A homogeneous high-DAR antibody-drug conjugate platform combining THIOMAB antibodies and XTEN polypeptides.

The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.

A Novel Depurination Methodology to Assess DNA Alkylation of Chloro-Bis-Seco-Cyclopropylbenzoindoles Allowed for Comparison of Minor-Groove Reactivity.

Duocarmycins [including cyclopropyl pyrroloindole (CPI) or cyclopropyl benzoindole (CBI)] are a class of DNA minor-groove alkylators and seco-CPI/CBIs are synthetic pro-forms that can spirocyclize to CPI/CBI. Bis-CPI/CBIs are potential drug candidates because of their enhanced cytotoxicity from DNA crosslinking, but it is difficult to analyze them for structure-activity correlation because of their DNA reactivity. To study their DNA alkylation, neutral thermal hydrolysis has been frequently applied to process depurination. However, unwanted side reactions under this condition have been reported, which could lead to poor correlation of DNA alkylation data with efficacy results, especially for bis-CPI/CBIs. In this study, an acidic depurination method was developed and applied for analysis of DNA alkylation and shown to be an easier and milder method than the traditional neutral thermal hydrolysis. DNA alkylation and stability of three bis-seco-CBIs were characterized in comparison with two mono-seco-CPIs. The results suggested that: 1) The acidic depurination method was capable of capturing a more representative population, sometimes a different population, of DNA adducts as they existed on DNA compared with the heat depurination method. 2) Di-adenine adducts were captured as expected for the CBI dimers, although the major type of adduct was still mono-adenine adducts. 3) The rate of DNA alkylation, DNA adduct profile, and relative amounts of di-adduct versus mono-adduct were significantly affected by the size, and possibly lipophilicity, of the nonalkylating part of the molecules. 4) Spirocyclization and amide hydrolysis represented two major pathways of degradation. Overall, by applying acidic depurination analyses, this study has illustrated DNA adduct characteristics of novel bis-seco-CBIs with dominating mono-alkylation and provides an alternative method for evaluating DNA minor-groove alkylators. These findings provide an effective analytical tool to evaluate DNA alkylators and to study the DNA alkylation that is a disposition mechanism of these compounds.

Developments in therapy with monoclonal antibodies and related proteins.

Monoclonal antibody therapeutics have been approved for over 30 targets and diseases, most commonly cancer. Antibodies have become the new backbone of the pharmaceutical industry, which previously relied on small molecules. Compared with small molecules, monoclonal antibodies (mAbs) have exquisite target selectivity and hence less toxicity as a result of binding other targets. The clinical value of both mAbs and ligand traps has been proven. New applications of mAbs are being tested and mAbs have now been designed to target two (bi-specific, eg TNF-α and IL-17) or more targets simultaneously, augmenting their therapeutic potential. Because of space limitations and the wide ranging scope of this review there are regrettably, but inevitably, omissions and missing citations. We have chosen to highlight the first successes in inflammatory diseases and cancer, but a broader overview of approved mAbs and related molecules can be found in Table 1.

The cryptophycins as potent payloads for antibody drug conjugates.

The cryptophycins are a potent class of cytotoxic agents that were evaluated as antibody drug conjugate (ADC) payloads. Free cryptophycin analog 1 displayed cell activity an order of magnitude more potent than approved ADC payloads MMAE and DM1. This potency increase was also reflected in the activity of the cryptophycin ADCs, attached via a either cleavable or non-cleavable linker.

Different mechanisms for resistance to trastuzumab versus lapatinib in HER2-positive breast cancers--role of estrogen receptor and HER2 reactivation.

