In vitro cytotoxicity of PF-06804103 across a panel of cancer cell lines.

ADC retention times by hydrophobic interaction chromatography (HIC).

Supplementary Methods, Table S1, and Figure S1

In vivo efficacy of site-specific anti-HER2 vc0101 4 DAR ADCs in N87 xenograft tumor model.

Immunohistochemical evaluation of HER2 expression in vivo cell line and patient-derived xenograft models.

Supplementary Figure S1: N87 and N87-TM cells are similarly sensitive to unconjugated DM1-SMe; Supplementary Figure S2: Trastuzumab binding to N87 and N87-TM cells; Supplementary Figure S3: Generation and characterization of T-DM1-resistant HCC1954-TM and BT474-TM cells; Supplementary Figure S4: Relative resistance profiles of N87-TM to N87 cells to T-ADCs, unconjugated payloads and standard-of-care chemotherapeutics; Supplementary Figure S5: Distribution of T-ADCs in N87-TM tumors; Supplementary Figure S6: N87-TM cells are cross-resistant to a T-ADC conjugated to a cleavable linker with a cysteine-capped auristatin analog; Supplementary Figure S7: N87-TM cells internalize T-ADC into non-low pH intracellular compartments; Supplementary Figure S8: CAV1-GFP expression causes intracellular caveolae formation capable of internalizing T-ADCs in N87 cells; Supplementary Figure S9: CAV1 knockdown is not sufficient to re-sensitize N87-TM cells to T-DM1; Supplementary Figure S10: Non-cleavable and cleavable T-ADCs co-localize with CAV1 similarly in N87 and N87-TM cells; Supplementary Table S1. Cytotoxicity values of Trastuzumab and isotype-control ADCs in HT29 cells, a HER2-negative cell line

Supplementary Methods, Table S1 S2, and Figure Legends. Supplementary Table S1: Properties of cell lines made resistant to TM-conjugate. Supplementary Table S2: Relative resistance of selected free payloads and Cys-capped-released species in H69 (no ABCC1) and H69AR (high ABCC1 expressing) cell lines.

Conjugation of vc0101 does not alter anti-HER2 mAb binding.

Nanoparticle (NP) technology holds significant promise to mediate targeted drug delivery to specific organs in the body. Understanding the 3D biodistribution of NPs in heterogeneous environments such as the tumor tissue can provide crucial information on efficacy, safety and potential clinical outcomes. Here we present a novel end-to-end workflow, VIOLA, which makes use of tissue clearing methodology in conjunction with high resolution imaging and advanced 3D image processing to quantify the spatiotemporal 3D biodistribution of fluorescently labeled ACCURIN® NPs. Specifically, we investigate the spatiotemporal biodistribution of NPs in three different murine tumor models (CT26, EMT6, and KPC-GEM) of increasing complexity and translational relevance. We have developed new endpoints to characterize NP biodistribution at multiple length scales. Our observations reveal that the macroscale NP biodistribution is spatially heterogeneous and exhibits a gradient with relatively high accumulation at the tumor periphery that progressively decreases towards the tumor core in all the tumor models. Microscale analysis revealed that NP extravasation from blood vessels increases in a time dependent manner and plateaus at 72 h post injection. Volumetric analysis and pharmacokinetic modeling of NP biodistribution in the vicinity of the blood vessels revealed that the local NP density exhibits a distance dependent spatiotemporal biodistribution which provide insights into the dynamics of NP extravasation in the tumor tissue. Our data represents a comprehensive analysis of NP biodistribution at multiple length scales in different tumor models providing unique insights into their spatiotemporal dynamics. Specifically, our results show that NPs exhibit a dynamic equilibrium with macroscale heterogeneity combined with microscale homogeneity.

