Targeted delivery of alcohol-containing payloads with antibody-drug conjugates.

We herein describe the cell-specific release of alcohol-containing payloads via a sulfatase-sensitive linker in antibody-drug conjugates (ADCs). The linker shows efficient sulfatase-mediated release and high stability in human and mouse plasma. In vitro evaluation demonstrates potent antigen dependent toxicity towards breast cancer cell lines.

Incorporation of Hydrophilic Macrocycles Into Drug-Linker Reagents Produces Antibody-Drug Conjugates With Enhanced in vivo Performance.

Antibody-drug conjugates (ADCs) have begun to fulfil their promise as targeted cancer therapeutics with ten clinical approvals to date. As the field matures, much attention has focused upon the key factors required to produce safe and efficacious ADCs. Recently the role that linker-payload reagent design has on the properties of ADCs has been highlighted as an important consideration for developers. We have investigated the effect of incorporating hydrophilic macrocycles into reagent structures on the in vitro and in vivo behavior of ADCs. Bis-sulfone based disulfide rebridging reagents bearing Val-Cit-PABC-MMAE linker-payloads were synthesized with a panel of cyclodextrins and crown ethers integrated into their structures via a glutamic acid branching point. Brentuximab was selected as a model antibody and ten ADCs with a drug-to-antibody ratio (DAR) of 4 were prepared for biological evaluation. In vitro, the ADCs prepared showed broadly similar potency (range: 16-34 pM) and were comparable to Adcetris® (16 pM). In vivo, the cyclodextrin containing ADCs showed greater efficacy than Adcetris® and the most efficacious variant (incorporating a 3'-amino-α-cyclodextrin component) matched a 24-unit poly(ethylene glycol) (PEG) containing comparator. The ADCs bearing crown ethers also displayed enhanced in vivo efficacy compared to Adcetris®, the most active variant (containing a 1-aza-42-crown-14 macrocycle) was superior to an analogous ADC with a larger 24-unit PEG chain. In summary, we have demonstrated that hydrophilic macrocycles can be effectively incorporated into ADC reagent design and offer the potential for enhanced alternatives to established drug-linker architectures.

Modulation of drug-linker design to enhance in vivo potency of homogeneous antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a promising class of anticancer agents which have undergone substantial development over the past decade and are now achieving clinical success. The development of novel site-specific conjugation technologies enables the systematic study of architectural features within the antibody conjugated drug linker that may affect overall therapeutic indices. Here we describe the results of a systematic study investigating the impact of drug-linker design on the in vivo properties of a series of homogeneous ADCs with a conserved site of conjugation, a monodisperse drug loading, a lysosomal release functionality and monomethyl auristatin E as a cytotoxic payload. The ADCs, which differed only in the relative position of certain drug-linker elements within the reagent, were first evaluated in vitro using anti-proliferation assays and in vivo using mouse pharmacokinetics (PK). Regardless of the position of a discrete polymer unit, the ADCs showed comparable in vitro potencies, but the in vivo PK properties varied widely. The best performing drug-linker design was further used to prepare ADCs with different drug loadings of 4, 6 and 8 drugs per antibody and compared to Adcetris® in a Karpas-299 mouse xenograft model. The most efficacious ADC showed complete tumor regression and 10/10 tumor free survivors at a single 0.5mg/kg dose. This study revealed drug-linker design as a critical parameter in ADC development, with the potential to enhance ADC in vivo potency for producing more efficacious ADCs.

In Vitro and In Vivo Evaluation of Cysteine Rebridged Trastuzumab-MMAE Antibody Drug Conjugates with Defined Drug-to-Antibody Ratios.

The conjugation of monomethyl auristatin E (MMAE) to trastuzumab using a reduction bis-alkylation approach that is capable of rebridging reduced (native) antibody interchain disulfide bonds has been previously shown to produce a homogeneous and stable conjugate with a drug-to-antibody ratio (DAR) of 4 as the major product. Here, we further investigate the potency of the DAR 4 conjugates prepared by bis-alkylation by comparing to lower drug loaded variants to maleimide linker based conjugates possessing typical mixed DAR profiles. Serum stability, HER2 receptor binding, internalization, in vitro potency, and in vivo efficacy were all evaluated. Greater stability compared with maleimide conjugation was observed with no significant decrease in receptor/FcRn binding. A clear dose-response was obtained based on drug loading (DAR) with the DAR 4 conjugate showing the highest potency in vitro and a much higher efficacy in vivo compared with the lower DAR conjugates. Finally, the DAR 4 conjugate demonstrated superior efficacy compared to trastuzumab-DM1 (T-DM1, Kadcyla), as evaluated in a low HER2 expressing JIMT-1 xenograft model.

A role for whey acidic protein four-disulfide-core 12 (WFDC12) in the regulation of the inflammatory response in the lung.

INTRODUCTION: Secretory leucocyte protease inhibitor and elafin are members of the whey acidic protein (WAP), or WAP four disulfide-core (WFDC), family of proteins and have multiple contributions to innate defence including inhibition of neutrophil serine proteases and inhibition of the inflammatory response to lipopolysaccharide (LPS). This study aimed to explore potential activities of WFDC12, a previously uncharacterised WFDC protein expressed in the lung. METHODS: Recombinant expression and purification of WFDC12 were optimised in Escherichia coli. Antiprotease, antibacterial and immunomodulatory activities of recombinant WFDC12 were evaluated and levels of endogenous WFDC12 protein were characterised by immunostaining and ELISA. RESULTS: Recombinant WFDC12 inhibited cathepsin G, but not elastase or proteinase-3 activity. Monocytic cells pretreated with recombinant WFDC12 before LPS stimulation produced significantly lower levels of the pro-inflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared with cells stimulated with LPS alone. Recombinant WFDC12 became conjugated to fibronectin in a transglutaminase-mediated reaction and retained antiprotease activity. In vivo WFDC12 expression was confirmed by immunostaining of human lung tissue sections. WFDC12 levels in human bronchoalveolar lavage fluid from healthy and lung-injured patients were quantitatively compared, showing WFDC12 to be elevated in both patients with acute respiratory distress syndrome and healthy subjects treated with LPS, relative to healthy controls. CONCLUSIONS: Together, these results suggest a role for this lesser known WFDC protein in the regulation of lung inflammation.

Bridging disulfides for stable and defined antibody drug conjugates.

To improve both the homogeneity and the stability of ADCs, we have developed site-specific drug-conjugating reagents that covalently rebridge reduced disulfide bonds. The new reagents comprise a drug, a linker, and a bis-reactive conjugating moiety that is capable of undergoing reaction with both sulfur atoms derived from a reduced disulfide bond in antibodies and antibody fragments. A disulfide rebridging reagent comprising monomethyl auristatin E (MMAE) was prepared and conjugated to trastuzumab (TRA). A 78% conversion of antibody to ADC with a drug to antibody ratio (DAR) of 4 was achieved with no unconjugated antibody remaining. The MMAE rebridging reagent was also conjugated to the interchain disulfide of a Fab derived from proteolytic digestion of TRA, to give a homogeneous single drug conjugated product. The resulting conjugates retained antigen-binding, were stable in serum, and demonstrated potent and antigen-selective cell killing in in vitro and in vivo cancer models. Disulfide rebridging conjugation is a general approach to prepare stable ADCs, which does not require the antibody to be recombinantly re-engineered for site-specific conjugation.

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments.