positive and CD30-negative cells, SGN-35C treatment induced cytotoxicity of the CD30-negative cells, demonstrating that SGN-35C can elicit bystander activity. A single dose of SGN-35C led to significant tumor growth inhibition in animals bearing lymphoma xenografts, including a BV-resistant model and a model of heterogeneous CD30 expression, demonstrating that SGN-35C can elicit bystander activity in vivo. SGN-35C demonstrates a tolerability profile in non-human primates that is consistent with camptothecins. A Phase 1, first-in-human study is planned to evaluate the safety and anti-tumor activity of SGN-35C in lymphoid malignancies.

Genes identified as critical for CD70 ADC function in Library 1 screen (S1); Genes identified as critical for CD70 ADC function in Library 2 screen (S2).

Antibody-drug conjugates (ADCs) employing the vedotin drug linker are effective anti-cancer agents in multiple indications including Hodgkin lymphoma, cervical cancer, and bladder cancer. While vedotin ADCs have demonstrated efficacy for the treatment of a wide variety of solid and hematologic cancers, some side effects, including dose-limiting neutropenia, are commonly observed. To improve the tolerability while leveraging the known activity of brentuximab vedotin, an ADC approved for the treatment of advanced classical Hodgkin lymphoma and other CD30-expressing lymphomas, alternative ADC structural components are under exploration. A novel cleavable linker containing a tripeptide molecule comprised of D-leucine-alanine-glutamate (DLAE) was developed and used to generate SGN-35T, an anti-CD30 ADC with the same mAb backbone and cytotoxic monomethyl auristatin E (MMAE) payload as brentuximab vedotin. SGN-35T was investigated for both anti-tumor activity and effects on hematopoiesis using in vitro and in vivo assays. SGN-35T displayed comparable activity to brentuximab vedotin against three lymphoma cell lines in vitro, and complete or near-complete tumor growth inhibition in five lymphoma xenograft models including a CD30-heterogeneous model. SGN-35T led to cell cycle arrest at the G2/M phase leading to apoptosis, in concordance with the established mechanism of action of MMAE. Introduction of the tripeptide linker in SGN-35T maintained the induction of immunogenic cell death hallmarks observed with brentuximab vedotin and other vedotin-based ADCs. SGN-35T demonstrated reduced potency on hematopoiesis compared to brentuximab vedotin in a human bone marrow colony-forming unit assay. In cynomolgus monkey toxicity studies, administration of SGN-35T had a reduced impact to neutrophils counts at matched dose levels and a two-fold improvement in tolerability compared to brentuximab vedotin, with a highest non-severely toxic dose (HNSTD) of 6 mg/kg versus an HNSTD of 3 mg/kg for brentuximab vedotin when administered Q3Wx4. Together, these data demonstrate that SGN-35T exhibits anti-tumor activity in preclinical models and may reduce the severity of hematopoietic toxicities while retaining anti-tumor activity observed with brentuximab vedotin. A Phase 1, first-in-human study is planned to evaluate the safety and anti-tumor activity of SGN-35T in lymphoid malignancies. Citation Format: Kevin J Hamblett, Steven Jin, Roma Yumul, Yufei Chen, John Kwon, Jessica Simmons, Clark Henderson, Hao Sun, Jason P Schrum, Andrea R Lim, Noah A Bindman, Melissa Conerly, Astrid Clarke. SGN-35T: A novel CD30-directed antibody-drug conjugate for the treatment of lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C132.

Supplementary Figure S1. Regression of U251vIII Human GBM Subcutaneous Xenografts Following AMG 595 Treatment

Abstract Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors. Citation Format: Stuart D. Barnscher, Andrea Hernández Rojas, Kevin J. Hamblett, Nichole Escalante. Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2633.

Supplementary Figure S2. Cell Growth inhibition in A431 cells (wild-type EGFR overexpressing cells)

Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors. Citation Format: Stuart D. Barnscher, Andrea Hernández Rojas, Kevin J. Hamblett, Nichole Escalante. Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2633.

