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Supplementary Figure S1. Comparable internalization rates for 5T4_0108 and the 5T4-Tub ADC;Supplementary Figure S2. Affinity optimization improves in vitro cytotoxicity of a 5T4-ADC;Supplementary Figure S3. Induction of apoptosis by 5T4-PBD and 5T4-Tub;Supplementary Figure S4. 5T4 is expressed on CSCs of multiple cancer cell lines;Supplementary Figure S5. Only PBD is capable of inducing cytotoxicity of CSC populations in vitro;Supplementary Table S1. Improved cytotoxic potency following affinity optimization of 5T4 antibody; Supplementary Table S2. Quantification of cell surface-associated 5T4 in various cancer cell lines;Supplementary Table S3. Tumorigenicity of CSC populations derived from NCI- N87 and MDA-MB-361 xenografts; Supplementary Materials & Methods

Per-patient ADA responses

Summary of mean (%CV) PK parameters of Total ADC and Active ADC after a single IV infusion of MEDI3726

CTC while on treatment (patients with at least one CTC measurement >50)

Mutation profiles in ctDNA: truncating and/or pathogenic mutations in DDR or DDR-related pathway

Purpose:MEDI3726 is an antibody–drug conjugate targeting the prostate-specific membrane antigen and carrying a pyrrolobenzodiazepine warhead. This phase I study evaluated MEDI3726 monotherapy in patients with metastatic castration-resistant prostate cancer after disease progression on abiraterone and/or enzalutamide and taxane-based chemotherapy.Patients and Methods:MEDI3726 was administered at 0.015–0.3 mg/kg intravenously every 3 weeks until disease progression/unacceptable toxicity. The primary objective was to assess safety, dose-limiting toxicities (DLT), and MTD/maximum administered dose (MAD). Secondary objectives included assessment of antitumor activity, pharmacokinetics, and immunogenicity. The main efficacy endpoint was composite response, defined as confirmed response by RECIST v1.1, and/or PSA decrease of ≥50% after ≥12 weeks, and/or decrease from ≥5 to Results:Between February 1, 2017 and November 13, 2019, 33 patients received MEDI3726. By the data cutoff (January 17, 2020), treatment-related adverse events (TRAE) occurred in 30 patients (90.9%), primarily skin toxicities and effusions. Grade 3/4 TRAEs occurred in 15 patients (45.5%). Eleven patients (33.3%) discontinued because of TRAEs. There were no treatment-related deaths. One patient receiving 0.3 mg/kg had a DLT of grade 3 thrombocytopenia. The MTD was not identified; the MAD was 0.3 mg/kg. The composite response rate was 4/33 (12.1%). MEDI3726 had nonlinear pharmacokinetics with a short half-life (0.3–1.8 days). The prevalence of antidrug antibodies was 3/32 (9.4%), and the incidence was 13/32 (40.6%).Conclusions:Following dose escalation, no MTD was identified. Clinical responses occurred at higher doses, but were not durable as patients had to discontinue treatment due to TRAEs.

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor β subunit (GMCSFRβ) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRβ KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.

Most strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent anti-tumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two.

ObjectiveImmune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development.MethodsVIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates.ResultsWe generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies.ConclusionsVIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases.

Purpose: MEDI3726 is an antibody–drug conjugate targeting the prostate-specific membrane antigen and carrying a pyrrolobenzodiazepine warhead. This phase I study evaluated MEDI3726 monotherapy in patients with metastatic castration-resistant prostate cancer after disease progression on abiraterone and/or enzalutamide and taxane-based chemotherapy. Patients and Methods: MEDI3726 was administered at 0.015–0.3 mg/kg intravenously every 3 weeks until disease progression/unacceptable toxicity. The primary objective was to assess safety, dose-limiting toxicities (DLT), and MTD/maximum administered dose (MAD). Secondary objectives included assessment of antitumor activity, pharmacokinetics, and immunogenicity. The main efficacy endpoint was composite response, defined as confirmed response by RECIST v1.1, and/or PSA decrease of ≥50% after ≥12 weeks, and/or decrease from ≥5 to Results: Between February 1, 2017 and November 13, 2019, 33 patients received MEDI3726. By the data cutoff (January 17, 2020), treatment-related adverse events (TRAE) occurred in 30 patients (90.9%), primarily skin toxicities and effusions. Grade 3/4 TRAEs occurred in 15 patients (45.5%). Eleven patients (33.3%) discontinued because of TRAEs. There were no treatment-related deaths. One patient receiving 0.3 mg/kg had a DLT of grade 3 thrombocytopenia. The MTD was not identified; the MAD was 0.3 mg/kg. The composite response rate was 4/33 (12.1%). MEDI3726 had nonlinear pharmacokinetics with a short half-life (0.3–1.8 days). The prevalence of antidrug antibodies was 3/32 (9.4%), and the incidence was 13/32 (40.6%). Conclusions: Following dose escalation, no MTD was identified. Clinical responses occurred at higher doses, but were not durable as patients had to discontinue treatment due to TRAEs.