Effects of a Conventional and a Site-Specific Conjugate Targeting HER2 on Indicators of Bone Marrow Toxicity and Serum ADC/Payload Exposures in Rats

Effects of Systemic Administration of Free Payload Compared with Conventional HER2-vc0101 on Indicators of Bone Marrow Toxicity and Bone Marrow Payload Levels in Rats

Includes Supplementary Materials and Methods, Data and Figure Legends

Supplementary Figures S1 to S11. Supplementary Figure S1: Schematic illustration of the approach used to create trastuzumab-maytansinoid ADC resistant cell lines. Supplementary Figure S2: Structures of payloads and ADCs. Supplementary Figure S3: 361-TM-resistant cells show differential responses to ADCs with modified linkers, payloads, and antibodies. Supplementary Figure S4: Ectoptic expression of Her2 in JIMT1-TM cells rescues sensitivity to TM-ADC. Supplementary Figure S5: Cell surface proteomic profiling of 361-TM resistant cells, with evidence of increased ABCC1. Supplementary Figure S6: Knockdown of ABCC1 (MRP1) in 361-TM cells sensitizes to ADCs. Supplementary Figure S7: Endosomal and lysosomal proteins altered in JIMT1-TM resistant cells as determined by proteomic profiling. Supplementary Figure S8: Immunoblot verification of elevated surface-associated proteins in JIMT1-TM resistant cells. Supplementary Figure S9: Co-localization analyses of ADC and lysosome via live-cell imaging. Supplementary Figure S10: Altered trafficking in JIMT1-TM cells are specific to TM-ADC and not transferrin receptor antibody. Supplementary Figure S11: LC-MRM analyses of released products from ADCs incubated with parental and resistant cell lines.

Abstract Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody–drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non–small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.

AbstractPurpose:Mortality due to acute myeloid leukemia (AML) remains high, and the management of relapsed or refractory AML continues to be therapeutically challenging. The reapproval of Mylotarg, an anti-CD33–calicheamicin antibody–drug conjugate (ADC), has provided a proof of concept for an ADC-based therapeutic for AML. Several other ADCs have since entered clinical development of AML, but have met with limited success. We sought to develop a next-generation ADC for AML with a wide therapeutic index (TI) that overcomes the shortcomings of previous generations of ADCs.Experimental Design:We compared the TI of our novel CD33-targeted ADC platform with other currently available CD33-targeted ADCs in preclinical models of AML. Next, using this next-generation ADC platform, we performed a head-to-head comparison of two attractive AML antigens, CD33 and CD123.Results:Our novel ADC platform offered improved safety and TI when compared with certain currently available ADC platforms in preclinical models of AML. Differentiation between the CD33- and CD123-targeted ADCs was observed in safety studies conducted in cynomolgus monkeys. The CD33-targeted ADC produced severe hematologic toxicity, whereas minimal hematologic toxicity was observed with the CD123-targeted ADC at the same doses and exposures. The improved toxicity profile of an ADC targeting CD123 over CD33 was consistent with the more restricted expression of CD123 in normal tissues.Conclusions:We optimized all components of ADC design (i.e., leukemia antigen, antibody, and linker-payload) to develop an ADC that has the potential to translate into an effective new therapy against AML.

Nucleic acid-sensing pathways play critical roles in innate immune activation through the production of type I interferon (IFN-I) and proinflammatory cytokines. These factors are required for effective antitumor immune responses. Pharmacological modulators of the pre-mRNA spliceosome splicing factor 3b subunit 1 (SF3B1) are under clinical investigation as cancer cytotoxic agents. However, potential roles of these agents in aberrant RNA generation and subsequent RNA-sensing pathway activation have not been studied. In this study, we observed that SF3B1 pharmacological modulation using pladienolide B (Plad B) induces production of aberrant RNA species and robust IFN-I responses via engagement of the dsRNA sensor retinoic acid-inducible gene I (RIG-I) and downstream interferon regulatory factor 3. We found that Plad B synergized with canonical RIG-I agonism to induce the IFN-I response. In addition, Plad B induced NF-kappa B responses and secretion of proinflammatory cytokines and chemokines. Finally, we showed that cancer cells bearing the hotspot SF3B1(K700E) mutation, which leads to global aberrant splicing, had enhanced IFN-I response to canonical RIG-I agonism. Together, these results demonstrate that pharmacological modulation of SF3B1 in cancer cells can induce an enhanced IFN-I response dependent on RIG-I expression. The study suggests that spliceosome modulation may not only induce direct cancer cell cytotoxicity but also initiate an innate immune response via activation of RNA-sensing pathways.