Antibody drug conjugate (ADC) therapies such as Kadcyla® and Adcetris® have significantly improved outcomes for patients. Despite these early advances, many ADCs have failed due to tolerability and efficacy concerns; therefore, there is a need to develop ADCs with a greater therapeutic window. We have previously reported the increased tolerability of a novel N-acyl sulfonamide auristatin payload conjugated to trastuzumab via a protease cleavable linker. In non-human primates (NHPs), the HNSTD for this ADC was 18 mg/kg compared to 3 mg/kg for the MMAE conjugate control. Separately, we have also reported that a biparatopic antibody targeting a tumor associated antigen (e.g. anti-HER2 bispecific antibody ZW25) can lead to enhanced receptor clustering and improved internalization, thereby increasing the efficiency of payload delivery. Our aim is to develop a series of novel biparatopic ADCs with expanded therapeutic windows against multiple targets. Here we present the proof-of-concept in vitro and in vivo characterization of benchmark ADCs against 3 different targets with improved tolerability and equivalent efficacy. Benchmark antibodies against 3 known clinical targets were conjugated to our N-acyl sulfonamide auristatin (mAb-ADCs) or to MMAE or DM4 controls (mAb-control ADCs) via cleavable linkers and were assessed for in vitro binding affinity and cytotoxicity. The therapeutic windows of mAb-ADCs and mAb-control ADCs were compared by assessing efficacy in mouse xenograft models and tolerability and pharmacokinetics in NHPs. mAb-ADCs had similar binding affinities to recombinant targets and/or to cancer cells expressing low to high levels of target antigen compared to the mAb-control ADCs. mAb-ADCs demonstrated similar in vitro cytotoxicity compared to mAb-control ADCs and this was recapitulated in vivo with similar tumor growth inhibition in mouse xenograft models. In a NHP tolerability/PK study, mAb-ADCs for all 3 targets were tolerated at doses up to 18 mg/kg (single dose IV infusion) compared to the mAb-control ADCs that showed severe to life-threatening neutropenia at lower doses. The increase in maximum tolerated dose for the mAb-ADCs over the mAb-control ADCs, together with comparable efficacy across 3 different targets, demonstrates the broad applicability of the novel N-acyl sulfonamide auristatin payload to expand the therapeutic window. This strategy, together with ongoing efforts to identify synergistic antibody paratopes that more efficiently deliver payload, could lead to next-generation biparatopic ADCs with improved activity. Citation Format: Rupert H. Davies, Stuart D. Barnscher, Peter W. Chan, Laurence Madera, Jamie R. Rich, Marylou Vallejo, Grant R. Wickman, Kevin Yin, Vincent Fung, Kevin J. Hamblett, Patrick G. Kaminker, John S. Babcook. Towards development of next-generation biparatopic ADCs using a novel linker-toxin with expanded therapeutic window [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3912.

Background: Antibody drug conjugates (ADCs) can offer significant benefit to patients suffering from a variety of solid and liquid tumors by combining the specificity of monoclonal antibodies and the cellular cytotoxicity of antineoplastic small molecules. While ADCs have tremendous potential, dose-limiting toxicity remains the largest barrier to robust patient responses. Through the utilization of proprietary protease cleavable N-acyl sulfonamide (NAcS) linked hemiasterlin and auristatin payloads, we have generated highly efficacious ADCs with improved therapeutic indices. Antibody drug conjugates (ADCs) can offer significant benefit to patients suffering from a variety of solid and liquid tumors by combining the specificity of monoclonal antibodies and the cellular cytotoxicity of antineoplastic small molecules. While ADCs have tremendous potential, dose-limiting toxicity remains the largest barrier to robust patient responses. Through the utilization of proprietary protease cleavable N-acyl sulfonamide (NAcS) linked hemiasterlin and auristatin payloads, we have generated highly efficacious ADCs with improved therapeutic indices. Methods: Antibodies against HER2 and three additional known clinical targets were conjugated with our N-acyl sulfonamide auristatin (NAcS-ADCs) or with MMAE or DM4 controls (control ADCs) via cleavable linkers and used to assess in vitro binding and cytotoxicity. The therapeutic window of NAcS-ADCs and control ADCs was compared by assessing efficacy in mouse xenograft models and tolerability/pharmacokinetics in non-human primates (NHPs). Results: NAcS-ADCs demonstrated similar in vitro binding and cytotoxicity compared to control ADCs. This activity was recapitulated in vivo with similar tumor growth inhibition in mouse xenograft models. In an NHP tolerability/pharmacokinetic study, the trastuzumab NAcS-ADC was well tolerated with a highest non-severely toxic dose (HNSTD) of 18 mg/kg, as compared to an HNSTD of 3 mg/kg for the trastuzumab MMAE control ADC. NAcS-ADCs for all three additional targets were well tolerated at doses of 18 mg/kg (single dose IV infusion) compared to their corresponding control ADCs that showed severe to life-threatening neutropenia at markedly lower doses. Conclusions: The increase in tolerability and comparable efficacy of the NAcS-ADCs vs. the control ADCs against HER2 as well as three additional known clinical targets highlight the broad applicability of our novel NAcS-linked auristatin payload and its ability to expand the therapeutic window of ADC therapy. Legal entity responsible for the study: Zymeworks Inc. Funding: Zymeworks Inc. Disclosure: J. Babcook, R. Davies, S. Barnscher, J. Rich, K. Yin, V. Fung, G. Winters, G. Garnett, P. Kaminker, K. Hamblett: Employee of Zymeworks Inc. and have a financial interest in the company in the form of stock or stock-options.

