Generation of a novel fully human non-superagonistic anti-CD28 antibody with efficient and safe T-cell co-stimulation properties

Abstract

Antibody-based therapeutics represent an important class of biopharmaceuticals in cancer immunotherapy. CD3 bispecific T-cell engagers activate cytotoxic T-cells and have shown remarkable clinical outcomes against several hematological malignancies. The absence of a costimulatory signal through CD28 typically leads to insufficient T-cell activation and early exhaustion. The combination of CD3 and CD28 targeting products offers an attractive strategy to boost T-cell activity. However, the development of CD28-targeting therapies ceased after TeGenero's Phase 1 trial in 2006 evaluating a superagonistic anti-CD28 antibody (TGN1412) resulted in severe life-threatening side effects. Here, we describe the generation of a novel fully human anti-CD28 antibody termed "E1P2" using phage display technology. E1P2 bound to human and mouse CD28 as shown by flow cytometry on primary human and mouse T-cells. Epitope mapping revealed a conformational binding epitope for E1P2 close to the apex of CD28, similar to its natural ligand and unlike the lateral epitope of TGN1412. E1P2, in contrast to TGN1412, showed no signs of in vitro superagonistic properties on human peripheral blood mononuclear cells (PBMCs) using different healthy donors. Importantly, an in vivo safety study in humanized NSG mice using E1P2, in direct comparison and contrast to TGN1412, did not cause cytokine release syndrome. In an in vitro activity assay using human PBMCs, the combination of E1P2 with CD3 bispecific antibodies enhanced tumor cell killing and T-cell proliferation. Collectively, these data demonstrate the therapeutic potential of E1P2 to improve the activity of T-cell receptor/CD3 activating constructs in targeted immunotherapeutic approaches against cancer or infectious diseases.

Keywords: CD28; CD3; bispecific antibodies; cancer immunotherapy; monoclonal antibodies; phage display technology; protein engineering; tumor targeting.

Figure 1.

Generation and characterization of E1P2 monoclonal antibody. (a) Schematic representation of the phage display selection strategy to isolate new mAbs toward human CD28. The recombinant human CD28 protein was engineered with a biotin tag close to the superagonistic region (C’’D loop), to favor the isolation of binders toward the apex of CD28. (b) IgG₄(E1P2) shows a pure protein with the expected molecular weight in the SEC (left) and SDS-PAGE (right). (c) Binding of IgG₄(E1P2) and TGN1412 to primary human and mouse T cells by flow cytometry. Only E1P2 bound to both human and mouse T cells (with 18-fold lower apparent affinity to mouse CD28). Data are presented as mean±SD of technical triplicates from three independent flow cytometry measurements. MFI was calculated, and the sigmoid curve for antibody concentration (x-axis) against relative MFI (y-axis) was plotted and fitted using a 4-parameter logistic (4PL) non-linear regression model to calculate the EC₅₀ values.

Figure 2.

Epitope mapping of E1P2 using SPOT technology. (a) The binding regions of E1P2 (highlighted in light and dark blue) are different from that of TGN1412 (highlighted in red). (b) A cellulose membrane with overlapping peptides covering the ECD of human CD28 was used to highlight the binding epitope. E1P2 binds to the apex of CD28 close to the natural binding site of CD80/CD86 (near the MYPPPY region), unlike TGN1412, which was previously shown to bind to a lateral epitope (PDB: 1YJD). The binding spots of E1P2 on the cellulose membrane show a conformational epitope since two different regions were positive. (c) Sequence alignment of human/cyno/mouse CD28 with the potential binding residues of E1P2 highlighted in blue. The non-identical sequence between human/cyno/mouse CD28 is indicated with an asterisk (*).

Figure 3.