INTRODUCTION: The human epidermal growth factor receptor 2 (HER2)-targeted therapies trastuzumab (T) and lapatinib (L) show high efficacy in patients with HER2-positive breast cancer, but resistance is prevalent. Here we investigate resistance mechanisms to each drug alone, or to their combination using a large panel of HER2-positive cell lines made resistant to these drugs. METHODS: Response to L + T treatment was characterized in a panel of 13 HER2-positive cell lines to identify lines that were de novo resistant. Acquired resistant lines were then established by long-term exposure to increasing drug concentrations. Levels and activity of HER2 and estrogen receptor (ER) pathways were determined by qRT-PCR, immunohistochemistry, and immunoblotting assays. Cell growth, proliferation, and apoptosis in parental cells and resistant derivatives were assessed in response to inhibition of HER or ER pathways, either pharmacologically (L, T, L + T, or fulvestrant) or by using siRNAs. Efficacy of combined endocrine and anti-HER2 therapies was studied in vivo using UACC-812 xenografts. RESULTS: ER or its downstream products increased in four out of the five ER+/HER2+ lines, and was evident in one of the two intrinsically resistant lines. In UACC-812 and BT474 parental and resistant derivatives, HER2 inhibition by T reactivated HER network activity to promote resistance. T-resistant lines remained sensitive to HER2 inhibition by either L or HER2 siRNA. With more complete HER2 blockade, resistance to L-containing regimens required the activation of a redundant survival pathway, ER, which was up-regulated and promoted survival via various Bcl2 family members. These L- and L + T-resistant lines were responsive to fulvestrant and to ER siRNA. However, after prolonged treatment with L, but not L + T, BT474 cells switched from depending on ER as a survival pathway, to relying again on the HER network (increased HER2, HER3, and receptor ligands) to overcome L's effects. The combination of endocrine and L + T HER2-targeted therapies achieved complete tumor regression and prevented development of resistance in UACC-812 xenografts. CONCLUSIONS: Combined L + T treatment provides a more complete and stable inhibition of the HER network. With sustained HER2 inhibition, ER functions as a key escape/survival pathway in ER-positive/HER2-positive cells. Complete blockade of the HER network, together with ER inhibition, may provide optimal therapy in selected patients.

Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer.

Trastuzumab (Herceptin(®)) is currently used as a treatment for patients whose breast tumors overexpress HER2/ErbB2. Trastuzumab-DM1 (T-DM1, trastuzumab emtansine) is designed to combine the clinical benefits of trastuzumab with a potent microtubule-disrupting drug, DM1 (a maytansine derivative). Currently T-DM1 is being tested in multiple clinical trials. The mechanisms of action for trastuzumab include inhibition of PI3K/AKT signaling pathway, inhibition of HER-2 shedding and Fcγ receptor mediated engagement of immune cells, which may result in antibody-dependent cellular cytotoxicity (ADCC). Here we report that T-DM1 retains the mechanisms of action of unconjugated trastuzumab and is active against lapatinib resistant cell lines and tumors.

Angiogenesis-independent endothelial protection of liver: role of VEGFR-1.

The vascular endothelium was once thought to function primarily in nutrient and oxygen delivery, but recent evidence suggests that it may play a broader role in tissue homeostasis. To explore the role of sinusoidal endothelial cells (LSECs) in the adult liver, we studied the effects of vascular endothelial growth factor (VEGF) receptor activation on mouse hepatocyte growth. Delivery of VEGF-A increased liver mass in mice but did not stimulate growth of hepatocytes in vitro, unless LSECs were also present in the culture. Hepatocyte growth factor (HGF) was identified as one of the LSEC-derived paracrine mediators promoting hepatocyte growth. Selective activation of VEGF receptor-1 (VEGFR-1) stimulated hepatocyte but not endothelial proliferation in vivo and reduced liver damage in mice exposed to a hepatotoxin. Thus, VEGFR-1 agonists may have therapeutic potential for preservation of organ function in certain liver disorders.

Inhibition of ligand-mediated HER2 activation in androgen-independent prostate cancer.

Hormone-independent tumor growth and metastasis are associated with increased mortality in human prostate cancer. In this study, we evaluate a potential role for ligand-mediated activation of HER2 receptor tyrosine kinase in androgen-independent prostate cancers. HER2, HER3, and epidermal growth factor receptor were detected in the androgen-independent cell line 22Rv1. Heregulin stimulation results in receptor phosphorylation and cell proliferation that is inhibited by increasing concentrations of anti-HER2 recombinant humanized monoclonal antibody (rhuMAb) 2C4. Furthermore, inhibition of tumor growth was observed in xenografts derived from 22Rv1 cells when treated with rhuMAb 2C4 in a dose-dependent manner. These studies provide a framework, both in vitro and in vivo, to examine the molecular mechanisms of ligand-driven HER2 activation in androgen-independent tumorigenesis.