Antibody Conjugates via Disulfide Bridging: Towards therapeutic and diagnostic applications

Antibodies play a prominent role in chemical and biological research and the largest application of chemical bioconjugation reagents is in the production of antibody conjugates. These conjugates provide a means of highly sensitive detection, for example in enzyme-linked immunosorbent assay (ELISA) systems. In therapeutics, such conjugates have enabled the development of bispecifics and antibody-enzyme directed prodrug therapy (ADEPT). Long-established chemical modification techniques for the conjugation of antibodies yield highly heterogeneous products. This heterogeneity is far from optimal and for therapeutic use antibody conjugates must be of a defined composition. Recently the site-specific introduction of chemical linkers has been reported through unnatural amino acid insertion. In this approach however, each protein must undergo successful mutation and expression prior to conjugation. To avoid this, an ideal site-directed conjugation technique would use residues natural to the protein. A new class of chemical bioconjugation reagents, the 3,4-substituted maleimides, allow the selective modification and bridging of naturally occurring protein disulfide bonds. In this thesis, the generation of homogeneous antibody-protein conjugates using 3,4-substituted maleimide based cross-linkers is investigated, with a focus on producing conjugates for ADEPT and bispecific therapeutics. A range of direct and indirect chemical cross-linking strategies via disulfide bridging are explored and the consequences of each approach examined. Ultimately, a new chemical platform to generate site-specific, homogeneous, antibody-antibody conjugates by targeting and bridging disulfide bonds was developed. A bispecific antibody construct was produced in good yield using a readily synthesised bis-dibromomaleimide cross-linker. Binding activity of antibodies was maintained, and in vitro binding of target antigens was observed. This technology is demonstrated through linking scFv and Fab antibody fragments, showing its potential for the construction of a diverse range of bispecifics. Finally, the ability of 3,4-substituted maleimide based reagents to functionalise antibodies for diagnostic applications is investigated. A strategy for the modification of a scFv-Fc construct with commercially available fluorophores is achieved and a synthetic route towards reagents suitable for immuno-PET applications determined

D-Brane Wess-Zumino Terms and U-Duality

We construct gauge-invariant and U-duality covariant expressions for Wess-Zumino terms corresponding to general Dp-branes (for any p<D) in arbitrary 2<D<11 dimensions. A distinguishing feature of these Wess-Zumino terms is that they contain twice as many scalars as the 10-D compactified dimensions, in line with doubled geometry. We find that for D<10 the charges of the higher-dimensional branes can all be expressed as products of the 0-brane charges, which include the D0-brane and the NS-NS 0-brane charges. We give the general expressions for these charges and show how they determine the non-trivial conjugacy class to which some of the higher-dimensional D-branes belong.Comment: 42 pages. Typos corrected, an error in table 6 corrected, comments in the conclusions adde

Counting supersymmetric branes

Maximal supergravity solutions are revisited and classified, with particular emphasis on objects of co-dimension at most two. This class of solutions includes branes whose tension scales with g_s^{-\sigma} for \sigma>2. We present a group theory derivation of the counting of these objects based on the corresponding tensor hierarchies derived from E11 and discrete T- and U-duality transformations. This provides a rationale for the wrapping rules that were recently discussed for \sigma<4 in the literature and extends them. Explicit supergravity solutions that give rise to co-dimension two branes are constructed and analysed.Comment: 1+33 pages. To the memory of Laurent Houart. v2: Published version with added reference

On "New Massive" 4D Gravity

We construct a four-dimensional (4D) gauge theory that propagates, unitarily, the five polarization modes of a massive spin-2 particle. These modes are described by a "dual" graviton gauge potential and the Lagrangian is 4th-order in derivatives. As the construction mimics that of 3D "new massive gravity", we call this 4D model (linearized) "new massive dual gravity". We analyse its massless limit, and discuss similarities to the Eddington-Schroedinger model.Comment: 17 pages, v2 : version published in JHE

Sigma models with off-shell N=(4,4) supersymmetry and noncommuting complex structures

We describe the conditions for extra supersymmetry in N=(2,2) supersymmetric nonlinear sigma models written in terms of semichiral superfields. We find that some of these models have additional off-shell supersymmetry. The (4,4) supersymmetry introduces geometrical structures on the target-space which are conveniently described in terms of Yano f-structures and Magri-Morosi concomitants. On-shell, we relate the new structures to the known bi-hypercomplex structures.Comment: 20 pages; v2: significant corrections, clarifications, and reorganization; v3: discussion of supersymmetry vs twisted supersymmetry added, relevant signs corrected

The effect of aspirin and eicosapentaenoic acid on urinary biomarkers of prostaglandin E2 synthesis and platelet activation in participants of the seAFOod polyp prevention trial