Antibody–drug conjugates (ADC) are targeted agents that have shown promise in treating cancer. A central challenge in development of ADCs is the relatively narrow therapeutic index observed in clinical studies. Patient selection strategies based on expression of the target in tumors have the potential to maximize benefit and provide the best chance of clinical success; however, implementation of biomarker-driven trials can be difficult both practically and scientifically. We conducted a survey of recent clinical experience from early-phase ADC trials completed between 2000 and 2019 to evaluate the different approaches to patient selection currently being used and assess whether there is evidence that target expression is associated with clinical activity. Our analysis of patient selection strategies indicates that optimal trial design for early-stage trials should be based on multiple factors, including prevalence and heterogeneity of target expression among intent-to-treat patients, as well as biological factors influencing expression of cell surface and soluble target. To ensure a high probability of success, early implementation of patient selection strategies centered around target expression are pivotal to development of ADCs. In this review, we propose a strategic approach that can be applied for optimization of trial design.

The regulatory approval process for new therapies in oncology involves costly clinical trials that can span multiple years and historically have had a low probability of success. To derive estimates of the probability of success and other related risk characteristics in oncology clinical trials, we searched Informa Pharma Intelligence's Trialtrove and Pharmaprojects databases for oncology-specific drug-indication programs between 2001 and 2020. In total, we identified 10,083 oncology-specific drug-indication programs, which included 2,800 therapeutic agents (excluding biosimilars) tested across 98 indications. The features we considered included indication and indication group, clinical phase, follow-on indication status, presence and type of patient selection biomarker, drug modality, and accelerated approval status. In several cases, clinical success rates were higher than the benchmark oncology historical average of the phase 1 to approval success rate of 4.1% found in our sample set. For example, the use of pre-selection biomarkers in early clinical development was associated with a greater than two-fold increase in probability of approval from phase 1. Moreover, drugs with prior approvals in another indication were more than three times more likely to be successful from phase 1 than a candidate targeting a novel mechanism. In addition, we found that monoclonal antibodies were numerically more successful than other modalities in the historical data, while oncolytic viruses, vaccines, and bispecific antibodies have been less successful. In all cases, concomitant with the emergence of genomic technologies, an improved scientific understanding of cancer biology, and an evolving regulatory environment that includes a higher number of accelerated approvals, success rates in oncology have improved during the time period of this analysis and particularly in the past 5 years. Citation Format: Mary Jane Hinrichs, Shomesh E. Chaudhuri, Robert Bowden, Andrew W. Lo, David W. Jenkins. Investigation into factors influencing probability of success in oncology clinical trials and trends over time [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 2623.

We previously tested HER2-targeted antibody drug conjugates (ADC) in immunocompromised (SCID) mice, precluding evaluation of host immunity, impact on cancer stem cells (CSCs), and potential benefit when combined with PD-L1 blockade. In this study, we tested HER2-targeted ADC in two immunocompetent mouse tumor models. HER2-targeted ADC specifically inhibited the growth of HER2-expressing tumors, prolonged animal survival, and reduced HER2(+) and PD-L1(+) cells. ADC plus anti-PD-L1 antibody augmented therapeutic efficacy, modulated immune gene signatures, increased the number and function of CD3(+) and CD19(+) tumor-infiltrating lymphocytes (TILs), induced tumor antigen specific immunological memory, stimulated B cell activation, differentiation, and IgG1 production both systemically and in the tumor microenvironment. In addition, ADC therapy modulated T cell subsets and their activation in TILs. Furthermore, HER2-targeted ADC reduced the number and tumorigenicity of ALDH(hi) CSCs. This study demonstrates that HER2-targeted ADC effectively targets ALDH(hi) CSCs and this effect is augmented by co-administration of anti-PD-L1 antibody.

