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Shortened Hinge Design of Fab x sdAb-Fc Bispecific Antibodies Enhances Redirected T-Cell Killing of Tumor Cells.

ABSTRACT: T cell engager (TCE) antibodies have emerged as promising cancer therapeutics that link cytotoxic T-cells to tumor cells by simultaneously binding to CD3E on T-cells and to a tumor-associated antigen (TAA) expressed by tumor cells. We previously reported a novel bispecific format, the IgG-like Fab x sdAb-Fc (also known as half-IG_VH-h-CH2-CH3), combining a conventional antigen-binding fragment (Fab) with a single domain antibody (sdAb). Here, we evaluated this Fab x sdAb-Fc format as a T-cell redirecting bispecific antibody (TbsAbs) by targeting mEGFR on tumor cells and mCD3E on T cells. We focused our attention specifically on the hinge design of the sdAb arm of the bispecific antibody. Our data show that a TbsAb with a shorter hinge of 23 amino acids (TbsAb.short) showed a significantly better T cell redirected tumor cell elimination than the TbsAb with a longer, classical antibody hinge of 39 amino acids (TbsAb.long). Moreover, the TbsAb.short form mediated better T cell-tumor cell aggregation and increased CD69 and CD25 expression levels on T cells more than the TbsAb.long form. Taken together, our results indicate that already minor changes in the hinge design of TbsAbs can have significant impact on the anti-tumor activity of TbsAbs and may provide a new means to improve their potency.

SUBMITTER: Huang S

PROVIDER: S-EPMC9599842 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Shortened Hinge Design of Fab x sdAb-Fc Bispecific Antibodies Enhances Redirected T-Cell Killing of Tumor Cells.

Huang Shuyu S Segués Aina A Waterfall Martin M Wright David D Vayssiere Charlotte C van Duijnhoven Sander M J SMJ van Elsas Andrea A Sijts Alice J A M AJAM Zaiss Dietmar M DM

Biomolecules 20220921 10

T cell engager (TCE) antibodies have emerged as promising cancer therapeutics that link cytotoxic T-cells to tumor cells by simultaneously binding to CD3E on T-cells and to a tumor-associated antigen (TAA) expressed by tumor cells. We previously reported a novel bispecific format, the IgG-like Fab x sdAb-Fc (also known as half-IG_VH-h-CH2-CH3), combining a conventional antigen-binding fragment (Fab) with a single domain antibody (sdAb). Here, we evaluated this Fab x sdAb-Fc format as a T-cell re ...[more]

PMID: 36291540

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Project description:Thymidine Kinase 1 (TK1) is primarily known as a cancer biomarker with good prognostic capabilities for both hematological and solid malignancies. However, recent studies targeting TK1 at protein and mRNA levels have shown that TK1 may be useful as a therapeutic target. In order to examine the use of TK1 as a therapeutic target, it is necessary to develop therapeutics specific for it. Single domain antibodies (sdAbs), represent an exciting approach for the development of immunotherapeutics due to their cost-effective production and higher tumor penetration than conventional antibodies. In this study, we isolated sdAb fragments specific to human TK1 from a human sdAb library. A total of 400 sdAbs were screened through 5 rounds of selection by monoclonal phage ELISA. The most sensitive sdAb fragments were selected as candidates for preclinical testing. The sdAb fragments showed specificity for human TK1 in phage ELISA, Western blot analysis and had an estimated limit of detection of 3.9 ng/ml for the antibody fragments 4-H-TK1_A1 and 4-H-TK1_D1. The antibody fragments were successfully expressed and used for detection of membrane associated TK1 (mTK1) through flow cytometry on cancer cells [lung (~95%), colon (~87%), breast (~53%)] and healthy human mononuclear cells (MNC). The most sensitive antibody fragments, 4-H-TK1_A1 and 4-H-TK1_D1 were fused to an engineered IgG1 Fc fragment. When added to cancer cells expressing mTK1 co-cultured with human MNCs, the anti-TK1-sdAb-IgG1_A1 and D1 were able to elicit a significant antibody-dependent cell-mediated cytotoxicity (ADCC) response against lung cancer cells compared to isotype controls (P<0.0267 and P<0.0265, respectively). To our knowledge this is the first time that the isolation and evaluation of human anti-TK1 single domain antibodies using phage display technology has been reported. The antibody fragments isolated here may represent a valuable resource for the detection and the targeting of TK1 on tumor cells.

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Shortened Hinge Design of Fab x sdAb-Fc Bispecific Antibodies Enhances Redirected T-Cell Killing of Tumor Cells.

Publications

Shortened Hinge Design of Fab x sdAb-Fc Bispecific Antibodies Enhances Redirected T-Cell Killing of Tumor Cells.

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