Ado-trastuzumab emtansine, referred to as trastuzumab-MCC-DM1 or T-DM1, was the first antibody drug conjugate (ADC) approved for HER2 positive metastatic breast cancer. This chapter reviews the development of trastuzumab-MCC-DM1, summarizes novel anti-HER2 antibody drug conjugate technologies in clinical trials, and discusses future directions of these technologies beyond targeting HER2. In an effort to improve the efficacy of trastuzumab a panel of drug linkers were conjugated to the anti-HER2 antibody and compared in preclinical experiments. In the hallmark phase III EMILIA trial treatment with trastuzumab-MCC-DM1 led to significantly longer median survival compared to the standard of care lapatinib and capecitabine in patients with 2nd line HER2 positive metastatic breast cancer. Subsequently, multiple anti-HER2 ADCs were generated with different ADC platforms allowing a comparison of different drug linkers, drug to antibody ratios, site-specific antibody drug conjugates, and biparatopic antibody drug conjugates. Anti-HER2 antibody drug conjugates currently in clinical testing are described. Promising early clinical data are emerging from some of the ADCs employing novel technologies. Future directions including bispecific antibody drug conjugates directed against HER2 and another target are discussed. Ultimately the goal is to generate clinical candidate ADCs that can improve patient outcomes. Comparison of anti-HER2 ADCs will inform how novel ADC technologies can be applied beyond HER2 to other cancer associated antigens.

Abstract Antibody drug conjugates (ADCs) combine the specificity of monoclonal antibodies with potent antineoplastic small molecules, and promise efficacy without the systemic toxicity of chemotherapy. Despite this tremendous potential, most clinical ADCs have failed to provide sufficient therapeutic benefit before the onset of off-target dose-limiting platform toxicities. Here we report the development of proprietary protease cleavable N-acyl sulfonamide linked hemiasterlin and auristatin payloads, Zymelink, that allow the generation of an efficacious ADC platform with improved tolerability. Both Zymelink drug-linkers were conjugated via maleimides to endogenous cysteines. As Zymelink drug-linkers are more polar than maleimide valine citrulline monomethyl auristatin E drug-linker, the resulting ADCs can be produced with potential advantages in PK, efficacy and safety. The resulting ADCs exhibit potent in vitro cytotoxicity. A trastuzumab-based ADC prepared with Zymelink drug-linker and demonstrated at least equivalent efficacy compared to a trastuzumab-based ADC prepared with MMAE, promoting durable complete regressions in a patient-derived xenograft model. Zymelink hemiasterlin and auristatin ADCs were tolerated at 5-6 fold higher doses compared to a MMAE ADC in cynomolgus monkeys. The maximum tolerated dose of the MMAE ADC was just 3 mg/kg based on severe neutropenia. Zymelink hemiasterlin ADC was tolerated at 15 mg/kg with no evidence of neutropenia or elevations in transaminases. Zymelink auristatin ADC was tolerated at 18 mg/kg based on increased levels of transaminases at 24 mg/kg. Moreover, Zymelink ADCs exhibited greater serum exposure at equivalent doses. These results suggest Zymelink ADCs have a greatly expanded therapeutic window compared to MMAE conjugates. Citation Format: Stuart Barnscher, John Babcook, Jamie Rich, Geoff Winters, Graham Garnett, Andrea Hernandez, Vincent Fung, Kevin Yin, Kevin Hamblett, Rupert Davies. Zymelink drug conjugate platform: redefining the therapeutic window for ADCs [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 61. doi:10.1158/1538-7445.AM2017-61

Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.

Abstract Epidermal growth factor receptor variant III (EGFRvIII) is a cancer-specific deletion mutant observed in approximately 25% to 50% of glioblastoma multiforme (GBM) patients. An antibody drug conjugate, AMG 595, composed of the maytansinoid DM1 attached to a highly selective anti-EGFRvIII antibody via a noncleavable linker, was developed to treat EGFRvIII-positive GBM patients. AMG 595 binds to the cell surface and internalizes into the endo-lysosomal pathway of EGFRvIII-expressing cells. Incubation of AMG 595 with U251 cells expressing EGFRvIII led to potent growth inhibition. AMG 595 treatment induced significant tumor mitotic arrest, as measured by phospho-histone H3, in GBM subcutaneous xenografts expressing EGFRvIII. A single intravenous injection of AMG 595 at 17 mg/kg (250 μg DM1/kg) generated complete tumor regression in the U251vIII subcutaneous xenograft model. AMG 595 mediated tumor regression in the D317 subcutaneous xenograft model that endogenously expresses EGFRvIII. Finally, AMG 595 treatment inhibited the growth of D317 xenografts orthotopically implanted into the brain as determined by magnetic resonance imaging. These results demonstrate that AMG 595 is a promising candidate to evaluate in EGFRvIII-expressing GBM patients. Mol Cancer Ther; 14(7); 1614–24. ©2015 AACR.