The continuing education course on integrating clinical and anatomical pathology data was designed to communicate the importance of using a weight of evidence approach to interpret safety findings in toxicology studies. This approach is necessary, as neither clinical nor anatomic pathology data can be relied upon in isolation to fully understand the relationship between study findings and the test article. Basic principles for correlating anatomic pathology and clinical pathology findings and for integrating these with other study end points were reviewed. To highlight these relationships, a series of case examples, presented jointly by a clinical pathologist and an anatomic pathologist, were used to illustrate the collaborative effort required between clinical and anatomical pathologists. In addition, the diagnostic utility of traditional liver biomarkers was discussed using results from a meta-analysis of rat hepatobiliary marker and histopathology data. This discussion also included examples of traditional and novel liver and renal biomarker data implementation in nonclinical toxicology studies to illustrate the relationship between discrete changes in biochemistry and tissue morphology.

Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.

Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody–drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient–derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176–86. ©2018 AACR.

Background Collectively, the cell surface Fc region of IgG receptors (FcγRs) engage soluble IgG and IgG containing immune complexes and trigger activation or inhibtory signals that play a critical role in the regulation of immune responses. The low affinity FcγRIIA (CD32A) is the most widely expressed activating FcγR in humans and appears to drive autoantibody and immune complex mediated autoimmune disorders. So far a therapeutic targeting this receptor has not been developed. Objectives To generate and characterize a novel humanized effector-deficient FcγRIIA antibody (MEDI9600) for clinical development. Methods The mode of action of MEDI9600 was assessed by confocal microscopy, whole blood internalization, and binding competition assays. Multiple cell based assays were used to measure autoantibody and immune complex mediated responses. The safety of MEDI9600 was assessed in in vitro by neutrophil migration, activation and opsonophagocytic killing assays. Safety and pharmacokinetics were examined in vivo in a single-dose PK/PD study in cynomolgus monkey. Results We generated a humanized effector-deficient FcγRIIA antibody (MEDI9600) that potently blocks both autoantibody and immuno complex-mediated proinflammatory responses from a variety of cell types. This includes the inhibition of Toll-like receptor stimulatory immune complexes that induce type I Interferons from pDC, and the inhibition of anti-neutrophil cytoplasmic antibody (ANCA) induced production of reactive oxygen species from neutrophils, which are associated with the pathogenesis of systemic lupus and ANCA vasculitis respectively. MEDI9600 specifically binds FcγRIIA and its suppressive activity is attributed to its capacity to block ligand engagement and to internalize the receptor from the cell surface. Moreover, in vivo studies indicate that MEDI9600 has a favorable pharmacokinetic and safety profile. Conclusions We have generated MEDI9600, a specific humanized antibody antagonist of FcγRIIA with null effector function that may provide a novel therapeutic approach in the treatment of immune complex mediated diseases. Disclosure of Interest None declared

Purpose: To use preclinical models to identify a dosing schedule that improves tolerability of highly potent pyrrolobenzodiazepine dimers (PBDs) antibody drug conjugates (ADCs) without compromising antitumor activity.Experimental Design: A series of dose-fractionation studies were conducted to investigate the pharmacokinetic drivers of safety and efficacy of PBD ADCs in animal models. The exposure–activity relationship was investigated in mouse xenograft models of human prostate cancer, breast cancer, and gastric cancer by comparing antitumor activity after single and fractionated dosing with tumor-targeting ADCs conjugated to SG3249, a potent PBD dimer. The exposure–tolerability relationship was similarly investigated in rat and monkey toxicology studies by comparing tolerability, as assessed by survival, body weight, and organ-specific toxicities, after single and fractionated dosing with ADCs conjugated to SG3249 (rats) or SG3400, a structurally related PBD (monkeys).Results: Observations of similar antitumor activity in mice treated with single or fractionated dosing suggests that antitumor activity of PBD ADCs is more closely related to total exposure (AUC) than peak drug concentrations (Cmax). In contrast, improved survival and reduced toxicity in rats and monkeys treated with a fractionated dosing schedule suggests that tolerability of PBD ADCs is more closely associated with Cmax than AUC.Conclusions: We provide the first evidence that fractionated dosing can improve preclinical tolerability of at least some PBD ADCs without compromising efficacy. These findings suggest that preclinical exploration of dosing schedule could be an important clinical strategy to improve the therapeutic window of highly potent ADCs and should be investigated further. Clin Cancer Res; 23(19); 5858–68. ©2017 AACR.

