Tuning the Hydrolytic Stability of Next Generation Maleimide Cross-Linkers Enables Access to Albumin-Antibody Fragment Conjugates and tri-scFvs Nafsika Forte, Maria Livanos, Enrique Miranda, Maurício Morais, Xiaoping Yang, et al. Bioconjugate Chemistry, 2018 We describe investigations to expand the scope of next generation maleimide cross-linkers for the construction of homogeneous protein-protein conjugates. Diiodomaleimides are shown to offer the ideal properties of rapid bioconjugation with reduced hydrolysis, allowing the cross-linking of even sterically hindered systems. The optimized linkers are exploited to link human serum albumin to antibody fragments (Fab or scFv) as a prospective half-life extension platform, with retention of antigen binding and robust serum stability. Finally, a triprotein conjugate is formed, by linking scFv antibody fragments targeting carcinoembryonic antigen. This tri-scFv is shown to infer a combination of greater antigen avidity and increased in vivo half-life, representing a promising platform for antibody therapeutic development.
Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors Frederick Arce Vargas, Andrew J.S. Furness, Isabelle Solomon, Kroopa Joshi, Leila Mekkaoui, et al. Immunity, 2017 &NA; CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti‐CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor‐infiltrating Treg cells in mice and humans. While existing anti‐CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (Fc&ggr;R) IIb at the tumor site prevented intra‐tumoral Treg cell depletion, which may underlie the lack of anti‐tumor activity previously observed in pre‐clinical models. Use of an anti‐CD25 antibody with enhanced binding to activating Fc&ggr;Rs led to effective depletion of tumor‐infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti‐programmed cell death protein‐1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune‐oncology. Graphical Abstract Figure. No caption available. HighlightsCD25 expression is largely restricted to Treg cells in mice and humansFc&ggr;RIIb inhibits anti‐CD25‐mediated depletion of intra‐tumoral Treg cellsFc‐optimized anti‐CD25 efficiently depletes intra‐tumoral Treg cellsAnti‐CD25 synergizes with PD‐1 blockade to reject established tumors &NA; Anti‐CD25 antibodies have displayed only modest therapeutic activity against established tumors. Arce Vargas et al. demonstrate that existing anti‐CD25 antibodies fail to deplete intra‐tumoral Treg cells due to upregulation of Fc&ggr;RIIb within tumors. Fc‐optimized anti‐CD25 mediates effective depletion of tumor‐infiltrating Treg cells and synergizes with PD‐1 blockade to promote tumor eradication.
A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy Antoine Maruani, Mark E.B. Smith, Enrique Miranda, Kerry A. Chester, Vijay Chudasama, et al. Nature Communications, 2015 Although recent methods for the engineering of antibody–drug conjugates (ADCs) have gone some way to addressing the challenging issues of ADC construction, significant hurdles still remain. There is clear demand for the construction of novel ADC platforms that offer greater stability, homogeneity and flexibility. Here we describe a significant step towards a platform for next-generation antibody-based therapeutics by providing constructs that combine site-specific modification, exceptional versatility and high stability, with retention of antibody binding and structure post-modification. The relevance of the work in a biological context is also demonstrated in a cytotoxicity assay and a cell internalization study with HER2-positive and -negative breast cancer cell lines.
Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging Robert Goldstein, Jane Sosabowski, Maria Livanos, Julius Leyton, Kim Vigor, et al. European Journal of Nuclear Medicine and Molecular Imaging, 2015 PurposeHuman epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.MethodsG3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with 125I, or with 111In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts.ResultsFor both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from 111In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from 125I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, 111In-labelled and 125I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with 111In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours.ConclusionRadiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.
Homogeneous bispecifics by disulfide bridging Elizabeth A. Hull, Maria Livanos, Enrique Miranda, Mark E. B. Smith, Kerry A. Chester, et al. Bioconjugate Chemistry, 2014 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.
Generation and Characterization of a Diabody Targeting the αvβ6 Integrin Heide Kogelberg, Enrique Miranda, Jerome Burnet, David Ellison, Berend Tolner, et al. Plos One, 2013 The αvβ6 integrin is up-regulated in cancer and wound healing but it is not generally expressed in healthy adult tissue. There is increasing evidence that it has a role in cancer progression and will be a useful target for antibody-directed cancer therapies. We report a novel recombinant diabody antibody fragment that targets specifically αvβ6 and blocks its function. The diabody was engineered with a C-terminal hexahistidine tag (His tag), expressed in Pichia pastoris and purified by IMAC. Surface plasmon resonance (SPR) analysis of the purified diabody showed affinity in the nanomolar range. Pre-treatment of αvβ6-expressing cells with the diabody resulted in a reduction of cell migration and adhesion to LAP, demonstrating biological function-blocking activity. After radio-labeling, using the His-tag for site-specific attachment of 99mTc, the diabody retained affinity and targeted specifically to αvβ6-expressing tumors in mice bearing isogenic αvβ6 +/− xenografts. Furthermore, the diabody was specifically internalized into αvβ6-expressing cells, indicating warhead targeting potential. Our results indicate that the new αvβ6 diabody has a range of potential applications in imaging, function blocking or targeted delivery/internalization of therapeutic agents.
Acid-cleavable thiomaleamic acid linker for homogeneous antibody–drug conjugation Lourdes Castañeda, Antoine Maruani, Felix F. Schumacher, Enrique Miranda, Vijay Chudasama, et al. Chemical Communications, 2013 Homogeneous antibody–drug conjugation is affected using a novel thiomaleamic acid linker that is stable at physiological temperature and pH, but quantitatively cleaves at lysosomal pH to release the drug payload.
Adenovirus-Mediated Sensitization to the Cytotoxic Drugs Docetaxel and Mitoxantrone Is Dependent on Regulatory Domains in the E1ACR1 Gene-Region Enrique Miranda, Hector Maya Pineda, Daniel Öberg, Gioia Cherubini, Zita Garate, et al. Plos One, 2012 Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922–947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922–947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics.