In vitro superagonistic assay on human PBMCs. (a) A graphical representation for the binding mode of conventional mAbs like E1P2 (top) and superagonistic mAbs like TGN1412 (bottom). Antibodies were wet coated on 6-well plates (6μg/well), and human PBMCs from a healthy donor were added (1.5 million/well). (b) A significant proliferation of PBMCs was observed using an MTS assay in the presence of TGN1412, but not E1P2, after 5 days. (c) Different pro-inflammatory cytokines (IL-2, IFN-γ, and TNF-α) were quantified by ELISA after 3 days. No cytokines were elevated after adding E1P2, unlike TGN1412. (d) The expression of an early activation marker (CD69) and a late activation marker (CD25) was assessed by flow cytometry on CD4⁺ and CD8⁺ T cells after 3 days. Activations markers were only elevated in the presence of TGN1412, but not E1P2. Data are presented as mean±SD. Each dot represents the value of a technical replicate. Statistical analysis of the data was performed by one-way ANOVA followed by Tukey’s multiple comparison test.

Figure 4.

In vitro co-stimulation assay with anti-CD3 antibodies. Freshly thawed human PBMCs from a healthy donor were used to test the activity of IgG₄(E1P2) in combination with either an anti-CD3 mAb (OKT3) or an anti-CD3/anti-EDB BiTE. (a) OKT3 was wet coated on 6-well plates (2μg/well), and antibodies (E1P2 IgG₄, TGN1412, or isotype control) were added to the solution (15μg/well) with human PBMCs. (b) A 2-fold increase in cell proliferation was observed when IgG₄(E1P2) was combined with OKT3, in comparison to when an isotype control was combined with OKT3. (c) Minimal secretion of cytokines was seen when adding OKT3 alone to human PBMCs, especially IL-2. A 100-fold increase in IL-2 secretion, a 10-fold increase in IFN-γ secretion, and a 5-fold increase in TNF-α were observed when combining IgG₄(E1P2) with OKT3. (d) EDB⁺ WI-38 cells were used as target cells to examine the synergistic effect in an in vitro killing assay. Human PBMCs were added (effector to target ratio of 5:1) in the presence of different concentrations of anti-CD3/anti-EDB BiTE, and in the presence or absence of IgG₄(E1P2) or controls. (e-h) A potent synergistic effect was observed as reflected in target cell lysis, CD25 expression, IFN-γ release, and cell proliferation. The activity seen with IgG₄(E1P2) was only in combination with CD3 bispecific antibodies, unlike the superagonistic TGN1412 antibody that showed nonspecific activation. Data are presented as mean±SD (n=3 from technical replicates). Statistical analysis of the data was performed by one-way ANOVA followed by Tukey’s multiple comparison test (B&C) or by two-way ANOVA followed by Dunnett’s multiple comparison test (E-H).

Figure 5.

In vivo safety study in humanized NSG mice. (a) The study design for the in vivo toxicity experiment highlighting the important milestones. NSG mice were humanized by engrafting 30x10 human PBMCs from two different healthy donors. IgG₄(E1P2), TGN1412, or PBS were injected, and a blood sample was taken after 6 hours to measure serum cytokines and to characterize the human CD45⁺ population. After 2 and 6 days, mice were sacrificed, and organs were examined for histopathological findings. (b) The TGN1412 group exhibited severe body weight loss upon treatment. (c) The human CD45⁺ population rapidly declined after the administration of TGN1412, but not E1P2. (d) Serum cytokines were quantified after 6 hours, and significant elevation was observed after injecting TGN1412, unlike E1P2. (e) Histopathological examination of vital organs after 6 days using H&E, Caspase3, and human CD45 staining. Necrotic tissues were observed in the TGN1412 group, but not in the E1P2 group. (f) The size of the spleen was measured by a ruler after 2 days, and splenomegaly was observed only after administering TGN1412. Representative data from two mice are shown in E and F. Data are presented as mean ± SD (n=3 from technical replicates, except in B in which n=4 from biological replicates). Statistical analysis of the data was performed by two-way ANOVA followed by Dunnett’s multiple comparison test (B-D). *p < 0.05; **p < 0.01; ***p < 0.001;****p < 0.0001. Scale bar: 100 µm.