https://orcid.org/0000-0002-0026-7069
Figures & data
Figure 1. Schematic diagram of the eight T-bsAb formats tested in the current study.
Illustration of the 8 bsAb formats using circles and lines. Each bsAb targets both HER2 and CD3 and are of different orientation and valency. Top left are 3 antibodies in the “1+1” format; Bottom left are 3 antibodies in the “2+1” format; Right are 2 antibodies in the “2+2” format.
Formats tested in this study include “1 + 1”, “2 + 2” and “2 + 1” mixed-valency bispecific antibodies enabled by either a homodimeric Fc or a heterodimeric Fc using the ‘Knob in hole’ technology. One arm of the scFv2 only contains the hinge, CH2, and CH3 domains. Green arms target HER2, and Blue arms target CD3.
Figure 2. Schematic diagram of the RMCE process and targeting vector design.
(Top) Diagram of the RMCE process, showing the replacement of the landing pad with the gene of interest using complementary FRT sites. (Bottom) Diagram of the components of the 8 bsAb formats.
(a) Overview of RMCE targeted integration platform for expressing the BsAbs in CHO cells. CHO-K1 cells were stably tagged with an Flp/FRT RMCE cassette (landing pad) via random integration. Cells bearing single landing pad was selected and used as master cell line(MCL). The targeting vector was designed to be promoter-less and consist of the FRT3/FRT sites (black and white triangles), gene-of-interest (GOI), and an IRES element to activate puromycin gene expression upon successful RMCE. (b) Schematic representation of targeting vectors carrying bsAb LC and HC genes linked by multiple wild-type EMCV IRES elements. hCMV: Human cytomegalovirus; EGFP; enhanced green fluorescence protein; NPT; neomycin phosphotransferase; pA: polyA tail; Pur: puromycin resistant gene; FLPe: flippase IRES: internal ribosome entry site; VH: variable heavy chain; VL: variable light chain; CHK: heavy chain-knob; FcH: fab-hole; DHFR: dihydrofolate reductase.
Figure 3. Comparison of titer, IVCD and qP of eight different HER2×CD3 T-bsAbs produced in CHO cells.
Bar Chart showing the titer. IVCD and qP of the bsAb cultures on day 14. The charts showed that all 8 bsAbs have similar IVCD however titer and qP is different for each format.
Comparison of the (a) titer, (b) IVCD and (c) qP of the 8 bsAb cultures harvested on day 14. Error bars show the standard error of the mean (SEM) of quadruplicate fed-batch cultures of four independent stable pools. IVCD: Integral viable cell concentration; qP: specific productivity, calculated by using day 14 titer/day 14 IVCD.
Figure 4. AKTA profile during CEX purification and SEC profile of eight different HER2×CD3 T-bsAbs after an initial protein a step.
AKTA and SEC chromatograms arranged according to valency in the following order: “1+1” (left), “2+1” (middle) and “2+2” (Right). AKTA chromatogram showed multiple peaks for all 8 bsAb. After CEX purification, all 8 bsAb SEC profiles displayed narrower peaks.
AKTA chromatograms were shown on the left of each graph. SEC profiles before CEX (black) and after CEX (green) were shown on the right of each graph.
Table 1. Aggregation profile of T-bsAbs of different formats post Protein a and post CEX.
Table 2. Effect of different format on Thermal Melting Temperatures (Tm1, Tm2, Tm3, and Tm4) of Trastuzumab and bispecific antibodies as measured by DSC. The Tm value indicated the midpoint temperature of the thermal unfolding transition of antibody.
Table 3. Binding affinity of different antibody formats to CD3E/D and Her2 via Octet and cell-based assays.
Figure 5. Flow cytometric analysis of antigen binding capacities of eight different HER2×CD3 T-bsAbs.
Flow cytometric analysis of CD3 binding (left) and HER2 binding (right) of the 8 bsAbs. CD3 binding of “1+1”, “2+1” and “2+2” formats were similar within the same group, with the 2+2 formats having the highest binding to CD3. The “2+1” and “2+2” formats have higher binding to HER2 as compared to “1+1” format.
The binding capacities of various HER2×CD3 T-bsAbs for antigens CD3 (a) and HER2 (b) antigens were determined by a competitive binding assay using flow cytometry. Jurkat T or SK-OV-3 cells were stained with diluted anti-human CD3 (clone UCHT1) or HER2 (Trastuzumab) antibodies conjugated with APC in the presence of various serially diluted T-bsAbs. The intensities of APC fluorescence were assayed on an LSR II (BD Pharmingen) and used for calculating the IC50 with a four-parameter analysis model of GraphPad Prism 6. The smallest and highest mean fluorescence intensity (MFI) for each data set was normalized as 0% and 100%, respectively.
Figure 6. T cell activation upon stimulation of various HER2×CD3 T-bsAbs in the presence of tumor cells.
Chart of CD25 and CD69 upregulation in CD4 and CD8 T cells. The symmetric (FabscFv-FcW)2 in the “2+2” format has the strongest CD25 and CD69-inducing efficacy among the bsAbs tested.
CD25 (a,c) and CD69 (b,d) cell surface expression in CD4+ and CD8+ T cells treated with different HER2×CD3 bsAbs at various concentrations and in the presence of SK-OV-3 cells. Purified human T cells were co-cultured with SK-OV3 cells at an E:T ratio of 1:1 in the presence of the indicated concentration of HER2×CD3 T-bsAbs for 16 h. The cells were harvested and stained with fluorescein-conjugated anti-human CD4, CD8, CD25, and CD69 antibodies and subjected to flow cytometric analysis. The percentages of CD25+ or CD69+ cells of the CD4+ or CD8+ T cells were analyzed by Flow Jo. The EC50 of CD25 and CD69 upregulation of the individual T-bsAb was calculated with a four-parameter logistic sigmoidal dose-response curve using GraphPad Prism. The data shown are representative of more than three independent experiments.
Table 4. Summary of results for cell-based functional characterization assays.
Figure 7. Tumor cell-killing and cytokine production by T cells induced by eight different HER2×CD3 T-bsAbs.
(Top) Chart of T cell-mediated killing showing maximum killing achieved at different bsAb concentrations. (Bottom) Bar chart showing IL2 and IFNγ production in T cells after stimulation with the 8 bsAbs in the presence of SK-OV-3 cells. (FabscFv-FcW)2 in the “2+2” format has the greatest killing efficacy and induced the highest production of IL2 and IFNγ in T cells.
(a) Analysis of T-bsAb-mediated killing of SK-OV-3 cells by T cells. Purified CD3+ human T cells were co-cultured with Her2+ SK-OV3-luciferase cells at an E:T ratio of 1:1 in the presence of the indicated concentration of various HER2×CD3 T-bsAb for 48 hours. The luciferase activity was used as the surrogate for cell viability and determined by measuring the luminescence in triplicates after adding the substrate luciferin. The EC50 of the tumor cell-killing of the T-bsAb was calculated with a four-parameter logistic sigmoidal dose-response curve using GraphPad Prism. (b,c) Production of IL-2 (b) and INFγ (c) by T cells upon stimulation of T-bsAbs. Human T cells were co-cultured with HER2+ SK-OV-3-luc cells in the presence of the indicated concentrations of various HER2×CD3 T-bsAb for 48 h. IL-2 and IFNγ in the culture supernatant were determined using ELISA in duplicates. The data shown are representative of more than three independent experiments.
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