Build-up functionalization of anti-EGFR × anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential

A Novel Bispecific Antibody against Human CD3 and Ephrin Receptor A10 for Breast Cancer Therapy.

Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3) was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (≤10(-1) μg/mL) and a lower effector to target (E/T) cell ratio (E/T = 2.5). Dimeric BsAb (EphA10/CD3) also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3), which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors

Creation of mouse TNFR2-selective agonistic TNF mutants using a phage display technique

Tumor necrosis factor-α (TNF), which is an immuno-modulatory cytokine, has been suggested to cause inflammatory responses as well as protection against tissue dysfunction by binding two types of TNF receptor (TNFR1/TNFR2). However, the physiological effects of TNFR2-specific activation remain unclear. We therefore aimed to generate a TNF mutant with full TNFR2-selective agonist activity as a functional analytical tool. In this study, we utilized a phage display technique to create mouse TNFR2 (mTNFR2)-selective TNF mutants that bind specifically to mTNFR2 and show full bioactivity compared with wild-type TNF. A new phage library displaying TNF mutants was created, in which nine amino acid residues at the predicted receptor-binding site were randomized. From this library, an agonistic TNF mutant exhibiting high binding selectivity and bioactivity to mTNFR2 was isolated. We propose that this TNF mutant would be a powerful tool with which to elucidate the functional roles of mTNFR2.•We generated a TNF mutant with full TNFR2-selective agonist activity.•This mutant was identified using a phage display technique.•This agonist exhibited high binding selectivity and bioactivity to mouse TNFR2.•This would be a powerful tool to elucidate the functional roles of mouse TNFR2

Dose-dependent effect of dimeric BsAb (EphA10/CD3) in BALB/c nu/nu mice.

<p>Each mouse (n = 6) was inoculated subcutaneously with a mixture of 10⁶ MDA-MB-435^EphA10 cells and 10⁶ human PBMC at an E/T ratio of 1 and the indicated doses of dimeric BsAb (EphA10/CD3) were administered intravenously on study days 0 to 3 (arrows). A) Mean values of tumor growth curves are shown for mice that were untreated (⬜) or only PBMC-treated (◇), or treated with PBMC and 1 μg dimeric BsAb (EphA10/CD3) (▲) or 10 μg dimeric BsAb (EphA10/CD3) (●). B) Mean values of tumor growth curves are shown for mice that were treated with PBMC and 10 μg anti-EphA10 IgG (■) or 10 μg anti-CD3 IgG (◆). C) Mean values of tumor growth curves are shown for mice that were only PBMC-treated (◇), or treated with PBMC and 10 μg dimeric BsAb (EphA10/CD3) (●) or 10 μg dimeric BsAb (HIs/CD3) (▲). Values represent mean tumor sizes (in mm³) ± SEM (n = 6 per group). Asterisks label readings that were statistically significant (unpaired Student’s T-test) from the Ab treated group and the untreated group (**: P<0.01, *: P<0.05). Daggers indicate that differences were statistically significant from BsAb (EphA10/CD3) and BsAb (His/CD3) (¶¶: P<0.01).</p

<i>In vitro</i> cytotoxicity of BsAb (EphA10/CD3) against MDA-MB-435^EphA10 and parental cells.

<p>The left panels are monomeric BsAb (A, B) and the right panels are dimeric BsAb (C, D). MDA-MB-435 parental cells (slashed column) and MDA-MB-435^EphA10 (black column) cells were co-cultured with human PBMC at E/T ratios of 2.5 (A, C) and 5 (B, D). Each point represents the mean of triplicate determinations; Error bars represent the standard deviations of triplicate determinations. Asterisks label readings that were statistically significant (unpaired Student’s T-test) from MDA-MB-435 and MDA-MB-435^EphA10 (***: P<0.001, **: P<0.01).</p

Th1 cytokine production from human PBMCs after BsAb (EphA10/CD3) reaction.

<p>Non-stimulated PBMC (E/T ratio of 5) were incubated in the presence of MDA-MB-435^EphA10 breast cancer cells (black column) or no target cells (slashed column) with various concentrations of each BsAb (EphA10/CD3) and control sample (BsAb (His/CD3), full IgG). The production of (A) IFN-γ, (B) IL-2 and (C) TNF-α in the presence of monomer (left panel; 1–10 μg/mL) or dimer (right panel; 0.1–1 μg/mL) was determined from the cell culture supernatants. Control samples were incubated with BsAb (His/CD3) (1 or 10 μg/mL) or anti-EphA10 IgG (1 or 10 μg/mL) or anti-CD3 IgG (1 or 10 μg/mL). Each point represents the mean of triplicate determinations; Error bars represent the standard deviations of triplicate determinations. Asterisks label readings that were statistically significant (unpaired Student's T-test) from PBMC with or without MDA-MB-435^EphA10 (***: P<0.001, **: P<0.01).</p

Characteristics of BsAb (EphA10/CD3) and BsAb (His/CD3).

<p>(A) Gene construct of operons encoding each BsAb in plasmid pcDNA3.1. (B) Gel filtration chromatography profile of BsAb (EphA10/CD3) and BsAb (His/CD3), which were purified with IMAC. (C) SDS-PAGE and Western blot analysis of dimer (lane D) and monomer (lane M) fractions. The parallel line shows molecular size standards with their apparent molecular weights in kiloDalton (kDa). The down arrow shows elution peak of monomeric and dimeric BsAb. Abbreviations: D is dimer, M is monomer.</p

Binding activity of monomeric and dimeric BsAbs.

<p>The left panels (A, B, C) are monomeric and dimeric BsAbs (EphA10/CD3) and the central panels (D, E, F) are monomeric and dimeric BsAbs (His/CD3) and the right panels (G, H, I) are control full IgG (anti-EphA10, anti-CD3). MDA-MB-435 parental cells, human EphA10 transfected cells (MDA-MB-435^EphA10) and Jurkat cells (CD3 positive) were used for flow cytometric analysis. Binding activities of each antibody sample were measured at 6 μg. Cell-binding proteins were detected via SureLight P3 conjugated anti-His tag or anti-mouse IgG mAb. Gray filled-in area is vehicle control (PBS). Line indicates each antibody sample (dotted lines are dimeric BsAb).</p