Anti-Extra Domain B Splice Variant of Fibronectin Antibody-Drug Conjugate Eliminates Tumors with Enhanced Efficacy When Combined with Checkpoint Blockade.

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).

NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells.

Aberrant NOTCH3 signaling and overexpression is oncogenic, associated with cancer stem cells and drug resistance, yet therapeutic targeting remains elusive. Here, we develop NOTCH3-targeted antibody drug conjugates (NOTCH3-ADCs) by bioconjugation of an auristatin microtubule inhibitor through a protease cleavable linker to two antibodies with differential abilities to inhibit signaling. The signaling inhibitory antibody rapidly induces ligand-independent receptor clustering and internalization through both caveolin and clathrin-mediated pathways.

PF-06804103, A Site-specific Anti-HER2 Antibody-Drug Conjugate for the Treatment of HER2-expressing Breast, Gastric, and Lung Cancers.

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.

Comparison of the molecular and cellular phenotypes of common mouse syngeneic models with human tumors.

Background: The clinical success of immune checkpoint inhibitors demonstrates that reactivation of the human immune system delivers durable responses for some patients and represents an exciting approach for cancer treatment. An important class of preclinical in vivo models for immuno-oncology is immunocompetent mice bearing mouse syngeneic tumors. To facilitate translation of preclinical studies into human, we characterized the genomic, transcriptomic, and protein expression of a panel of ten commonly used mouse tumor cell lines grown in vitro culture as well as in vivo tumors.

Natural Product Bis-Intercalator Depsipeptides as a New Class of Payloads for Antibody-Drug Conjugates.

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.

Establishing in vitro-in vivo correlation for antibody drug conjugate efficacy: a PK/PD modeling approach.

The objective of this manuscript was to establish in vitro-in vivo correlation (IVIVC) between the in vitro efficacy and in vivo efficacy of antibody drug conjugates (ADCs), using a PK/PD modeling approach. Nineteen different ADCs were used to develop IVIVC. In vitro efficacy of ADCs was evaluated using a kinetic cell cytotoxicity assay.

Caveolae-Mediated Endocytosis as a Novel Mechanism of Resistance to Trastuzumab Emtansine (T-DM1).

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2 metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2 tumors, are not well understood. We used HER2 cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an "on-off" routine until a T-DM1-resistant population was generated.

Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate.

Antibody drug conjugates (ADCs) are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates.

Optimization of Tubulysin Antibody-Drug Conjugates: A Case Study in Addressing ADC Metabolism.

As part of our efforts to develop new classes of tubulin inhibitor payloads for antibody-drug conjugate (ADC) programs, we developed a tubulysin ADC that demonstrated excellent in vitro activity but suffered from rapid metabolism of a critical acetate ester. A two-pronged strategy was employed to address this metabolism. First, the hydrolytically labile ester was replaced by a carbamate functional group resulting in a more stable ADC that retained potency in cellular assays.

Found in Translation: Maximizing the Clinical Relevance of Nonclinical Oncology Studies.

The translation of nonclinical oncology studies is a subject of continuous debate. We propose that translational oncology studies need to optimize both pharmacokinetic (drug exposure) and pharmacodynamic (xenograft model) aspects. While improvements in pharmacodynamic translatability can be obtained by choosing cell lines or patient-derived xenograft models closer to the clinical indication, significant ambiguity and variability exists when optimizing the pharmacokinetic translation of small molecule and biotherapeutic agents.

Natural Product Splicing Inhibitors: A New Class of Antibody-Drug Conjugate (ADC) Payloads.

There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells.

Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy.

Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition.

Preclinical Development of an anti-5T4 Antibody-Drug Conjugate: Pharmacokinetics in Mice, Rats, and NHP and Tumor/Tissue Distribution in Mice.

The pharmacokinetics of an antibody (huA1)-drug (auristatin microtubule disrupting MMAF) conjugate, targeting 5T4-expressing cells, were characterized during the discovery and development phases in female nu/nu mice and cynomolgus monkeys after a single dose and in S-D rats and cynomolgus monkeys from multidose toxicity studies. Plasma/serum samples were analyzed using an ELISA-based method for antibody and conjugate (ADC) as well as for the released payload using an LC-MS/MS method. In addition, the distribution of the Ab, ADC, and released payload (cys-mcMMAF) was determined in a number of tissues (tumor, lung, liver, kidney, and heart) in two tumor mouse models (H1975 and MDA-MB-361-DYT2 models) using similar LBA and LC-MS/MS methods.

Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells to Result in Sustained Tumor Regressions.

Purpose: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival.

Experimental Design: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined.

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2.

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice.

Tumor cells chronically treated with a trastuzumab-maytansinoid antibody-drug conjugate develop varied resistance mechanisms but respond to alternate treatments.

Antibody-drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab-maytansinoid ADC (TM-ADC) at IC80 concentrations followed by recovery.

Mild method for succinimide hydrolysis on ADCs: impact on ADC potency, stability, exposure, and efficacy.

The stability of the connection between the antibody and the toxin can have a profound impact on ADC safety and efficacy. There has been increasing evidence in recent years that maleimide-based ADCs are prone to payload loss via a retro-Michael type reaction. Herein, we report a mild method for the hydrolysis of the succinimide-thioether ring which results in a "ring-opened" linker.

Advances in patient-derived tumor xenografts: from target identification to predicting clinical response rates in oncology.

Most oncology compounds entering clinical development have passed stringent preclinical pharmacology evaluation criteria. However, only a small fraction of experimental agents induce meaningful antitumor activities in the clinic. Low predictability of conventional preclinical pharmacology models is frequently cited as a main reason for the unusually high clinical attrition rates of therapeutic compounds in oncology.

A priori prediction of tumor payload concentrations: preclinical case study with an auristatin-based anti-5T4 antibody-drug conjugate.

The objectives of this investigation were as follows: (a) to validate a mechanism-based pharmacokinetic (PK) model of ADC for its ability to a priori predict tumor concentrations of ADC and released payload, using anti-5T4 ADC A1mcMMAF, and (b) to analyze the PK model to find out main pathways and parameters model outputs are most sensitive to. Experiential data containing biomeasures, and plasma and tumor concentrations of ADC and payload, following A1mcMMAF administration in two different xenografts, were used to build and validate the model. The model performed reasonably well in terms of a priori predicting tumor exposure of total antibody, ADC, and released payload, and the exposure of released payload in plasma.

Long-term tumor regression induced by an antibody-drug conjugate that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells.

Antibody-drug conjugates (ADC) represent a promising therapeutic modality for the clinical management of cancer. We sought to develop a novel ADC that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells (TIC), which comprise the most aggressive cell population in the tumor. We optimized an anti-5T4 ADC (A1mcMMAF) by sulfydryl-based conjugation of the humanized A1 antibody to the tubulin inhibitor monomethylauristatin F (MMAF) via a maleimidocaproyl linker.

Identification of 2-oxatriazines as highly potent pan-PI3K/mTOR dual inhibitors.

We recently described several highly potent, triazine (1) and triazolopyrimidine (2) scaffold-based, dual PI3K/mTOR-inhibitors (e.g., 1, PKI-587) that were efficacious in both in vitro and in vivo models.

Discovery of a stable macrocyclic o-aminobenzamide Hsp90 inhibitor which significantly decreases tumor volume in a mouse xenograft model.

An extension of our previously reported series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. Addition of a second methyl group to the tether provided analogs that show increased potency in binding as well as cell-proliferation assays and, more importantly, are stable toward microsomes. We wish to disclose the discovery of a macrocycle which showed impressive biomarker activity 24-h post dosing and which demonstrated prolonged exposure in tumors.

Discovery of a macrocyclic o-aminobenzamide Hsp90 inhibitor with heterocyclic tether that shows extended biomarker activity and in vivo efficacy in a mouse xenograft model.

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. In continuation of our research, heterocycle-containing tethers were explored with the intent to further improve potency and minimize hERG liabilities. This effort culminated in the discovery of compound 10, which efficiently suppressed proliferation of HCT116 and U87 cells.

Macrocyclic lactams as potent Hsp90 inhibitors with excellent tumor exposure and extended biomarker activity.

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. In continuation of our research in this area, macrocyclic amides and lactams were explored to reduce the risk of hERG liabilities. This effort culminated in the discovery of compound 38, which showed a favorable in vitro profile, and efficiently suppressed proliferation of several relevant cell lines.

Antitumor efficacy of PKI-587, a highly potent dual PI3K/mTOR kinase inhibitor.

Purpose: The aim of this study was to show preclinical efficacy and clinical development potential of PKI-587, a dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor.

Experimental Design: In vitro class 1 PI3K enzyme and human tumor cell growth inhibition assays and in vivo five tumor xenograft models were used to show efficacy.

Results: In vitro, PKI-587 potently inhibited class I PI3Ks (IC(50) vs.