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. 2023 Jan-Dec;15(1):2231129.
doi: 10.1080/19420862.2023.2231129.

Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies

Han Ping Loh  1 , Farouq Bin Mahfut  1 , Serene W Chen  1 , Yuhan Huang  2 , Jianxin Huo  2 , Wei Zhang  1 , Kong Peng Lam  2   3 , Shengli Xu  2   4 , Yuansheng Yang  1
Affiliations

Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies

Han Ping Loh et al. MAbs. 2023 Jan-Dec.

Abstract

T-cell-engaging bispecific antibodies (T-bsAbs) are promising immunotherapies for cancer treatment due to their capability of redirecting T-cells toward destroying tumor cells. Numerous T-bsAb formats have been developed, each with advantages and disadvantages in terms of developability, immunogenicity, effector functions, and pharmacokinetics. Here, we systematically compared T-bsAbs produced using eight different formats, evaluating the effect of molecular design of T-bsAbs on their manufacturability and functionality. These eight T-bsAb formats were constructed using antigen-binding fragments (Fabs) and single-chain variable fragments (scFvs) of antibodies linked to the crystallizable fragment (Fc) domain of immunoglobulin G. To ensure a fair comparison of growth and production data, we used recombinase-mediated cassette exchange technology to generate the T-bsAb-producing CHO cell lines. The produced T-bsAbs were assessed for their purification profile and recovery, binding capability, and biological activities. Our findings indicated that the manufacturability of bsAbs was adversely affected with increased number of scFv building blocks, while the functionality was affected by the combination of multiple factors, including the binding affinity and avidity of targeting moieties and the flexibility and geometry of formats. These results provide valuable insights into the impact of the format design on the optimal production and function of T-bsAbs.

Keywords: CHO cell; avidity; bispecific antibody; functionality; manufacturability; targeted integration; thermal stability.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

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.
Figure 1.
Schematic diagram of the eight T-bsAb formats tested in the current study.
(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.
Figure 2.
Schematic diagram of the RMCE process and targeting vector design.
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.
Figure 3.
Comparison of titer, IVCD and qP of eight different HER2×CD3 T-bsAbs produced in CHO cells.
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.
Figure 4.
AKTA profile during CEX purification and SEC profile of eight different HER2×CD3 T-bsAbs after an initial protein a step.
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.
Figure 5.
Flow cytometric analysis of antigen binding capacities of eight different HER2×CD3 T-bsAbs.
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.
Figure 6.
T cell activation upon stimulation of various HER2×CD3 T-bsAbs in the presence of tumor cells.
(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.
Figure 7.
Tumor cell-killing and cytokine production by T cells induced by eight different HER2×CD3 T-bsAbs.
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