Modular design of bi- and multi-specific knob domain fusions.

Introduction: The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively. Methods: Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights. Results: Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference. Discussion: Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.

Discovery of ginisortamab, a potent and novel anti-gremlin-1 antibody in clinical development for the treatment of cancer.

Gremlin-1, a high-affinity antagonist of bone morphogenetic proteins (BMP)-2, -4, and -7, is implicated in tumor initiation and progression. Increased gremlin-1 expression, and therefore suppressed BMP signaling, correlates with poor prognosis in a range of cancer types. A lack of published work using therapeutic modalities has precluded the testing of the hypothesis that blocking the gremlin-1/BMP interaction will provide benefits to patients. To address this shortfall, we developed ginisortamab (UCB6114), a first-in-class clinical anti-human gremlin-1 antibody, currently in clinical development for the treatment of cancer, along with its murine analog antibody Ab7326 mouse immunoglobulin G1 (mIgG1). Surface plasmon resonance assays revealed that ginisortamab and Ab7326 mIgG1 had similar affinities for human and mouse gremlin-1, with mean equilibrium dissociation constants of 87 pM and 61 pM, respectively. The gremlin-1/Ab7326 antigen-binding fragment (Fab) crystal structure revealed a gremlin-1 dimer with a Fab molecule bound to each monomer that blocked BMP binding. In cell culture experiments, ginisortamab fully blocked the activity of recombinant human gremlin-1, and restored BMP signaling pathways in human colorectal cancer (CRC) cell lines. Furthermore, in a human CRC - fibroblast co-culture system where gremlin-1 is produced by the fibroblasts, ginisortamab restored BMP signaling in both the CRC cells and fibroblasts, demonstrating its activity in a relevant human tumor microenvironment model. The safety and efficacy of ginisortamab are currently being evaluated in a Phase 1/2 clinical trial in patients with advanced solid tumors (NCT04393298).

Serum albumin binding knob domains engineered within a VH framework III bispecific antibody format and as chimeric peptides.

Background: Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering. Methods: Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion. Results: By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity. Conclusions: This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.

A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF.

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.

Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies.

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.

The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization.

Abstract Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.

Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection.

T-dependent Ab responses are characterized by parallel extrafollicular plasmablast growth and germinal center (GC) formation. This study identifies that, in mice, the Ab response against Salmonella is novel in its kinetics and its regulation. It demonstrates that viable, attenuated Salmonella induce a massive early T-dependent extrafollicular response, whereas GC formation is delayed until 1 mo after infection. The extrafollicular Ab response with switching to IgG2c, the IgG2a equivalent in C57BL/6 mice, is well established by day 3 and persists through 5 wk. Switching is strongly T dependent, and the outer membrane proteins are shown to be major targets of the early switched IgG2c response, whereas flagellin and LPS are not. GC responses are associated with affinity maturation of IgG2c, and their induction is associated with bacterial burden because GC could be induced earlier by treating with antibiotics. Clearance of these bacteria is not a consequence of high-affinity Ab production, for clearance occurs equally in CD154-deficient mice, which do not develop GC, and wild-type mice. Nevertheless, transferred low- and high-affinity IgG2c and less efficiently IgM were shown to impede Salmonella colonization of splenic macrophages. Furthermore, Ab induced during the infection markedly reduces bacteremia. Thus, although Ab does not prevent the progress of established splenic infection, it can prevent primary infection and impedes secondary hemogenous spread of the disease. These results may explain why attenuated Salmonella-induced B cell responses are protective in secondary, but not primary infections.

Distribution of the serine protease autotransporters of the Enterobacteriaceae among extraintestinal clinical isolates of Escherichia coli.

Urinary tract infections continue to be among the most common extraintestinal diseases. Cystitis in women is by far the most common urinary tract infection; pyelonephritis in both sexes and prostatitis in men are more severe but less frequent complaints. Escherichia coli is by far the most common cause of urinary tract infection. It is believed that uropathogenic E. coli is adept at colonizing the urinary tract via the production of specific virulence factors. Recently, a novel virulence determinant, Vat, was described for the prototypical uropathogenic E. coli strain CFT073. Vat is a member of the SPATE (serine protease autotransporters of the Enterobacteriaceae) subfamily of the autotransporters. Previously, SPATEs have been described for all pathovars of E. coli, but until recently their presence had been noticeably absent in nonpathogenic E. coli. In this report we describe the prevalence and phylogenetic distribution of the SPATEs among uropathogenic E. coli and the ECOR collection, demonstrating an association between the presence of the SPATEs, including Vat, and uropathogenic E. coli phylogroups. In addition, we describe the distribution of SPATEs among nonpathogenic E. coli.

Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications.

Clostridium botulinum neurotoxins are potently toxic proteins of 150 kDa with specific endopeptidase activity for SNARE proteins involved in vesicle docking and release. Following treatment with trypsin, a fragment of botulinum neurotoxin serotype A that lacks the C-terminal domain responsible for neuronal cell binding, but retains full catalytic activity, can be obtained. Known as the LH(N) fragment, we report the development of a recombinant expression and purification scheme for the isolation of comparable fragments of neurotoxin serotypes B and C. Expressed as maltose-binding protein fusions, both have specific proteolytic sites present between the fusion tag and the light chain to facilitate removal of the fusion, and between the light chain endopeptidase and the H(N) translocation domains to facilitate activation of the single polypeptide. We have also used this approach to prepare a new variant of LH(N)/A with a specific activation site that avoids the need to use trypsin. All three LH(N)s are enzymatically active and are of low toxicity. The production of specifically activatable LH(N)/A, LH(N)/B, and LH(N)/C extends the opportunities for exploitation of neurotoxin fragments. The potential utility of these fragments is discussed.