Abstract The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody–drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug–antibody ratio (DAR) ADC with this linker–payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site–specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo–acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.

Novel neolymphostin-based antibody-drug conjugate (ADC) precursors were synthesized either through amide couplings between both cleavable and non-cleavable linkers and neolymphostin derivatives, or through Cu(I)-catalyzed acetylene-azide click cycloadditon between non-cleavable linkers and neolymphostin acetal derivatives. These precursors were site-specifically conjugated to cysteine mutant trastuzumab-A114C to provide neolymphostin-based ADCs. Preliminary in vitro data indicated that the corresponding ADCs were active against HER2-expressing tumor cell lines, thus providing a proof-of-concept for using neolymphostin as ADC-based anticancer agents.

A potent class of DNA-damaging agents, natural product bis-intercalator depsipeptides (NPBIDs), was evaluated as ultrapotent payloads for use in antibody-drug conjugates (ADCs). Detailed investigation of potency (both in cells and via biophysical characterization of DNA binding), chemical tractability, and in vitro and in vivo stability of the compounds in this class eliminated a number of potential candidates, greatly reducing the complexity and resources required for conjugate preparation and evaluation. This effort yielded a potent, stable, and efficacious ADC, PF-06888667, consisting of the bis-intercalator, SW-163D, conjugated via an N-acetyl-lysine-valine-citrulline- p-aminobenzyl alcohol- N, N-dimethylethylenediamine (AcLysValCit-PABC-DMAE) linker to an engineered variant of the anti-Her2 mAb, trastuzumab, catalyzed by transglutaminase.

Abstract Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice and represent personalized murine ‘avatars' of those tumors. PDXs are valuable tumor models for drug development since they recapitulate the complexity of the human tumor microenvironment more extensively than cell line xenografts (CLXs). Unlike CLXs, PDXs are never passaged in vitro, and therefore they more faithfully recapitulate native tumor biology and response to therapeutics. Thus, PDXs may more accurately predict clinical activity of therapeutic compounds than traditional CLXs. We are developing a next generation (NG)-HER2 antibody-drug conjugate (ADC), PF-06804103, that employs a proprietary site-specific conjugation technology that chemically links the clinically-validated linker-payload “ValCit-Aur0101” to an anti-HER2 antibody. The site-specific conjugation enables enhanced exposures and reduced off-target toxicities as previously described. We utilized our PDX collection to evaluate the breadth-of-efficacy of PF-06804103 versus trastuzumab emtansine (T-DM1), an FDA-approved ADC for metastatic breast cancer patients with high HER2 expression. Efficient cleavage of the ‘ValCit' linker and payload release in the early endosome is not impacted by the rapid recycling properties of the HER2 receptor, unlike T-DM1 which requires catabolism in the within the lysosomal milieu to efficiently release its payload. To date, we have enrolled >20 HER2-expressing breast, gastric and non-small cell lung cancer PDXs, with varying low to high HER2 expression levels, in a head-to-head ‘mouse avatar clinical trial' comparing activity of a single cycle of T-DM1 (6 mg/kg) to PF-06804103 (3 mg/kg). Impressively, PF-06804103 had more durable complete responses and a higher objective response rate (ORR) than T-DM1 (84% vs. 4%), including in low-moderate HER2 expressers. PF-06804103 prolonged median overall survival (OS) of mice with HER2-expressing tumors compared to T-DM1 (100 vs 45 days). Biomarker analysis showed that tumors at all levels of HER2 expression were more likely to receive more benefit with PF-06804103 than T-DM1 (HR < 0.45). By leveraging our novel PDX ‘avatar' clinic, we were able to demonstrate that PF-06804103 displays superior in vivo breadth-of-efficacy compared to T-DM1. Citation Format: Matthew S. Sung, Christine Hopf, Erik Upeslacis, Jonathon Golas, Mark Kaplan, Kiran Khandke, Manoj Charati, Frank Kotch, Frank Loganzo, Ken Geles, Judy Lucas, Hans-Peter Gerber, Puja Sapra, Edward Rosfjord. NG-HER2 ADC (PF-06804103) is superior to trastuzumab emtansine in a mouse 'avatar' head-to-head clinical trial [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 818.