BackgroundThe secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the potential to avoid this overload by matching expression levels to the host secretion capacity.ResultsHere we report the application of the RiboTite gene expression control system to achieve this by finely controlling cellular expression levels. The level of control afforded by this system allows cell viability to be maintained, permitting production of high-quality, active product with enhanced volumetric titres.ConclusionsThe methods and systems reported expand the tools available for the production of disulfide-bond containing proteins, including antibody fragments, in bacterial hosts.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1047-z) contains supplementary material, which is available to authorized users.

Background Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. Methods CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. Results CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. Conclusions CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.

The recent advances over the past 5 years in the utilisation of fluorescent proteins in microbial biotechnology applications, including recombinant protein production, food processing, and environmental biotechnology, are reviewed. We highlight possible areas where fluorescent proteins currently used in other bioscience disciplines could be adapted for use in biotechnological applications and also outline novel uses for recently developed fluorescent proteins.

Agonistic monoclonal antibodies targeting CD137/4-1BB have shown much preclinical promise but their clinical development has been slowed due to a poor therapeutic index, in particular liver toxicity. CB307 is a novel half-life extended bispecific Humabody VH targeting CD137 (4-1BB) and prostate specific membrane antigen (PSMA). The design of CB307 enables agonism of CD137 selectively in the presence of PSMA positive tumour cells and in this way enable tumour selective T cell activation whilst minimising systemic activation. The molecular weight of CB307 is less than 50 kDa (around a third of the size of a standard IgG) and it does not contain an Fc domain, thereby avoiding interaction with Fc receptors. Half life extension is achieved through the inclusion of a VH domain with specificity for human serum albumin (HSA). The identification of CB307 using the Crescendo Mouse™ which develops fully human VH domains in a background devoid of light chains will be described along with characterisation of the key properties of the molecule in in vitro and in vivo models. In dual target binding assays CB307 shows potent co-binding to both PSMA and CD137 targets and mediates CD137 signalling in an NFKB cell reporter assay in the presence of PSMA positive cells but not PSMA negative cells. Co-incubation of primary human T-cells from healthy individuals or cancer patients together with PSMA positive tumour cells and CD3 stimulation induces T-cell activation and cytokine release. In an in vivo model using NSG mice engrafted with human PBMCs the growth of PSMA positive DU145 prostate tumour cells is inhibited by a surrogate bispecific. Together these data support progression of CB307 into clinical development. Citation Format: James W. Legg, Brian McGuinness, Sophie Archer, Phil Bland-Ward, Verena Brucklacher, Jenny Craigen, Carolyn Edwards, Emma Hames, Jay Majithiya, pavel Pisa, Bhindu Revi, Nikki Royale, Angelica Sette, Yumin Teng, Lorraine Thompson, Wembin Wang, Chris Wilson, Chris Wyre, Chris Rossant. A novel T-cell costimulatory Humabody® VH therapeutic for PSMA-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 565.

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