A Bispecific Protein Capable of Engaging CTLA-4 and MHCII Protects Non-Obese Diabetic Mice from Autoimmune Diabetes

<div><p>Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) with a bispecific fusion protein (BsB) comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3) has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3⁺ regulatory T cells (Tregs) in an allogenic mixed lymphocyte reaction (MLR). Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD) female mice between 9–12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D). However, a longer course of treatment (10 weeks) of 4–13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing β-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing T1D.</p></div

Longer-term treatment of NOD mice with BsB significantly delayed the onset of T1D in NOD mice.

<p>(A) The cumulative incidences of overt diabetes in BsB-treated (n = 16) and untreated mice (n = 16). BsB treatment significantly reduced the incidence of T1D compared with mice treated with saline (<i>p</i><0.01). (B) A histopathological analysis of pancreatic tissues from animals treated with saline or BsB. Panels a through c represent the sections from saline-treated mice that remained non-diabetic with H&E, an antibody to insulin (pink), or anti-CD3 (brown) and Foxp3 (pink), respectively. Similar observations were noted in BsB-treated NOD mice that remained disease-free. No evidence of infiltration or insulitis was noted in any of the sections; a few Foxp3⁺ Tregs may be present (arrows in panel c). Panels d through f represent the pancreatic sections from diabetic NOD animals. Invasive insulitis was clearly evident, and the insulin-producing β-cells were completely destroyed (e). Several CD3⁺ T cell infiltrations were also detected along with a few Tregs and many non-T cell leukocytes with blue nuclei (f). Panels g through i show that the islets of BsB treated animals that remained non-diabetic but exhibited characteristic peri-insulitis. Leukocyte infiltrations were noted but were restricted to the periphery of the islets. Moreover, there was no notable destruction of the insulin-producing β-cells. Most of the leukocytes at the periphery were non-T cells (with blue nuclei). The enlarged inset (panel j, represents the red square in i) indicated Foxp3⁺ Tregs (yellow arrow head) were intermixed with other CD3⁺ T cells and non-T cell leukocytes (with blue nuclei) at the periphery of islets. The images were acquired with a 40× objective; the inset was acquired with a 60× objective, which was then further enlarged 3× digitally.</p

Analysis of asparagine-linked glycosylation on BsB.

<p>The amino acid sequence of BsB was submitted to the NetNGlyc 1.0 Server for the prediction of asparagine-linked glycosylation sites. A total of 10 asparagine-linked glycosylation sites (denoted N) were predicted; other amino acids are presented as dots. The monosaccharide composition of BsB was also performed to determine the composition of the glycans. Fucose (Fuc), N-acetylglucosamine (GlcNAc), galactose (Gal), mannose (Man), sialic acid, N-acetylneuraminic acid. A sialic acid:galactose ratio of 0.68 indicates that approximately one third of the galactose residues are available for binding to the ASGPR. Numbers represent mean ± std.</p

Treatment of NOD mice with BsB modestly delayed the onset of T1D in a late prevention treatment paradigm.

<p><b>(</b>A) The levels of Foxp3⁺ Tregs in the blood of BsB-treated (closed circles, n = 15) and saline-treated control mice (closed triangles, n = 14). There was a moderate but significant increase in the number of Tregs in the BsB-treated animals over the number of Tregs noted in the control animals. (B) The cumulative incidences of overt diabetes in animals treated with BsB (filled circles) or saline (filled triangles).</p

BsB-mediated induction of antigen-specific Tregs in vitro.

<p>(A) The in vitro induction of Ova_233–339-specific Tregs. Naïve OT-II T cells were mixed with LPS-activated and irradiated syngeneic APCs in the presence of 0.5 µg/ml Ova_233–239 peptide. Control mIgG2a, BsB, and BsB plus an anti-TGB-β antibody (αTGF-β) were then added and tested as indicated (left panels). The cells were cultured for 5 days and then labeled with anti-CD25 and anti-Foxp3 antibodies before being analyzed by flow cytometry. IL-2, IL-10 and TGF-β levels in the culture media were assayed by ELISA. (B) The monitoring of induced Tregs proliferation. The studies were conducted as in (A) except naïve OT-II T cells were pre-labeled with CFSE before being mixed with APCs. The cells were gated on Foxp3 and CFSE fluorescent channels.</p

Pharmacokinetics of BsB in vivo and biochemical analysis.

<p>(A) The pharmacokinetic profiles of BsB in mice. Normal C57BL/6 mice (n = 5) were dosed intraperitoneally with 20 mg/kg of BsB. Blood samples were collected at the different time points indicated, and the levels of BsB were determined using an ELISA. Data represent mean ± sem. (B) Comparison of the binding of BsB and mouse IgG2a to FcRn. The FcRn were immobilized to a Biacore chip as described in the material and methods. BsB or control mouse IgG2a was loaded onto the chip at various concentrations, and the signals were then recorded.</p

High-Affinity VEGF Antagonists by Oligomerization of a Minimal Sequence VEGF-Binding Domain

Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu²⁺ oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ∼50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ∼2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (∼5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfide-linked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc-independent VEGF antagonists with ultrahigh affinity, high stability, and a range of hydrodynamic radii for application to multiple therapeutic targets