Novel mesothelin antibodies enable crystallography of the intact mesothelin ectodomain and engineering of potent, T cell-engaging bispecific therapeutics

Mesothelin is a glypiated, cell-surface glycoprotein expressed at low levels on normal mesothelium but overexpressed by many cancers. Implicated in cell adhesion and multiple signaling pathways, mesothelin’s precise biological function and overall structure remain undefined. Antibodies targeting mesothelin have been engineered into immunotoxins, antibody-drug conjugates, CAR-T cells, or bispecific T cell engagers as candidate therapeutics but most face challenges, including binding epitopes that are not optimal for selected modalities. Here we describe the isolation and characterization of a novel anti-mesothelin antibody, 1A12, including crystallographic mapping of the 1A12 epitope in relation to other antibodies (amatuximab, anetumab). 1A12 possesses uniquely favorable properties, including a membrane-proximal epitope, and enabled structure determination of the complete mesothelin ectodomain. We incorporated 1A12 into two different bispecific T cell engaging architectures with various anti-CD3 co-targeting elements as candidate therapeutics, demonstrating in vitro functionality and potency

Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20–100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications

Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

<div><p>Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.</p> </div

Stably-induced expression of Scn does not drive apoptosis in FL5.12 cells.

<p>(A) FL5.12 cells were transduced with the pCVL-SFFV-muScn-IRES-GFP lentivirus and GFP mean fluorescence intensity was determined one-week post-transduction by cytometry, confirming transgene functionality. (B) A Western blot of supernatants, concentrated from 32 µL, from FL5.12 cells shows that the transduced cells constitutively express Scn, while parental cells in the presence or absence of IL-3 do not secrete detectable amounts of Scn after 72 h in culture. (C) Transduced FL5.12 were incubated with a variety of siderophores in order to assess the role of exogenous siderophores on cell viability (NT: no treatment). The hexadentate chelators DFO and Ent at 100 µM produce robust apoptosis, while the bidentate chelators at 300 µM do not affect viability.</p

Scn does induce apoptosis in murine 32D.3 or FL5.12 cells.

<p>FL5.12 (A) and 32D.3 (C) cells were incubated with 10 µM Scn and DFO for 48 h (NT: no treatment; -IL-3: in the absence of added IL-3). Apoptosis was assayed by annexin V-FITC staining and DAPI was used as a vital stain; percentages of cells positive for annexin staining are indicated. Average annexin V-positivity from three independent experiments are shown for FL5.12 (B) and 32D.3 (D) cells; error was calculated as the standard deviation of three replicates. Statistical significance is indicated as *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. Note that while the effect of adding Scn was significant, the effect was <i>anti</i>-apoptotic.</p

Steric clashes imposed by the Scn calyx preclude binding of ferric SA and GA complexes.

<p>(A) Hexadentate siderophore structures are shown with iron liganding atoms colored blue. (B) Structures of 2,3-DHBA, GA (2,5-DHBA), 3,4-DHBA and SA (2-hydroxybenzoic acid) are shown in the left column and complexes with iron in the center column (only two of three bidentate groups are shown for clarity). The Scn calyx is represented at top by a gray cylinder and the size constraint imposed by the calyx diameter is represented by dashed lines, schematically showing clashes with all iron complexes except 2,3-DHBA. (C) A section of the Scn/carboxymycobactin complex structure (PDB accession code 1X89) showing a GA moiety superimposed on the phenolate ring of carboxymycobactin. The steric clash of the 5-OH is indicated by penetrating the molecular surface of Scn (dashed red circle) and the short distance to neighboring atoms (green line).</p

BOCT N-and C-terminal domains do not bind Scn.

<p>Predicted BOCT membrane topologies are shown, either as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043696#pone.0043696-Devireddy2" target="_blank">[15]</a> (A) or calculated here (B), with transmembrane-spanning helices shown as blue cylinders. The sequence lengths of the NTD (green), CTD (red) and connecting loops are indicated; loops synthesized as peptides for binding analyses are indicated with numbered black circles, corresponding to the numbering in the <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043696#s4" target="_blank">Materials & Methods</a></b> section. The amino termini of fragments used to originally identify BOCT as a Scn receptor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043696#pone.0043696-Devireddy2" target="_blank">[15]</a> are indicated with orange arrows in (A). PAGE analyses of bacterially-expressed soluble, purified NTD (C) and CTD (D) are shown. SEC analysis of NTD/Scn is shown in (E). Complex formation would have been indicated by a shift in the Scn+NTD peak to lower elution volumes; in this case, the Scn/NTD mixture runs as the simple summation of the Scn and NTD alone peaks, indicating no binding under these conditions. (F) SPR analysis of Scn/CTD binding, with Scn analyte concentrations indicated. The bar indicates the sample injection period (association phase); gaps in the sensorgrams cover transients associated with injections.</p