Urinary prostaglandin (PG) E metabolite (PGE-M) and 11-dehydro (d)-thromboxane (TX) B2 are biomarkers of cyclooxygenase-dependent prostanoid synthesis. We investigated (1) the effect of aspirin 300 mg daily and eicosapentaenoic acid (EPA) 2000 mg daily, alone and in combination, on urinary biomarker levels and, (2) whether urinary biomarker levels predicted colorectal polyp risk, during participation in the seAFOod polyp prevention trial. Urinary PGE-M and 11-d-TXB2 were measured by liquid chromatography-tandem mass spectrometry. The relationship between urinary biomarker levels and colorectal polyp outcomes was investigated using negative binomial (polyp number) and logistic (% with one or more polyps) regression models. Despite wide temporal variability in PGE-M and 11-d-TXB2 levels within individuals, both aspirin and, to a lesser extent, EPA decreased levels of both biomarkers (74% [P ≤.001] and 8% [P ≤.05] reduction in median 11-d-TXB2 values, respectively). In the placebo group, a high (quartile [Q] 2-4) baseline 11-d-TXB2 level predicted increased polyp number (incidence rate ratio [IRR] [95% CI] 2.26 [1.11,4.58]) and risk (odds ratio [95% CI] 3.56 [1.09,11.63]). A low (Q1) on-treatment 11-d-TXB2 level predicted reduced colorectal polyp number compared to placebo (IRR 0.34 [0.12,0.93] for combination aspirin and EPA treatment) compared to high on-treatment 11-d-TXB2 values (0.61 [0.34,1.11]). Aspirin and EPA both inhibit PGE-M and 11-d-TXB2 synthesis in keeping with shared in vivo cyclooxygenase inhibition. Colorectal polyp risk and treatment response prediction by 11-d-TXB2 is consistent with a role for platelet activation during early colorectal carcinogenesis. The use of urinary 11-d-TXB2 measurement for a precision approach to colorectal cancer risk prediction and chemoprevention requires prospective evaluation

Plasma and rectal mucosal oxylipin levels during aspirin and eicosapentaenoic acid treatment in the seAFOod polyp prevention trial

BACKGROUND: Aspirin and eicosapentaenoic acid (EPA) have colorectal polyp prevention activity, alone and in combination. This study measured levels of plasma and rectal mucosal oxylipins in participants of the seAFOod 2 × 2 factorial, randomised, placebo-controlled trial, who received aspirin 300 mg daily and EPA 2000 mg free fatty acid, alone and in combination, for 12 months. METHODS: Resolvin (Rv) E1, 15-epi-lipoxin (LX) A4 and respective precursors 18-HEPE and 15-HETE (with chiral separation) were measured by ultra-high performance liquid chromatography-tandem mass spectrometry in plasma taken at baseline, 6 months and 12 months, as well as rectal mucosa obtained at trial exit colonoscopy at 12 months, in 401 trial participants. RESULTS: Despite detection of S- and R- enantiomers of 18-HEPE and 15-HETE in ng/ml concentrations, RvE1 or 15‑epi-LXA4 were not detected above a limit of detection of 20 pg/ml in plasma or rectal mucosa, even in individuals randomised to both aspirin and EPA. We have confirmed in a large clinical trial cohort that prolonged (12 months) treatment with EPA is associated with increased plasma 18-HEPE concentrations (median [inter-quartile range] total 18-HEPE 0.51 [0.21-1.95] ng/ml at baseline versus 0.95 [0.46-4.06] ng/ml at 6 months [P<0.0001] in those randomised to EPA alone), which correlate strongly with respective rectal mucosal 18-HEPE levels (r = 0.82; P<0.001), but which do not predict polyp prevention efficacy by EPA or aspirin. CONCLUSION: Analysis of seAFOod trial plasma and rectal mucosal samples has not provided evidence of synthesis of the EPA-derived specialised pro-resolving mediator RvE1 or aspirin-trigged lipoxin 15‑epi-LXA4. We cannot rule out degradation of individual oxylipins during sample collection and storage but readily measurable precursor oxylipins argues against widespread degradation

Homogeneous Bispecifics by Disulfide Bridging

We report on a chemical platform to generate site-specific, homogeneous, antibody-antibody conjugates by targeting and bridging disulfide bonds. A bispecific antibody construct was produced in good yield through simple reduction and bridging of antibody fragment disulfide bonds, using a readily synthesized bis-dibromomaleimide cross-linker. Binding activity of antibodies was maintained, and in vitro binding of target antigens was observed. This technology is demonstrated through linking scFv and Fab antibody fragments, showing its potential for the construction of a diverse range of bispecifics