Antibody–drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576–87. ©2017 AACR.

The continuing education course on integrating clinical and anatomical pathology data was designed to communicate the importance of using a weight of evidence approach to interpret safety findings in toxicology studies. This approach is necessary, as neither clinical nor anatomic pathology data can be relied upon in isolation to fully understand the relationship between study findings and the test article. Basic principles for correlating anatomic pathology and clinical pathology findings and for integrating these with other study end points were reviewed. To highlight these relationships, a series of case examples, presented jointly by a clinical pathologist and an anatomic pathologist, were used to illustrate the collaborative effort required between clinical and anatomical pathologists. In addition, the diagnostic utility of traditional liver biomarkers was discussed using results from a meta-analysis of rat hepatobiliary marker and histopathology data. This discussion also included examples of traditional and novel liver and renal biomarker data implementation in nonclinical toxicology studies to illustrate the relationship between discrete changes in biochemistry and tissue morphology.

HER2-targeted antibody drug conjugate (ADC) was only tested in immunocompromised (SCID) host, which doesn’t allow the evaluation of the ADC-induced host immune responses. In addition, no potential impact of ADC on cancer stem cells (CSCs) has been reported. Furthermore, tumor cells with PD-L1 expression may escape immune attack by converting PD-1 positive immune effector cells into anergy. We hypothesized that HER2-targeted ADC could modulate host immune response, target HER2high CSCs, and the efficacy of ADC could be enhanced by anti-mPD-L1. To test this hypotheses, we used two immunocompetent murine models: mouse breast tumor D2/F2 and TNBC 4T1 with enforced expression of HER2, D2F2/E2 and HER2-4T1 respectively. Both D2F2/E2 and HER2-4T1 could generate tumor in immunocompetent Balb/c mice, which allowed us to test the efficacy and specificity of HER2-targeted ADC, and to evaluate host immune responses. In D2F2/E2 mouse model, we found that HER2-targeted ADC significantly inhibited D2F2/E2 tumor growth, but not the parental D2F2 tumors, in a dose dependent manner. The specificity was confirmed in HER2-4T1 model. In parallel, we observed that HER2-targeted ADC treatment reduced the number of HER2 positive cells and ALDHhighCSCs. We found that anti-mPD-L1 mAb significantly augmented the therapeutic efficacy of HER2-targeted ADC in both D2F2/E2 and HER2-4T1 tumor models. Mechanistically, HER2-targted ADC combined with anti-PD-L1 significantly modulated the gene signature of multiple immune factors in tumor microenvironment. At the cellular level, anti-mPD-L1 plus HER2-targeted ADC increased the number of CD3+ and CD19+ tumor infiltrating lymphocytes (TILs). Expanded CD3+ TILs from the tumor subjected to combined treatment killed tumor cells significantly more than mono-treatment. Expanded CD19+ TILs from tumor subjected to the combined therapy produced more IgG than all the controls. Importantly, the IgG could specifically bind to tumor cells, resulting in cytotoxicity of the tumor cells via ADCC. As a result, ADC plus anti-PD-L1 further reduced the number of HER2 positive cells. Importantly, the combined therapy not only reduced the number of ALDHhigh CSCs in the residual tumor, but also significantly paralyzed their tumoriginicity. Together, our data indicate that HER2-targeted ADC could induce significant host immune responses, which was evidenced by the regulation of gene signature and detection of anti-tumor CD3+ and CD19+ TILs. We first show that HER2-targeted ADC could target ALDHhigh cancer stem cells, and such effect was significantly enhanced by anti-mPD-L1 administration. Citation Format: Leiming Xia, Lu Wen, Gang Wu, Tao Zhang, Frank Comer, Mary Jane Masson Hinrichs, Michael Oberst, Steven R. Coats, Alfred E. Chang, Yuanyuan Liu, Yangyi Bao, Max Wicha, Qiao Li. HER2-targeted antibody drug conjugates (ADCs) induce host immunity against cancer stem cells and are enhanced by anti-PD-L1 [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 4981.

