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.

Molecular mechanisms underlying neurodevelopmental disorders, ADHD and autism.

Neurodevelopmental disorders such as attention deficit hyperactivity disorder and autism represent a significant economic burden, which justify vigorous research to uncover its genetics and developmental clinics for a diagnostic workup. The urgency of addressing attention deficit hyperactivity disorder comorbidities is seen in the chilling fact that attention deficit hyperactivity disorder (ADHD), mood disorders, substance use disorders and obesity each increase the risk for mortality. However, data about comorbidity is mainly descriptive, with mechanistic studies limited to genetic epidemiological studies that document shared genetic risk factors among these conditions. Autism and intellectual disability affects 1.5 to 2% of the population in Western countries with many individuals displaying social-emotional agnosia and having difficulty in forming attachments and relationships. Underlying mechanisms include: (i) dysfunctions of neuronal miRNAs; (ii) deletions in the chromosome 21, subtelomeric deletions, duplications and a maternally inherited duplication of the chromosomal region 15q11-q13; (iii) microdeletions in on the long (q) arm of the chromosome in a region designated q21.1 increases the risk of delayed development, intellectual disability, physical abnormalities, and neurological and psychiatric problems associated with autism, schizophrenia, and epilepsy and weak muscle tone (hypotonia); (iv) interstitial duplications encompassing 16p13.11.

Age-related hypoxia in CNS pathology.

Although neuropathological conditions differ in the etiology of the inflammatory response, cellular and molecular mechanisms of neuroinflammation are probably similar in aging, hypertension, depression and cognitive impairment. Moreover, a number of common risk factors such as obesity, hypertension, diabetes and atherosclerosis are increasingly understood to act as "silent contributors" to neuroinflammation and can underlie the development of disorders such as cerebral small vessel disease (cSVD) and subsequent dementia. On the other hand, acute neuroinflammation, such as in response to traumatic or cerebral ischemia, aggravates the acute damage and can lead to a number of pathological such as depression, post-stroke dementia and potentially neurodegeneration. All of those sequelae impair recovery and most of them provide the ground for further cerebrovascular events and a vicious cycle develops. Therefore, understanding the mechanisms associated with vascular dementia, stroke and related complications is of paramount importance in improving current preventive and therapeutic interventions. Likewise, understanding of molecular factors and pathways associated with neuroinflammation will eventually enable the discovery and implementation of new diagnostic and therapeutic strategies indicated in a wide range of neurological conditions.

Up-regulation of serotonin receptor 2B mRNA and protein in the peri-infarcted area of aged rats and stroke patients.

Despite the fact that a high proportion of elderly stroke patients develop mood disorders, the mechanisms underlying late-onset neuropsychiatric and neurocognitive symptoms have so far received little attention in the field of neurobiology. In rodents, aged animals display depressive symptoms following stroke, whereas young animals recover fairly well. This finding has prompted us to investigate the expression of serotonin receptors 2A and 2B, which are directly linked to depression, in the brains of aged and young rats following stroke. Although the development of the infarct was more rapid in aged rats in the first 3 days after stroke, by day 14 the cortical infarcts were similar in size in both age groups i.e. 45% of total cortical volume in young rats and 55.7% in aged rats. We also found that the expression of serotonin receptor type B mRNA was markedly increased in the perilesional area of aged rats as compared to the younger counterparts. Furthermore, histologically, HTR2B protein expression in degenerating neurons was closely associated with activated microglia both in aged rats and human subjects. Treatment with fluoxetine attenuated the expression of Htr2B mRNA, stimulated post-stroke neurogenesis in the subventricular zone and was associated with an improved anhedonic behavior and an increased activity in the forced swim test in aged animals. We hypothesize that HTR2B expression in the infarcted territory may render degenerating neurons susceptible to attack by activated microglia and thus aggravate the consequences of stroke.

Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal.

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA.

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.

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.

A new reagent for stable thiol-specific conjugation.

Many clinically used protein therapeutics are modified to increase their efficacy. Example modifications include the conjugation of cytotoxic drugs to monoclonal antibodies or poly(ethylene glycol) (PEG) to proteins and peptides. Monothiol-specific conjugation can be efficient and is often accomplished using maleimide-based reagents. However, maleimide derived conjugates are known to be susceptible to exchange reactions with endogenous proteins. To address this limitation in stability, we have developed PEG-mono-sulfone 3, which is a latently reactive, monothiol selective conjugation reagent. Comparative reactions with PEG-maleimide and other common thiol-selective PEGylation reagents including vinyl sulfone, acrylate, and halo-acetamides show that PEG-mono-sulfone 3 undergoes more efficient conjugation under mild reaction conditions. Due to the latent reactivity of PEG-mono-sulfone 3, its reactivity can be tailored and, once conjugated, the electron-withdrawing ketone is easily reduced under mild conditions to prevent undesirable deconjugation and exchange reactions from occurring. We describe a comparative stability study demonstrating a PEG-maleimide conjugate to be more labile to deconjugation than the corresponding conjugate obtained using PEG-mono-sulfone 3.

Flexible dosing with paliperidone ER in the treatment of patients with acutely exacerbated schizophrenia: results from a single-arm, open-label study.

OBJECTIVE: To extend findings from fixed-dose, double-blind, placebo-controlled clinical trials in selected patient populations by using flexibly-dosed oral paliperidone extended-release (ER) in a more naturalistic setting. METHODS: Adults hospitalized with an acute exacerbation of schizophrenia were prospectively treated with open-label flexibly-dosed paliperidone ER 3-12 mg/day for 6 weeks. RESULTS: Overall, 294 patients were treated. The primary endpoint, defined as ≥30% improvement in Positive and Negative Syndrome Scale total scores from baseline to endpoint, was achieved by 66.3% of patients. The percentage of patients rated as at least 'markedly ill' in Clinical Global Impression of Severity scale decreased from baseline (74.1%) to endpoint (20.0%). Patient functioning, assessed by the Personal and Social Performance scale, improved significantly from 50.0 ± 14.3 at baseline to 63.6 ± 14.9 at endpoint (p < 0.0001). Concomitant benzodiazepines were newly initiated in 191 patients (65.0%), and new concomitant medications other than benzodiazepines were started after baseline for 133 patients (45.2%), most frequently paracetamol, zolpidem, and zopiclone. No unexpected adverse events were identified. CONCLUSIONS: These data support findings in more selected patient populations treated with fixed-dose paliperidone ER. Flexibly-dosed paliperidone ER administered in a naturalistic hospital setting to a more representative patient population experiencing an acute episode of schizophrenia, was associated with clinically meaningful treatment response. Strength of conclusions is limited by the open-label design and lack of a comparator group. Furthermore, some of the improvements observed may in part be associated with increased attention provided to patients and concomitant use of psychotropic medications, such as benzodiazepines, during this study.

Site-specific PEGylation at histidine tags.

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.

Receptors for auxin: will it all end in TIRs?

The role of TIR1 in ubiquitination and regulated degradation of Aux/IAA transcription factors has been recognized for some years, but recent results have shown that TIR1 itself is also the binding site for auxin. The affinity and specificity of TIR1 match properties anticipated of a nuclear auxin receptor and we look at how they compare with the properties of ABP1. We also consider the mechanism of auxin action via TIR1 and the likelihood that the TIR1 family could account for all auxin responses. It seems likely that the TIR1 system can account for a large part of the repertoire of auxin-mediated responses, but maybe not all.