The neutral amino acid transporter, ASCT2, is frequently overexpressed in several cancers to sustain "glutamine addiction" of cancer cells. High expression of ASCT2 is often associated with poor disease prognosis. Immuno-histochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue samples revealed high prevalence of membranous ASCT2 expression in several hematological cancers, including MM, AML, and DLBCL summarized in Table1. ASCT2 expression was predominantly on the plasma membrane of the carcinoma cells. ASCT2 staining was observed in almost all the samples with high positivity (>2+ and in >50% of tumor cells) in 98, 74 and 55% of MM, AML and DLBCL samples respectively. Additionally, flow cytometry analyses suggest significantly high expression of ASCT2 in bone marrow (BM) samples from AML and MM patients in comparison to BM from healthy donors. Also, our data suggest relatively less expression of ASCT2 in LT-HSC (long term hematopoietic stem cells) in comparison to prominent myeloid-associated marker CD33. In contrast, we found similar expression profile of ASCT2 and CD33 in downstream lineage-committed progenitor cells, such as MPP (multi potent progenitors) and CP (common progenitors). Furthermore, IHC evaluated across normal tissues suggest restricted ASCT2 expression in the normal tissues of vital organs. Broad expression across various unmet cancers and restricted expression in normal tissues warrant ASCT2 as an attractive candidate for an antibody drug conjugate. MEDI7247 is a novel investigational antibody-drug conjugate (ADC) comprising an anti-ASCT2 human monoclonal antibody site-specifically conjugated to pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker, with a drug to antibody ratio (DAR) of 2. MEDI7247 specifically binds to the cell surface ASCT2 while exhibiting no affinity to the other members of the family, including ASCT1. Following binding to the extracellular region of ASCT2 antigen, MEDI7247 is internalized and trafficked to the lysosomes to subsequently release the PBD warhead. The release of PBD induces DNA damage and results in tumor cell death. MEDI7247 shows potent in vitro cytotoxic killing in several heme cancer cell lines (IC50 range of 0.05 ng/ml to ~65 ng/ml) with variable levels of ASCT2 expression (e.g. H929 (high) ~ 1.1 X106 copies/cell; Nomo (low) ~ 1.3 X105 copies/cell). In vivo anti-tumor activity of MEDI7247 was evaluated in a panel of hematological tumor models (CDx) representing AML, MM, DLBCL, cALL, and Burkitt's lymphoma. Briefly, MEDI7247 (dosed: single dose or Q1Wx4) demonstrated a significant survival advantage in disseminated or subcutaneous tumor models, when compared to the untreated control or standard of care (SOC) at the various dose levels examined: 0.05, 0.1 and 0.4 mg/kg, respectively. Likewise, MEDI7247 was also tested in disseminated AML PDX (ASCT2-low) models. Significant improvement in survival was observed at both 0.1 and 0.4 mg/kg with the higher dose level extending survival by >80 days and PDX models showed robust antitumor effect and significant survival benefit compared to standard of care (SOC). We used flow cytometry sorting of CD34+ and CD38+ populations from AML BM and ran colony forming assays in methocult culture conditions to confirm that CD34+CD38+ cells are leukemic stem cells (LSC) of AML BM samples. Furthermore, we confirmed high expression of ASCT2 in LSC and in the bulk population of AML BM. Similar analyses suggest relatively high expression of ASCT2 in MM plasma cells (CD138+CD19-) and modest to low expression in MM stem cells (CD19+ CD138-). This may yield opportunity for more durable clinical response in AML, genetically heterogeneous diseases. Furthermore, MEDI7247 demonstrated acceptable safety profile in toxicity studies with non-human primates to support first in human trials. In conclusion, based on its combined efficacy and safety, MEDI7247, a first-in class ADC is currently being evaluated in clinic for the treatment of ASCT2 positive hematological malignancies (NCT03106428). Disclosures Pore: Medimmune: Employment. Schifferli:Medimmune: Employment. Monks:Medimmune: Employment. Tammali:Medimmune: Employment. Borrok:Medimmune: Employment. Hurt:Medimmune: Employment. Flynn:Medimmune: Employment. Rebelatto:Medimmune: Employment. Townsley:Medimmue: Employment. Hinrichs:Medimmune: Employment. Dixit:Medimmune: Employment. Coats:Medimmune: Employment. Herbst:Medimmune: Employment. Tice:Medimmune: Employment.

Prostate specific membrane antigen (PSMA), a type II membrane glycoprotein, is highly expressed in nearly all prostate cancers, with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence, increased surface expression in prostate tumors, and constitutive internalization make PSMA an attractive target for an antibody-drug conjugate (ADC) approach in anticancer treatment of patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of anti-PSMA antibody J591, site-specifically conjugated with DNA cross-linking pyrrolobenzodiazepine (PBD) dimer SG3249 for targeting prostatic cancer cells. Using prostate cancer cell line models, we have previously shown that MEDI3726 specifically binds to the extracellular domain of PSMA and, once internalized, releases the PBD dimer to cross link DNA and achieve potent in vitro and in vivo cytotoxicity. Here we investigated the in vivo activity of MEDI3726 in a series of LuCaP prostate cancer patient-derived xenograft (PDX) models. The selected LuCaP models had varying PSMA expression and heterogeneous genetic and phenotypic backgrounds. In agreement with the earlier cell line xenograft data, dose-dependent antitumor activity was observed in PSMA-positive PDX models with durable tumor regressions in models with high PSMA expression. In the PSMA-negative LuCaP 35CR PDX model, MEDI3726 did not have significant antitumor activity, thus highlighting target-mediated in vivo activity. Increased phosphorylation of histone H2AX was observed in xenografts dosed with MEDI3726, confirming DNA damage induced by interstrand cross-linking PBD dimer as the mechanism of antitumor activity of MEDI3726. In summary, MEDI3726 demonstrated potent and specific in vivo antitumor activity, concurrent with DNA damage, in clinically relevant prostate cancer PDX models. MEDI3726 is being evaluated in phase 1 clinical trial as an anticancer treatment in patients with metastatic castrate-resistant prostate cancer (NCT02991911). Citation Format: Song Cho, Francesca Zammarchi, Noel R. Monks, Kapil Vashisht, Ravinder Tammali, Kevin Schifferli, Patrick Strout, Wanda King, Karma Dacosta, Ryan Fleming, David G. Williams, Karin Havenith, Mary Jane Masson Hinrichs, Simon Chivers, Nazzareno Dimasi, Phil W. Howard, John A. Hartley, Steve Coats, Ronald Herbst, Patrick H. van Berkel, David A. Tice. MEDI3726 (ADCT-401), a novel antibody-drug conjugate targeting PSMA, has potent in vivo antitumor activity in prostate cancer patient-derived xenograft models [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A004.

Antibody drug conjugates (ADCs) are biopharmaceutical molecules consisting of a cytotoxic small molecule covalently linked to a targeted protein carrier via a stable cleavable or noncleavable linker. The process of conjugation yields a highly complex molecule with biochemical properties that are distinct from those of the unconjugated components. The impact of these biochemical differences on the safety and pharmacokinetic (PK) profile of the conjugate must be considered when determining the types of nonclinical safety studies required to support clinical development of ADCs. The hybrid nature of ADCs highlights the need for a science-based approach to safety assessment that incorporates relevant aspects of small and large molecule testing paradigms. This thinking is reflected in current regulatory guidelines, where sections pertaining to conjugates allow for a flexible approach to nonclinical safety testing. The aim of this article is to review regulatory expectations regarding early assessment of nonclinical safety considerations and discuss how recent advances in our understanding of ADC-mediated toxicity can be used to guide the types of nonclinical safety studies needed to support ADC clinical development. The review will also explore nonclinical testing strategies that can be used to streamline ADC development by assessing the safety and efficacy of next generation ADC constructs using a rodent screen approach.

Background. First-in-human (FIH) trials of low-molecular-weight anticancer agents conventionally derive a safe start dose (SD) from one-tenth the severely toxic dose in 10% of rodents or one-sixth the highest nonseverely toxic dose (HNSTD) in nonrodent species. No consensus has been reached on whether this paradigm can be safely applied to biotechnology-derived products (BDPs). Materials and Methods. A comprehensive search was conducted to identify all BDPs (excluding immune checkpoint inhibitors and antibody drug conjugates) with sufficient nonclinical and clinical data to assess the safety of hypothetical use of one-sixth HNSTD in an advanced cancer FIH trial. Results. The search identified 23 BDPs, of which 21 were monoclonal antibodies. The median ratio of the maximum tolerated or maximum administered dose (MTD or MAD) to the actual FIH SD was 36 (range, 8–500). Only 2 BDPs reached the MTD. Hypothetical use of one-sixth HNSTD (allometrically scaled to humans) would not have exceeded the MTD or MAD for all 23 BDPs and would have reduced the median ratio of the MTD or MAD to a SD to 6.1 (range, 3.5–55.3). Pharmacodynamic (PD) markers were included in some animal toxicology studies and were useful to confirm the hypothetical SD of one-sixth HNSTD. Conclusion. One-sixth HNSTD would not have resulted in unacceptable toxicities in the data available. Supporting its use could reduce the number of dose escalations needed to reach the recommended dose. A low incidence of toxicities in animals and humans underscores the need to identify the pharmacokinetic and PD parameters to guide SD selection of BDPs for FIH cancer trials. Implications for Practice: Start dose (SD) for biotechnology-derived products (BDPs) can be safely derived from one-sixth the highest nonseverely toxic dose in nonrodent species and may reduce the number of dose escalations needed to reach the recommended dose in first-in-human studies while limiting unnecessary exposure to high drug levels in humans. The use of this type of SD could improve the design of phase I studies of BDPs by making them more efficient. The role of preclinical pharmacodynamic markers was useful in confirming the hypothetical SD, and attempts should be explored in future animal studies to identify such parameters.

Prostate-specific membrane antigen (PSMA) is an attractive target for an ADC approach as it is over-expressed by virtually all prostate cancers and its expression is highest in poorly-differentiated, metastatic and castration-resistant cases. PSMA has limited expression in non-prostatic tissues, it is not secreted or cleaved by PSMA-expressing cells, and it is constitutively internalized, a process that may be accelerated by specific antibody binding. ADCT-401/MEDI3726 is an ADC composed of a monoclonal antibody (J591), directed against human PSMA, site-specifically conjugated (drug-to-antibody ratio of 1.8) to a highly cytotoxic DNA cross-linking PBD dimer with a valine-alanine dipeptide linker. In vitro, ADCT-401/MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-expressing prostate cancer cell lines, whereas its activity was greatly reduced in PSMA-negative cell lines. In vivo, ADCT-401/MEDI3726 showed strong antitumor activity against CWR22Rv1 and LNCaP human-derived prostate cancer xenograft models. In the CWR22Rv1 model, a tumor with low, heterogeneous PSMA expression, ADCT-401/MEDI3726 achieved dose-dependent antitumor activity when administered as single dose at either 0.33 or 1 mg/kg, which resulted in significant increase in survival compared to the vehicle-treated animals. Moreover, a single dose of ADCT-401/MEDI3726 showed remarkable superior antitumor activity compared to multiple doses of another PSMA-targeted ADC stochastically conjugated to the auristatin payload vcMMAE with a DAR of ~4. In the LNCaP model, ADCT-401/MEDI3726 resulted in dose-dependent antitumor activity when dosed once at 0.11, 0.33 or 1 mg/kg. In the PSMA-negative prostate cancer-derived PC3 xenograft model, ADCT-401/MEDI3726 and an isotope-control ADC showed limited tumor growth inhibition highlighting target-mediated antitumor activity. ADCT-401/MEDI3726 demonstrated potent and specific in vitro and in vivo antitumor activity in prostate cancer-derived models of differing levels of PSMA and this warrants further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, Simon Chivers, Karin Havenith, David G. Williams, Lauren Adams, Maria Mellinas-Gomez, Simon Corbett, Peter Tyrer, Francois D'Hooge, Song Cho, Nazzareno Dimasi, Mary Jane Hinrichs, Phil W. Howard, John A. Hartley, Patrick H. van Berkel. ADCT-401/MEDI3726, a novel pyrrolobenzodiazepine (PBD)-based antibody-drug conjugate (ADC) targeting PSMA-expressing prostate cancers [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 3111A. doi:10.1158/1538-7445.AM2017-3111A

Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potent cytotoxicity of small molecule drugs and have shown to provide therapeutic options for various cancers. We report herein the discovery of a HER2-targeting ADC MEDI4276 that showed potent cell killing activity in vitro in cancer cell lines that express the HER2 receptor. The observed in vitro activity translated into in vivo tumor growth inhibition in various xenograft mouse models. MEDI4276 is a homogeneous molecule with precise control of drug loading following site specific conjugation of a cytotoxic drug. The drug in MEDI4276 is MMETA, a fully synthetic analog of the tubulysin family that showed pM potency in a panel of cancer cell lines. MMETA was conjugated to the antibody via engineered cysteines with a maleimide-bearing mc-Lys protease cleavable linker. The antibody in MEDI4276 is a bivalent biparatopic antibody targeting two distinct non-overlapping epitopes on HER2 that leads to antibody-receptor clustering following binding and thus promoting internalization, lysosomal trafficking and degradation. The combination of enhanced internalization and potent cytotoxic drug allows for this ADC to kill tumor cell populations with a broader range of HER2 expression. Preclinical studies showed that MEDI4276 induced tumor regression in HER2-positive tumor models that had developed acquired resistance to T-DM1 and in a number of models with lower HER2 expression that are refractory to T-DM1 treatment. Overall, our findings underscore the potential application of MEDI4276 to treat a large patient population that is ineligible for or relapsed/refractory to current HER2-targeted therapies. MEDI4276 is currently being investigated in a Phase I clinical trial. Citation Format: John Li, Dorin Toader, Samuel R. Perry, Vanessa Muniz-Medina, Leslie Wetzel, Marlon C. Rebelatto, Mary Jane Masson Hinrichs, Ryan Fleming, Binyam Bezabeh, Pamela Thompson, Nazzareno Dimasi, Brandon Lam, Xian-Qing Yu, Changshou Gao, Rakesh Dixit, Steven Coats, Jane Osbourn, Herren Wu. MEDI4276, a HER2-targeting antibody tubulysin conjugate, displays potent in vitro and in vivo activity in preclinical studies. [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 2970.

Antibody-drug conjugates (ADCs) selectively deliver cytotoxic agents to tumors and provide the potential for increased clinical benefit to cancer patients. We have developed an ADC consisting of an antibody against a target expressed on the surface of tumor cells that is site-specifically conjugated with a pyrrolobenzodiazepine (PBD) dimer payload (PBD-ADC). The tumor-associated antigen is overexpressed in many carcinomas, on both bulk tumor cells and cancer stem cells (CSCs), while normal tissue expression is limited, thus making it an ideal ADC target. The PBD payload was selected due to its picomolar potency and because, unlike tubulin inhibitors, it could be cytotoxic to CSCs as well as bulk tumor cells. The PBD-ADC is significantly efficacious in target-positive in vivo tumor models of gastric, breast and lung carcinomas, inducing long-term tumor regressions and demonstrates greater potency compared to ADCs conjugated with either tubulysin or auristatin payloads, both tubulin inhibitors. Additionally, the PBD-ADC significantly reduced the CSC population of tumors in vivo, unlike ADCs bearing tubulin inhibitor payloads. The PBD-ADC appears to be efficacious against tumors that have acquired resistance to a tubulysin-based ADC, however possible mechanisms of resistance to this PBD-ADC are being investigated. Collectively, this novel ADC represents a promising therapy that may deliver durable clinical responses in indications with unmet medical need. Citation Format: Jay W. Harper, Chris Lloyd, Shenlan Mao, Xin Lin, Nazzareno Dimasi, Phil Howard, Ellen O'Connor, David Farkas, Christopher Barton, Jeff Smith, Helen Zhong, Tracy Chen, Mary Jane Hinrichs, Marlon Rebelatto, Xiang-Qing Yu, Susan Spitz, Andrew Pierce, Dominic Lai, Hurt Elaine, Lilian van Vlerken-Ysla, Jalla Sanjoo, Katy Miller, Karen McCullough, Priya Kannan, Simon Thompson, Parthiv Mahadevia, Chris Martin, Robert Hollingsworth, Adeela Kamal. A novel antibody-drug conjugate that induces long-term tumor regression and anticancer stem cell activity. [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 948. doi:10.1158/1538-7445.AM2015-948